Binding of Hg(I) and Hg(II) to amyloid-beta (Aβ) peptide variants: effects on structure and aggregation

Binding of Hg(I) and Hg(II) to amyloid-beta (Aβ) peptide variants: effects on structure and aggregation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Binding of Hg(I) and Hg(II) to amyloid-beta (Aβ) peptide variants: effects on structure and aggregation Elina Berntsson, Andra Noormägi, Kärt Padari, Jüri Jarvet, Astrid Gräslund, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5888115/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Mercury (Hg) exposure is a possible risk factor for Alzheimer´s disease (AD), and some studies have found higher Hg levels in AD patients. Yet, the evidence is inconclusive, and a mechanism linking Hg exposure to AD neuropathology remains to be found. The hallmark of AD brains is deposits of insoluble amyloid plaques consisting mainly of aggregated amyloid-β (Aβ) peptides. Here, we use transmission electron microscopy (TEM) and biophysical spectroscopy techniques to study in vitro interactions between inorganic Hg and the pathologically relevant Aβ(1-40) and Aβ(4-40) variants and the Aβ(1-40)(H6A, H13A, H14A) mutant. For the first time, the effect on Aβ aggregation of both Hg(I) and Hg(II) is compared. Hg(II) binds to Aβ(1-40) with an apparent binding affinity of 28±8 µM. The N-terminal His6, His13 and His14 residues are involved in binding coordination. Hg(II) binding induces structural alterations (coil-coil interactions) in Aβ monomers positioned in membrane-mimicking SDS micelles. Equimolar amounts of either Hg(I) or Hg(II) inhibit normal Aβ fibrillation by directing the aggregation process towards formation of large amorphous aggregates. All these structural rearrangements may be relevant for the harmful Aβ aggregation processes involved in AD brain pathology. Inducing protein misfolding and aggregation might be a general toxic mechanism of mercury. Biophysics General Biochemistry Chemical Biology Inorganic mercury Alzheimer’s disease Amyloid aggregation Metal-protein binding Neurodegeneration Heavy metal toxicity Spectroscopy Transmission electron microscopy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction A possible connection between Alzheimer´s disease (AD) and environmental mercury (Hg) exposure has been debated for a long time (Mutter et al., 2004 ; Rooney, 2007 ; Mutter et al., 2010 ; Bjørklund et al., 2019 ; Siblerud et al., 2019 ; Wallin et al., 2019 ; Babic Leko et al., 2023 ; Pamphlett and Bishop, 2023 ; Althobaiti, 2025 ). Hg is a heavy metal with established neurotoxic properties (Oliveira et al., 2018 ; Nordberg and Costa, 2022 ), and some studies have found higher Hg levels in AD patients (Xu et al., 2018 ; Siblerud et al., 2019 ; Babic Leko et al., 2023 ). But the evidence is inconclusive, and a number of studies have reported normal Hg levels in both blood and brain of AD patients (Babic Leko et al., 2023 ; Kooshki et al., 2024 ). A complicating factor is that AD and mercury intoxication share a common genetic risk factor, namely the APOE-ε4 gene variant (Mutter et al., 2004 ). That is, individuals with APOE-ε4 have an increased probability of developing AD (Belloy et al., 2023 ), and also suffer more severe consequences from Hg exposure (Arrifano et al., 2018a; Arrifano et al., 2018b; Santos-Sacramento et al., 2021 ). As anthropogenic emissions of mercury continue to increase, mainly due to mining activities and human combustion of coal and oil (UNEP, 2019), it becomes important to investigate how Hg might contribute to AD pathology (Bjørklund et al., 2019 ). Hg is a neurotoxic and genotoxic heavy metal that induces damage to organs such as the brain and the kidneys (Bernhoft, 2012 ; Rice et al., 2014 ; Arrifano et al., 2018b; Nordberg and Costa, 2022 ). Exposure can lead to permanent health problems and even death (Bernhoft, 2012 ; Rice et al., 2014 ; Santos-Sacramento et al., 2021 ), with different outcomes depending on dose level and exposure time (James et al., 2022 ). The molecular mechanisms underlying Hg toxicity are not fully known (Bjørklund et al., 2019 ), but include toxic molecular mimicry (Bridges and Zalups, 2005 ) and disruption of antioxidant activity (Yang et al., 2020 ; Kang et al., 2024 ) especially in the mitochondria (Carocci et al., 2014 ). The different forms of Hg, e.g., metallic, inorganic Hg(I) and Hg(II) species, and organo-metallic complexes such as methyl-Hg and ethyl-Hg, have different chemical properties, toxicity profiles and species distributions (Leermakers et al., 2005 ; Oliveira et al., 2018 ; Nordberg and Costa, 2022 ). For inorganic Hg(I) and Hg(II) species, the retention time in the human brain is several years, or even decades (Rooney, 2014 ). AD is a progressive, irreversible, and currently incurable chronic neurodegenerative disorder, and also the leading cause of dementia worldwide (Scheltens et al., 2021 ; Alzheimer's_Association, 2024 ). Pathological hallmarks of AD include brain atrophy, with extensive deposits of amyloid plaques and neurofibrillary Tau tangles occurring years before symptom manifestation (Scheltens et al., 2021 ; Alzheimer's_Association, 2024 ). The insoluble plaques consist mainly of amyloid-β (Aβ) peptides (Glenner and Wong, 1984 ) aggregated into fibrils that display a characteristic cross-β structure (Sunde and Blake, 1998 ). The plaques are the end-product of an aggregation process (Luo et al., 2016 ; Selkoe and Hardy, 2016 ; Lee et al., 2017 ) that involves formation of extra- and intracellular intermediates such as soluble neurotoxic Aβ oligomers (Glabe, 2008 ). As the latter are considered one of the main toxic species in AD brains (Ghosh et al., 2023 ), it becomes imperative to investigate factors that may influence Aβ aggregation and oligomer formation (Owen et al., 2019 ). The Aβ peptides are intrinsically disordered in monomeric form and soluble in water (Wärmländer et al., 2013 ). The central and C-terminal Aβ segments are hydrophobic and can interact with membranes or fold into a hairpin conformation that likely is required for aggregation (Abelein et al., 2014 ), while the anionic N-terminal segment is hydrophilic and readily interacts with metal ions (Wärmländer et al., 2013 ; Wärmländer et al., 2019 ). Inorganic Hg(II) has previously been shown to bind to Aβ peptides and interfere with the Aβ aggregation mechanisms (Meleleo et al., 2019 ; Wallin et al., 2019 ). Specifically, Hg(II) has been reported to increase Aβ 42 toxicity, promote formation of high-molecular-weight soluble aggregates, and reduce the propensity of Aβ 42 to form harmful ion channels in membranes (Meleleo et al., 2020 ). However, no study has so far investigated Aβ interactions with monovalent Hg(I), even though both forms exist in vivo . It is still not clear if the formation and/or activity of toxic Aβ oligomers in AD brains occurs extracellularly, where the oxidizing environment yields Hg(II), or/and intracellularly, where the reducing environment yields Hg(I). In this study, we use transmission electron microscopy (TEM) imaging together with fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to study in vitro interactions between Aβ peptides and inorganic Hg ions. The focus is on Hg binding properties and Hg binding effects on Aβ structure and aggregation. Both Hg(I) and Hg(II) are investigated and compared for the first time. The studied Aβ peptides are the pathologically relevant Aβ(1–40) and Aβ(4–40) variants, together with the Aβ(1–40)(H6A, H13A, H14A) mutant that is used to investigate the importance of the Aβ His residues for Hg binding. 2. Materials and Methods 2.1 Materials Recombinantly produced wild-type (wt) Aβ(1–40) peptide, abbreviated as Aβ 40 , with the primary sequence DAEFR 5 HDSGY 10 EVHHQ 15 KLVFF 20 AEDVG 25 SNKGA 30 IIGLM 35 VGGVV 40 , were purchased as lyophilized powder from AlexoTech AB (Umeå, Sweden). Recombinantly produced truncated Aβ(4–40) peptide, abbreviated as Aβ 4−40 , Aβ(1–40)(H6A, H13A, H14A) mutant peptide, abbreviated as Aβ NoHis , and uniformly 15 N-labeled Aβ 40 peptide were also purchased from AlexoTech AB. All Aβ peptide variants were stored at -80°C until use, when they were dissolved to monomeric form in 10 mM NaOH. The fresh solutions were sonicated for 5 mins in an ice-bath to dissolve possible pre-formed aggregates. Then, phosphate buffer or 2-(N-Morpholino)ethanesulfonic acid hydrate (MES) buffer was added. All preparation steps were performed on ice. The peptide concentrations were first estimated from the weight of the dry powder, and then more accurately determined by NanoDrop measurements of the solutions. 2.2 Transmission electron microscopy (TEM) imaging Negative staining TEM images were recorded for 10 µM Aβ 40 peptide in 20 mM MES buffer, pH 7.3, that had been incubated for 20 hours on a thermo shaker operating at 37 o C and 300 rpm, together with either 0 µM, 0.5 µM, 1 µM, 3 µM, or 10 µM of Hg(NO 3 ) 2 . Then, 5 µL of each incubated sample were put on copper grids of 200 µm mesh size. The grids were covered with Pioloform film upon which a carbon layer had first been deposited and then glow-discharged using a Leica EM ACE600 carbon coater (Leica Microsystems, Germany). The Aβ 40 samples were absorbed to the grids for 5 mins, rinsed with Milli-Q water two times, and then stained for 2 mins with 2% aqueous solution of uranyl acetate. The excess stain was removed with filter paper, and then the samples were left to air-dry. A digital Orius SC1000 camera was used to record TEM images in a FEI Tecnai G2 Spirit electron microscope (FEI, The Netherlands) operating at 120 kV accelerating voltage. To investigate the effects of both monovalent Hg(I) and divalent Hg(II) on Aβ aggregation, all samples were prepared and incubated in both oxidizing and reducing conditions, respectively. Standard oxidizing conditions yield the Hg(II) form, which typically exists as free Hg 2+ ions. A reducing condition, roughly corresponding to the chemical environment inside the cell cytosol, was obtained by adding 1 mM of the reducing agent TCEP to the samples. This yields monovalent Hg(I), which typically exists as diatomic Hg 2 2+ ions (Neisler and Pitzer, 1987 ; Argent et al., 2006 ). 2.3 Fluorescence spectroscopy Fluorescence measurements were carried out with a Jobin Yvon Horiba Fluorolog 3 fluorescence spectrometer (Longjumeau, France). Emission intensities at 306 nm (excitation 276 nm) were recorded at room temperature for samples of 20 µM Aβ peptide (Aβ 40 , Aβ NoHis , or Aβ 4−40 ) in 20mM MES buffer, pH 5.1 or 7.3, using a quartz cuvette with 4mm path length and a total volume of 600 µL. For Aβ 40 at pH 7.3, measurements were also conducted in the presence of 50 mM SDS (sodium dodecyl sulfate) surfactant. Small aliquots of Hg(NO 3 ) 2 were titrated to the samples (less than 3% of total volume added) using stock solutions of 2 mM, 3 mM, and 10 mM Hg(NO 3 ) 2 , respectively. The measured tyrosine fluorescence intensities were then plotted against the concentration of Hg ions. Dissociation constants (K D ) for the Aβ·Hg complexes were calculated by fitting the data curves to Eq. 1, the Morrison equation (Kuzmic, 2015 ): Eq. 1: $$\:\text{I}={\text{I}}_{0}+\frac{{\text{I}}_{{\infty\:}}-{\text{I}}_{0}}{2·\left[\text{A}{\beta\:}\right]}·\left(\left({\text{K}}_{\text{D}}+\left[\text{H}\text{g}\right]+\left[\text{A}{\beta\:}\right]\right)-\sqrt{{\left({\text{K}}_{\text{D}}+\left[\text{H}\text{g}\right]+\left[\text{A}{\beta\:}\right]\right)}^{2}-4·\left[\text{H}\text{g}\right]·\left[\text{A}{\beta\:}\right]}\right)$$ Here, I 0 is the initial fluorescence intensity with no added Hg ions, I ∞ is the steady-state intensity at the end of the titration, [Hg] is the concentration of added Hg ions, K D is the dissociation constant, and [Aβ] is the peptide concentration. This version of the equation assumes that the peptide has a single binding site for Hg ions. Adding a linear term for the fluorescence quenching effect of free Hg ions appeared unnecessary (Lindgren et al., 2013 ). In this study no corrections were made for possible interactions between the buffer and the Hg ions. The derived dissociation constants should therefore be considered as apparent, i.e. K D App . However, the buffer effects are likely small, as MES is a Good buffer devised to have minimal interactions with metal ions and other cations (Good et al., 1966 ). For each condition three separate samples were prepared and measured, allowing mean K D App values to be calculated. 2.4 Nuclear magnetic resonance (NMR) spectroscopy Nuclear magnetic resonance (NMR) spectroscopy measurements were performed on a 700 MHz Bruker Avance spectrometer equipped with a cryoprobe. Two-dimensional 1 H, 15 N-HSQC spectra were recorded at 5°C for 92 µM monomeric 15 N-labelled Aβ 40 peptide in 20 mM MES buffer at pH 5.1, (90/10 H 2 O/D 2 O), before and after addition of 92 µM Hg(NO 3 ) 2 . The Topspin v.3.6.2 software was used to process the NMR spectra, and the crosspeaks were assigned according to previously published data at both neutral pH (Danielsson et al., 2006 ; Yamaguchi et al., 2011 ; Roche et al., 2016 ) and at acidic pH (Ghalebani et al., 2012 ). 2.5 CD spectroscopy Circular dichroism (CD) spectra were recorded between 195 nm and 250 nm, with 0.5 nm step size, using a Chirascan CD instrument (Applied Photophysics Ltd., U.K.) operating at 20°C. A quartz cuvette with 2 mm pathlength was used to hold 600 µL of either 10 µM Aβ 40 peptide or 10 µM Aβ 4−40 peptide in 20 mM phosphate buffer, pH 7.3. Both samples also contained 50 mM SDS surfactant. As this concentration is much higher than the critical micelle concentration (cmc) of SDS, which in pure water at 25°C is 8.2 mM (Dominguez et al., 1997 ), the SDS forms micelles that constitute a simple but efficient membrane model (Österlund et al., 2019 ). As the SDS micelle concentration is much higher than the Aβ concentration, there should be no more than one Aβ peptide in each micelle. To both Aβ variants, i.e. Aβ 40 and Aβ 4−40 , the dissolved mercury salt Hg(NO 3 ) 2 was added in steps of 16 µM, 56 µM, 156 µM and 256 µM. The recorded spectra were processed with the Chirascan Pro-Data v.4.4.1 software (Applied Photophysics Ltd., U.K.), including smoothing with an eight points Savitzky-Golay smoothing filter. 3. Results 3.1 Transmission electron microscopy (TEM) imaging TEM images were recorded to study the morphologies of aggregates of 10 µM Aβ 40 peptide, incubated for 20 hours together with different concentrations of Hg(NO 3 ) 2 . The incubations were carried out either in MES buffer only (Fig. 1 ), or together with 1 mM of the reducing agent TCEP (Fig. 2 ). Under standard oxidizing conditions, i.e. without TCEP present, the aggregated control sample of Aβ 40 peptide without added mercury displays slender fibrils that are several microns long, together with smaller aggregate particles that might be protofibrils (Fig. 1 A). This is in line with previous in vitro studies of the size and shape of self-aggregated Aβ 40 fibrils (Wallin et al., 2017 ), which appear to grow out from globular protofibrillar assemblies (Luo et al., 2014 ). A similar fibril morphology is observed for the Aβ 40 samples incubated with 0.5 µM Hg(II) ions (Fig. 1 B). The Aβ 40 sample with 1 µM Hg(II) ions is somewhat different, displaying a mixture of slender fibrils, small aggregate particles, and larger amorphous aggregates (Fig. 1 C). The Aβ 40 samples with 3 µM and 10 µM Hg(II) contain no fibrils, but only small and large aggregate particles (Figs. 1 D and 1 E). The sample with 10 µM Hg(II) is dominated by large aggregated particles, roughly half a micron across (Fig. 1 E). This shows that Hg(II) ions promote formation of large amorphous aggregates rather than slender fibrils. The effect is clearly concentration-dependent, and the formation of long fibrils is completely inhibited already at sub-stoichiometric mercury concentrations, i.e. with 3 µM Hg(NO 3 ) 2 to 10 µM Aβ 40 peptide (Fig. 1 D). For the samples incubated under reducing conditions, i.e. with 1 mM TCEP present, the Aβ 40 control sample without added mercury again displays numerous amyloid fibrils, several microns long, together with small aggregate particles (Fig. 2 A). The Aβ 40 samples incubated together with 0.5 µM, 1 µM, and 3 µM Hg(I) display similar combinations of amyloid fibrils and small amorphous aggregates (Figs. 2 B, 2 C, and 2 D). At 10 µM added Hg(I) the Aβ 40 peptide no longer forms slender fibrils, but rather amorphous aggregates in various sizes (Fig. 2 E). This shows that Hg(I) has a similar, albeit weaker, concentration-dependent effect on Aβ 40 aggregation as Hg(II). For example, complete inhibition of Aβ 40 fibrillation is achieved by Hg(I) at stoichiometric concentrations (1:1 Hg(I):Aβ 40 ratio; Fig. 2 E), but by Hg(II) already at sub-stoichiometric concentrations (3:10 Hg(II):Aβ 40 ratio; Fig. 1 D). 3.2 Fluorescence measurements of Hg(II) binding affinity Hg ions have previously been shown to be able to quench the intrinsic fluorescence of Tyr residues (Berntsson et al., 2022 ), similar to e.g. Ag(I), Cu(II), and UO 2 (II) ions (Alies et al., 2013 ; Lindgren et al., 2013 ; Tiiman et al., 2016 ; Berntsson et al., 2023a; Lakela et al., 2025 ). The effect of added Hg(II) on the fluorescence of Tyr10, the only natural fluorophore in Aβ peptides, was therefore used to evaluate binding affinities for Hg(II)·Aβ complexes in different conditions (Fig. 3 ), using three replicates for each Aβ variant and/or condition, For titrations with Hg(II) at pH 7.3, the fluorescence intensity curves can be fitted to Eq. 1 with good accuracy (Fig. 3 ). This shows that the binding interactions are dominated by a single binding site. As seen in Table 1 , Hg(II) displays the strongest binding to the Aβ 4−40 variant (3.5 ± 1.1 µM), weakest binding to Aβ NoHis (60 ± 18 µM), and intermediate binding affinity to wild-type Aβ 40 (28 ± 8 µM). The apparent dissociation constant for the Hg(II)·Aβ 40 complex in the presence of SDS micelles is 20 ± 9 µM, which is very similar to the 28 ± 8 µM value observed in buffer only (Fig. 3 ; Table 1 ). Although Aβ peptides are known to interact with SDS micelles (Jarvet et al., 2007 ; Österlund et al., 2018 )., which are a very simple membrane model, these interactions apparently have no significant effect on the Hg(II) binding affinity. At pH 5.1 the overall fluorescence quenching effect is smaller, possibly suggesting generally weaker binding at acidic pH (Fig. 3 ). Due to this smaller effect, the K D App values derived at pH 5.1 have larger errors than those at pH 7.3 (Table 1 ). The Hg(II) affinity values at pH 5.1 are 45 ± 31 µM for Aβ 40 , 8 ± 5 µM for Aβ 4−40 , and 11 ± 5 µM for Aβ NoHis (Table 1 ). Both Aβ 40 and Aβ 4−40 display weaker binding to Hg(II) at pH 5.1 than at pH 7.3. However, for neither peptide the difference is statistically significant, mainly due to the larger errors at pH 5.1 (Fig. 3 ; Table 1 ). For Aβ NoHis , the Hg(II) binding is instead somewhat stronger at lower pH (Table 1 ). The most reasonable explanation for weaker Hg(II) binding to Aβ 40 and Aβ 4−40 at acidic pH is protonation of His residues in Aβ, as this would disfavour binding of cations. This implies that His6, His13 and/or His14 are involved as binding ligands, which is supported by Aβ NoHis displaying weaker Hg(II) binding than Aβ 40 and Aβ 4−40 at neutral pH (Table 1 ). Also, as there are no His residues that can be protonated in Aβ NoHis , this peptide variant does not show weaker Hg(II) binding at acidic pH (Table 1 ). Binding titrations were also carried out with Hg(I), i.e. under reducing conditions obtained by addition of 1 mM TCEP to the samples. These titrations were not successful, as they did not produce meaningful binding curves (data not shown). Table 1 Apparent dissociation constants (K D App ) in µM for Aβ · Hg(II) complexes, measured at different conditions and with different Aβ peptide variants. Three separate samples were prepared and measured for each condition, labelled 1, 2 and 3 in the table. The K D App values were obtained by fitting Eq. 1 to the binding curves shown in Fig. 3 . 1 2 3 Mean K D App Aβ 40 pH 7.3 38.8 22.9 23.6 28 ± 8 Aβ 40 pH 5.1 14.6 87.2 32.9 45 ± 31 Aβ 4−40 pH 7.3 4.9 2.5 3.0 4 ± 2 Aβ 4−40 pH 5.1 16.9 10.3 6.4 8 ± 5 Aβ NoHis pH 7.3 70.3 74.8 35.2 60 ± 18 Aβ NoHis pH 5.1 5.8 13.2 3.5 11 ± 5 Aβ 40 pH 7.3 + 50 mM SDS 30.1 19.8 10.3 20 ± 9 3.3 NMR spectroscopy High-resolution NMR experiments were performed to investigate possible residue-specific molecular interactions between Hg(II) and monomeric Aβ 40 peptide (Fig. 4 ). The experiments were conducted at pH 5.1, where histidine residues are protonated. Figure 4 A shows 2D 1 H, 15 N-HSQC spectra for the amide cross-peak region of the Aβ 40 peptide, recorded before and after addition of Hg(II) in a 1:1 Hg(II):Aβ 40 ratio. Although there is a general loss of signal intensity for all residues after added Hg(NO 3 ) 2 , the signal loss is most pronounced in the N-terminal segment (Fig. 4 B). This shows that there are specific binding interactions between Hg(II) and certain N-terminal residues. Because Hg(II) is diamagnetic with a 5d 10 electronic configuration, the increased loss of cross-peak intensity for these amino acids is not caused by paramagnetic effects. Instead, it likely results from intermediate chemical exchange on the NMR time-scale (Farrar and Becker, 1971 ), between the Hg(II) · Aβ 40 complex and free Aβ 40 peptide. This could be similar to the effects previously observed when Aβ peptide interacts with diamagnetic Zn(II) ions (Danielsson et al., 2007 ; Fawzi et al., 2010 ; Wallin et al., 2020 ). 3.4 Circular dichroism (CD) spectroscopy CD spectroscopy was used to monitor possible effects of Hg(II) on the secondary structure of monomeric Aβ 40 and Aβ 4−40 peptides, investigated in a membrane-mimicking environment consisting of SDS micelles. In this environment and without added Hg(NO 3 ) 2 , both peptides display CD spectra characteristic for α-helix structure (Fig. 5 ), i.e. with typical minima around 222 and 208 nm (Wärmländer et al., 2025 ). The spectra of the two peptides are however not identical, as they differ in the ratios between the CD intensities at 208 and 222 nm (Table 2 ). This shows that the two peptide variants have slightly different α-helix conformations in micelles, which is consistent with earlier studies showing Aβ 4−40 in SDS micelles to have a higher 222/208 ratio than Aβ 40 (Berntsson et al., 2023b ). Furthermore, the overall α-helix CD signal is stronger for Aβ 40 (-6300 at 208 nm) than for Aβ 4−40 (-3900 at 208 nm), which is in line with earlier measurements of these two peptides (Berntsson et al., 2023b ). Addition of Hg(II) induces similar concentration-dependent structural transitions in both peptide variants (Fig. 5 ). For the Aβ 4−40 peptide, the start and end spectra have similar characteristic minima around 208 and 222 nm, and there is an isodichroic point around 200 nm (Fig. 5 B). This show that the transition is from one well-defined α-helical conformation to another. The main difference between the start and end spectra is the 222/208 ratio, which increases from 0.67 without Hg(II) to 0.84 with 256 µM added Hg(II) (Table 2 ). Such spectral changes are known to correspond to changes in helix supercoiling, i.e. when two or more α-helices form coiled coils via hydrophobic interactions (Lau et al., 1984 ; Zhou et al., 1992b ; a ). In such structures, a 222/208 ratio close to 1 reflects large amounts of superhelicity (Barbar and Nyarko, 2014 ). For the Aβ 40 peptide, addition of Hg(II) induces a structural transition (Fig. 5 A) that is similar but not identical to the one observed for the Aβ 4−40 peptide (Fig. 5 B). That is, for Aβ 40 there is no clear isodichroic point (Fig. 5 A), and the 222/208 ratios remain around 0.5 during the titration (Table 2 ). It is therefore less clear how bound Hg(II) ions modulate the α-helical structure of wildtype Aβ 40 peptide. Table 2 CD signal intensities at 208 nm and 222 nm for the Aβ 40 and Aβ 4−40 peptide variants, as a function of added Hg(II). The values are derived from the CD spectra shown in Fig. 5 . Wavelength (nm) 0 µM Hg(II) 16 µM Hg(II) 56µM Hg(II) 156 µM Hg(II) 256 µM Hg(II) Aβ 40 208 -6332 -6510 -6123 -4965 -4055 222 -3406 -3447 -3093 -2865 -2522 222/208 ratio 0.538 0.538 0.529 0.505 0.577 Aβ 4−40 208 -3974 -3351 -2827 -2357 -2100 222 -2679 -2446 -2169 -1851 -1772 222/208 ratio 0.67 0.73 0.77 0.78 0.84 4. Discussion Harmful metal exposure has for a long time been suspected to contribute to neurodegenerative diseases (Pamphlett and Kum Jew, 2018 ; Koski et al., 2021 ; Squitti et al., 2021 ; Åström and Roos, 2022 ; Babic Leko et al., 2023 ; Pamphlett and Bishop, 2023 ; Roos and Wärmländer, 2024 ; Althobaiti, 2025 ). Thus, both endogenous metals such as Cu, Fe and Zn, and numerous exogenous metals have been investigated in relation to AD, either by measuring metal concentrations in blood and brains of AD patients (Xu et al., 2018 ; Babic Leko et al., 2023 ; Scolari Grotto and Glaser, 2024 ), or by in vitro studies of metal ion interactions with disease-related molecules such as Aβ and tau (Faller and Hureau, 2009 ; Wärmländer et al., 2013 ; Kim et al., 2018 ; Ahmadi et al., 2019 ; Lermyte et al., 2019 ; Wärmländer et al., 2019 ; Di Natale et al., 2022 ). Our TEM images show that both Hg(I) and Hg(II) affect Aβ 40 peptide aggregation, by inhibiting fibril formation and instead promoting formation of large amorphous clump-like aggregates (Figs. 1 and 2 ). The effect is somewhat stronger for Hg(II), which completely inhibits fibril formation already at sub-stoichiometric concentrations (Fig. 1 D), while Hg(I) inhibits fibrillation at stoichiometric amounts (Fig. 2 E). These results support earlier studies on the effect of Hg(II) on Aβ fibrillation (Wallin et al., 2019 ). Similar effects on Aβ aggregation have also been observed for ions of the heavy metals Ag, Cd, Pb and U (Wallin et al., 2017 ; Wallin et al., 2020 ; Berntsson et al., 2023a; Lakela et al., 2025 ). As Hg has been found to induce harmful aggregation of other proteins such as γ-crystallins (Dominguez-Calva et al., 2018 ) and ovalbumin (Mathew et al., 2021 ), it is possible that one toxic mechanism of mercury, and perhaps of heavy metals in general, could be to induce misfolding and aggregation of proteins and peptides (Sharma et al., 2008 ; Tamas et al., 2014 ). It therefore appears very likely that the aggregation-modulating properties of Hg(I) and Hg(II) would affect the structure and toxicity of Aβ oligomers, which are considered a main toxic species in AD neuropathology (Glabe, 2008 ; Ghosh et al., 2023 ). However, due to the intrinsic Hg toxicity, it is difficult to compare the toxicities of Aβ aggregates formed in the presence or absence of Hg. It has still not been clarified if harmful Aβ aggregation occurs intracellularly or extracellularly in AD brains. Because the Hg(II) form exists in oxidizing environments, i.e. extracellularly, and the Hg(I) form exists in reducing environments, i.e. intracellularly, the TEM images in Figs. 1 and 2 prove that Hg ions can interfere with Aβ aggregation both inside and outside neural cells. As discussed above in section 2.2 , Hg(II) usually occurs as a free Hg 2+ ion, while Hg(I) typically occurs as the diatomic Hg 2 2+ ion (Neisler and Pitzer, 1987 ; Argent et al., 2006 ). The fluorescence quenching experiments show that Hg(II) binds Aβ 40 peptide with apparent dissociation constants in the micromolar range (Fig. 1 ; Table 1 ). At neutral pH, Hg(II) binds wildtype Aβ 40 peptide with an apparent dissociation constant of 28 ± 8 µM. Because the values in Table 1 have not been corrected for buffer effects, the true affinity values should be somewhat lower (Alies et al., 2013 ). The Hg(II) binding affinity is similar to Aβ 40 binding to Ag(I), Ni(II) and Zn(II) (Danielsson et al., 2007 ; Berntsson et al., 2023b ; Lakela et al., 2025 ), stronger than binding to Li(I), Mn(II) and uranyl ions (Wallin et al., 2016 ; Berntsson et al., 2021 ; Berntsson et al., 2023a), but weaker than binding to Cu(II) (Alies et al., 2013 ; Lindgren et al., 2013 ). The somewhat stronger binding of Hg(II) to Aβ 4−40 than to wildtype Aβ 40 (4 ± 2 µM vs. 28 ± 8 µM; Table 1 ) suggests that Hg(II) could display strong binding to ATCUN (amino-terminal Cu,Ni-binding) motifs. These motifs consist of an N-terminal tripeptide with a His residue at position three, i.e. X-Z-His, and are known to strongly bind metal ions such as Cu(II) and Ni(II) (Stefaniak et al., 2021 ; Lefevre et al., 2022 ; Noormägi et al., 2023 ). Truncating the first three residues in Aβ 40 produces an ATCUN motif, as His6 then becomes residue number three. Because Aβ 4−40 and other N-terminally truncated peptide variants are abundant in both healthy and AD brains (Wirths et al., 2019 ; Stefaniak et al., 2021 ), it has been argued that the observed stronger Cu(II) binding to Aβ 4−40 peptide could be of importance in AD progression (Stefaniak and Bal, 2019 ; Stefaniak et al., 2021 ; Lefevre et al., 2022 ). Cu(II) and Ni(II) have the electron configurations [Ar]3d 9 and [Ar]3d 8 , respectively, and usually prefer square planar or tetrahedral binding configurations with a coordination number of four (Flowers et al., 2024 ). Hg(II) has the electron configuration [Xe]4f 14 5d 10 , and can adopt a wide range of coordination geometries, such as octahedral, T-shaped, square planar, linear, and tetrahedral (Argent et al., 2006 ; Khavasi and Azhdari Tehrani, 2013 ; Khavasi and Mir Mohammad Sadegh, 2014 ). Thus, it is conceivable that Hg(II) might benefit from the advantageous binding properties of ATCUN binding sites, just like Cu(II) and Ni(II). The stronger binding to the ATCUN motif in Aβ 4−40 implies that His6 is involved as a binding ligand for Hg(II). Hg ions often bind to proteins via thiol or selenol groups (Suzuki et al., 1998 ; Rooney, 2007 ; Riccardi et al., 2013 ; Kang et al., 2024 ), which are not present in Aβ peptides. Instead, earlier studies have suggested that the three N-terminal His residues, i.e. His6, His13 and His4, could be involved in coordinating Hg(II) to Aβ (Wallin et al., 2019 ). This is supported by the weaker Hg(II) binding affinity to the Aβ NoHis mutant, i.e. 60 ± 18 uM at pH 7.3, compared to the 28 ± 8 µM observed for wildtype Aβ 40 (Table 1 ). Additional evidence is provided by the weaker binding of Hg(II) to both Aβ 40 and Aβ 4−40 at acidic pH, compared to the affinities at neutral pH (Table 1 ). The most reasonable explanation for these results is protonation of the three Aβ His residues, which have pKa values around 6 (Gielnik et al., 2023 ). Protonated binding ligands are of course less effective at binding cations. However, our NMR results show that also at acidic pH, Hg(II) still displays residue-specific binding to the N-terminal region up to His14 (Fig. 4 ). Earlier work has demonstrated that Cu(II) binding to Aβ peptides occurs via multiple binding configurations, which involve different combinations of His6, His13, His14 and anionic residues such as Asp1 and Glu11 (Faller and Hureau, 2009 ). It is quite possible that a similar ensamble of different binding configurations exists also for Hg(II) binding to Aβ 40 peptide, especially given the propensity of Hg(II) to adopt many different coordination geometries (Argent et al., 2006 ; Khavasi and Azhdari Tehrani, 2013 ; Khavasi and Mir Mohammad Sadegh, 2014 ). We have so far not been able to obtain binding affinities for Hg(I) binding to Aβ. A recent study demonstrated weaker Hg(I) binding and stronger Hg(II) binding to the ApoE protein, where the binding sites appear to involve His residues (Berntsson et al., 2022 ). This suggests that also Aβ peptides might display weaker binding to Hg(I) than to Hg(II), although this is something that should be further investigated in future studies. The ApoE protein study furthermore demonstrated almost identical Hg(II) binding affinities to the three protein variants ApoE2, ApoE3 and ApoE4 (Berntsson et al., 2022 ). This result refuted the idea that the missing cysteines in ApoE4 would make it a worse Hg transporter than ApoE2 and ApoE3, which previously was considered the most likely explanation as to why ApoE4 is a risk factor in Hg exposure (Mutter et al., 2004 ; Mutter et al., 2010 ; Arrifano et al., 2018a; Arrifano et al., 2018b). Now, other explanations need to be considered and investigated. The binding affinity of the Hg(II)·Aβ 40 complex is not much changed when micelles of 50 mM SDS are added to the sample, i.e. from 28 ± 8 µM to 20 ± 9 µM (Table 1 ). The 50 mM of SDS molecules corresponds to slightly less than 1 mM of micelles, which is much higher than 10 µM Aβ peptide. Consequently, there is arguably no more than one Aβ peptide in each micelle. It is well established that the central and C-terminal hydrophobic Aβ segments insert themselves into SDS micelles, where they adopt α-helical conformations (Jarvet et al., 2007 ; Österlund et al., 2019 ). The hydrophilic N-terminal Aβ segment is hanging outside the membrane surface, where it is free to e.g. bind metal ions. Thus, the current results are in line with previous studies showing that Aβ N-terminal metal binding is not much affected when the peptide binds to membranes or membrane mimetica (Lindgren et al., 2013 ). These results also suggest that Hg(II) binding should be very similar for Aβ 40 and the slightly longer Aβ 42 peptide, as the two additional hydrophobic C-terminal residues in Aβ 42 should not affect the N-terminal metal binding properties. The CD results show that addition of Hg(II) induces concentration-dependent changes in the α-helical structure of Aβ peptides positioned in SDS micelles (Fig. 5 ). For Aβ 4−40 , the structural transition involves Hg(II)-induced formation of coil-coil interactions (Fig. 5 B), similar to previously observed effects of e.g. Cu(II) and Ni(II) ions (Tiiman et al., 2016 ; Berntsson et al., 2023b ). For Aβ 40 , the nature of the structural changes are less clear, and may involve other secondary structures than α-helix (Fig. 5 A). The observed structural rearrangements of Aβ peptides in a membrane-mimicking environment could be biologically relevant, as membrane disruption is one likely toxic mechanism of Aβ oligomers (Wärmländer et al., 2019 ). Structural alterations are also a likely explanation for the previously reported observation that binding of Hg(II) reduces the propensity of Aβ 42 to form ion channels in membranes (Meleleo et al., 2020 ). 5. Conclusions Hg(II) binds to the N-terminal segment of biologically relevant Aβ peptide variants. The Hg(II) binding affinity is in the low µM range, with the three N-terminal Aβ His residues involved as binding ligands. Hg(II) binding induces structural alterations in Aβ monomers positioned in membrane-mimicking SDS micelles. Equimolar amounts of either Hg(I) or Hg(II) inhibit normal Aβ fibrillation by instead directing the aggregation process towards formation of large amorphous aggregates. As Aβ aggregation is considered a major toxic mechanism in AD progression, the observed Hg-induced structural rearrangements are likely relevant for AD neuropathology. The capacity to induce misfolding and aggregation of proteins and peptides might be a general toxic mechanism of mercury. Declarations Conflict of interest : AG, JJ, MP, and SW are shareholders in CellPept Sweden AB. Neither this company, nor the funding organizations, had any role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Funding: This work was supported by grants from the Estonian Research Council to MP (no. PRG1506) and to PP (no. PRG1289), from the Magnus Bergvall Foundation to SW, and from the Swedish Brain Foundation and the Swedish Natural Science Research Council to AG. Data availability: The data that support the findings of this study are available from the corresponding author upon reasonable request. References Abelein A, Abrahams JP, Danielsson J, Gräslund A, Jarvet J, Luo J, Tiiman A, Wärmländer SK (2014) The hairpin conformation of the amyloid beta peptide is an important structural motif along the aggregation pathway. 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Sci Total Environ 841:156672 Österlund N, Kulkarni YS, Misiaszek AD, Wallin C, Krüger DM, Liao Q, Mashayekhy Rad F, Jarvet J, Strodel B, Wärmländer SKTS, Ilag LL, Kamerlin SCL, Gräslund A (2018) Amyloid-beta Peptide Interactions with Amphiphilic Surfactants: Electrostatic and Hydrophobic Effects. ACS Chem Neurosci 9(7):1680–1692 Österlund N, Luo J, Wärmländer SKTS, Gräslund A (2019) Membrane-mimetic systems for biophysical studies of the amyloid-beta peptide. Biochim Biophys Acta Proteins Proteom 1867(5):492–501 Additional Declarations The authors declare potential competing interests as follows: Conflict of interest: AG, JJ, MP, and SW are shareholders in CellPept Sweden AB. Neither this company, nor the funding organizations, had any role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5888115","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":406139593,"identity":"ca1bd4e6-5b8b-4924-af82-141de62e20d4","order_by":0,"name":"Elina Berntsson","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Elina","middleName":"","lastName":"Berntsson","suffix":""},{"id":406139594,"identity":"d6549363-b6b1-489b-a8cf-c9e8ab1d030f","order_by":1,"name":"Andra Noormägi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Andra","middleName":"","lastName":"Noormägi","suffix":""},{"id":406139595,"identity":"37f56712-24a1-4a75-90c6-3fed36d42f2b","order_by":2,"name":"Kärt Padari","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kärt","middleName":"","lastName":"Padari","suffix":""},{"id":406139596,"identity":"cfbd95ee-af9c-44fd-9769-c494714f4601","order_by":3,"name":"Jüri Jarvet","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jüri","middleName":"","lastName":"Jarvet","suffix":""},{"id":406139597,"identity":"18d7a1a5-93d7-4be6-91f7-46ebe15f2b20","order_by":4,"name":"Astrid Gräslund","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Astrid","middleName":"","lastName":"Gräslund","suffix":""},{"id":406139598,"identity":"a7fb03d5-8d87-4466-8c8c-70220b91a09f","order_by":5,"name":"Peep Palumaa","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Peep","middleName":"","lastName":"Palumaa","suffix":""},{"id":406139599,"identity":"06cd51a0-e3b8-45cd-ba12-f279a88e6a3a","order_by":6,"name":"Sebastian Wärmländer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYFACHsYHQJKxgRQtzAYMCSAtzMRrYZMgTYt8e++xyp8/Dsv2S/cfYPzZxmDPT0iLwZlzabd5Eg4bz5xzmIGZt40hcWYDIS0SOWa3GRIOJ264kczAzNjGkGBwgJDD5r8xK/wB1LIfqAXsMHtCWhhu8Jgx8IBskUhmYAA6jHEDYb/kJUvzpKUbz7iRbHCY55xE4gyCDms/e/DjDxtr2f4ZiQ8f/iizsedvIGQNMgCaL0GK+lEwCkbBKBgFuAAA6D4+e6YyCHsAAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Wärmländer","suffix":""}],"badges":[],"createdAt":"2025-01-23 11:53:23","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5888115/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5888115/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74680444,"identity":"bbbdf0d8-b9fe-4de9-adde-8fd42e2356eb","added_by":"auto","created_at":"2025-01-24 15:43:36","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":451154,"visible":true,"origin":"","legend":"\u003cp\u003eNegative staining TEM images for aggregates of 10 µM Aβ\u003csub\u003e40\u003c/sub\u003e peptide in 20 mM MES buffer, pH 7.3, incubated for 20 hours on a thermo shaker at 37 \u003csup\u003eo\u003c/sup\u003eC and 300 rpm, together with different amounts of Hg(II). A: 0 µM Hg(II). B: 0.5 µM Hg(II). C: 1.0 µM Hg(II). D: 3.0 µM Hg(II). E: 10 µM Hg(II). The white scale bars are 1 µm. Images by K.P.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5888115/v1/4aae1260ef1c1560fb543ede.jpeg"},{"id":74680446,"identity":"4b49b4c4-09a9-4c5e-a464-daf8178e9ba6","added_by":"auto","created_at":"2025-01-24 15:43:36","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":435063,"visible":true,"origin":"","legend":"\u003cp\u003eNegative staining TEM images for aggregates of 10 µM Aβ\u003csub\u003e40\u003c/sub\u003e peptide in 1 mM TCEP and 20 mM MES buffer, pH 7.3, incubated for 20 hours on a thermo shaker at 37 \u003csup\u003eo\u003c/sup\u003eC and 300 rpm, together with different amounts of Hg(I). A: 0 µM Hg(I). B: 0.5 µM Hg(I). C: 1.0 µM Hg(I). D: 3.0 µM Hg(I). E: 10 µM Hg(I). The white scale bars are 500 nm. Images by K.P.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5888115/v1/7d80b826a1dd3eabf7ffe14e.jpeg"},{"id":74680445,"identity":"538401c9-6f28-42fd-aaaf-698fc8031289","added_by":"auto","created_at":"2025-01-24 15:43:36","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":114114,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in intrinsic fluorescence of Tyr10 in Aβ peptide variants upon addition of Hg(II). All measurements were carried out with 20 µM Aβ peptide and 20 mM MES buffer at 20 °C. Left column: pH 7.3. Right column: pH 5.1. A and E: Aβ\u003csub\u003e40\u003c/sub\u003e. B and F: Aβ\u003csub\u003e1-40\u003c/sub\u003eNoHis. C and G: Aβ\u003csub\u003e4-40\u003c/sub\u003e. D: Aβ\u003csub\u003e40\u003c/sub\u003e with 50 mM SDS.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5888115/v1/215c38ee66a66a281957a6c5.jpeg"},{"id":74680454,"identity":"4e8d0705-4e91-4da7-a984-b04b4bebe19b","added_by":"auto","created_at":"2025-01-24 15:43:37","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":168271,"visible":true,"origin":"","legend":"\u003cp\u003eA: NMR spectra (2D \u003csup\u003e1\u003c/sup\u003eH,\u003csup\u003e15\u003c/sup\u003eN-HSQC) of 92 μM monomeric \u003csup\u003e15\u003c/sup\u003eN-labelled Aβ\u003csub\u003e40\u003c/sub\u003e peptide in 20 mM MES buffer, pH 5.1, recorded at 5 °C before (blue crosspeaks) and after (red crosspeaks) addition of 92 μM Hg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. B: Relative crosspeak intensities for individual Aβ\u003csub\u003e40\u003c/sub\u003e residues, i.e. I/I\u003csub\u003e0\u003c/sub\u003e, showing the effect of added Hg(II) at a 1:1 ratio.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5888115/v1/ec2b4a4983a300ee50ff87f9.jpeg"},{"id":74680452,"identity":"3a09219b-eae2-4451-b9b6-47c7f2d78cf8","added_by":"auto","created_at":"2025-01-24 15:43:37","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":170779,"visible":true,"origin":"","legend":"\u003cp\u003eCD spectra of 10 µM Aβ\u003csub\u003e40\u003c/sub\u003e peptide (A) and 10 µM Aβ\u003csub\u003e4-40\u003c/sub\u003e peptide (B) in 20 mM sodium phosphate buffer, pH 7.3, titrated with Hg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e at 20 °C in the presence of SDS (50 mM) micelles. Black: Aβ peptide only. Red: 16 µM Hg(II). Purple: 56 µM Hg(II). Green: 156 µM Hg(II). Blue: 256 µM Hg(II).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5888115/v1/d5e5bb6e2730c9d7a7db5b0a.jpeg"},{"id":74681728,"identity":"f9fcb4ca-c93c-430c-86fd-228accc241fb","added_by":"auto","created_at":"2025-01-24 15:59:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2261189,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5888115/v1/17391ca6-8088-4448-9f82-4284a8d6ac6d.pdf"}],"financialInterests":"The authors declare potential competing interests as follows: Conflict of interest: AG, JJ, MP, and SW are shareholders in CellPept Sweden AB. Neither this company, nor the funding organizations, had any role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. ","formattedTitle":"\u003cp\u003e\u003cstrong\u003eBinding of Hg(I) and Hg(II) to amyloid-beta (Aβ) peptide variants: effects on structure and aggregation\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eA possible connection between Alzheimer\u0026acute;s disease (AD) and environmental mercury (Hg) exposure has been debated for a long time (Mutter et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Rooney, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Mutter et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Bj\u0026oslash;rklund et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Siblerud et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wallin et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Babic Leko et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Pamphlett and Bishop, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Althobaiti, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Hg is a heavy metal with established neurotoxic properties (Oliveira et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nordberg and Costa, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and some studies have found higher Hg levels in AD patients (Xu et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Siblerud et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Babic Leko et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). But the evidence is inconclusive, and a number of studies have reported normal Hg levels in both blood and brain of AD patients (Babic Leko et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kooshki et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A complicating factor is that AD and mercury intoxication share a common genetic risk factor, namely the APOE-ε4 gene variant (Mutter et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). That is, individuals with APOE-ε4 have an increased probability of developing AD (Belloy et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and also suffer more severe consequences from Hg exposure (Arrifano et al., 2018a; Arrifano et al., 2018b; Santos-Sacramento et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As anthropogenic emissions of mercury continue to increase, mainly due to mining activities and human combustion of coal and oil (UNEP, 2019), it becomes important to investigate how Hg might contribute to AD pathology (Bj\u0026oslash;rklund et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHg is a neurotoxic and genotoxic heavy metal that induces damage to organs such as the brain and the kidneys (Bernhoft, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Rice et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Arrifano et al., 2018b; Nordberg and Costa, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Exposure can lead to permanent health problems and even death (Bernhoft, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Rice et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Santos-Sacramento et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), with different outcomes depending on dose level and exposure time (James et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The molecular mechanisms underlying Hg toxicity are not fully known (Bj\u0026oslash;rklund et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), but include toxic molecular mimicry (Bridges and Zalups, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) and disruption of antioxidant activity (Yang et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) especially in the mitochondria (Carocci et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The different forms of Hg, e.g., metallic, inorganic Hg(I) and Hg(II) species, and organo-metallic complexes such as methyl-Hg and ethyl-Hg, have different chemical properties, toxicity profiles and species distributions (Leermakers et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Oliveira et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nordberg and Costa, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For inorganic Hg(I) and Hg(II) species, the retention time in the human brain is several years, or even decades (Rooney, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAD is a progressive, irreversible, and currently incurable chronic neurodegenerative disorder, and also the leading cause of dementia worldwide (Scheltens et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Alzheimer's_Association, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Pathological hallmarks of AD include brain atrophy, with extensive deposits of amyloid plaques and neurofibrillary Tau tangles occurring years before symptom manifestation (Scheltens et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Alzheimer's_Association, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The insoluble plaques consist mainly of amyloid-β (Aβ) peptides (Glenner and Wong, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1984\u003c/span\u003e) aggregated into fibrils that display a characteristic cross-β structure (Sunde and Blake, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The plaques are the end-product of an aggregation process (Luo et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Selkoe and Hardy, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) that involves formation of extra- and intracellular intermediates such as soluble neurotoxic Aβ oligomers (Glabe, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). As the latter are considered one of the main toxic species in AD brains (Ghosh et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), it becomes imperative to investigate factors that may influence Aβ aggregation and oligomer formation (Owen et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Aβ peptides are intrinsically disordered in monomeric form and soluble in water (W\u0026auml;rml\u0026auml;nder et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The central and C-terminal Aβ segments are hydrophobic and can interact with membranes or fold into a hairpin conformation that likely is required for aggregation (Abelein et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), while the anionic N-terminal segment is hydrophilic and readily interacts with metal ions (W\u0026auml;rml\u0026auml;nder et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; W\u0026auml;rml\u0026auml;nder et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Inorganic Hg(II) has previously been shown to bind to Aβ peptides and interfere with the Aβ aggregation mechanisms (Meleleo et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Wallin et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Specifically, Hg(II) has been reported to increase Aβ\u003csub\u003e42\u003c/sub\u003e toxicity, promote formation of high-molecular-weight soluble aggregates, and reduce the propensity of Aβ\u003csub\u003e42\u003c/sub\u003e to form harmful ion channels in membranes (Meleleo et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, no study has so far investigated Aβ interactions with monovalent Hg(I), even though both forms exist \u003cem\u003ein vivo\u003c/em\u003e. It is still not clear if the formation and/or activity of toxic Aβ oligomers in AD brains occurs extracellularly, where the oxidizing environment yields Hg(II), or/and intracellularly, where the reducing environment yields Hg(I).\u003c/p\u003e \u003cp\u003eIn this study, we use transmission electron microscopy (TEM) imaging together with fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to study \u003cem\u003ein vitro\u003c/em\u003e interactions between Aβ peptides and inorganic Hg ions. The focus is on Hg binding properties and Hg binding effects on Aβ structure and aggregation. Both Hg(I) and Hg(II) are investigated and compared for the first time. The studied Aβ peptides are the pathologically relevant Aβ(1\u0026ndash;40) and Aβ(4\u0026ndash;40) variants, together with the Aβ(1\u0026ndash;40)(H6A, H13A, H14A) mutant that is used to investigate the importance of the Aβ His residues for Hg binding.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eRecombinantly produced wild-type (wt) Aβ(1\u0026ndash;40) peptide, abbreviated as Aβ\u003csub\u003e40\u003c/sub\u003e, with the primary sequence DAEFR\u003csub\u003e5\u003c/sub\u003eHDSGY\u003csub\u003e10\u003c/sub\u003eEVHHQ\u003csub\u003e15\u003c/sub\u003eKLVFF\u003csub\u003e20\u003c/sub\u003eAEDVG\u003csub\u003e25\u003c/sub\u003eSNKGA\u003csub\u003e30\u003c/sub\u003eIIGLM\u003csub\u003e35\u003c/sub\u003eVGGVV\u003csub\u003e40\u003c/sub\u003e,\u003c/p\u003e \u003cp\u003ewere purchased as lyophilized powder from AlexoTech AB (Ume\u0026aring;, Sweden). Recombinantly produced truncated Aβ(4\u0026ndash;40) peptide, abbreviated as Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e, Aβ(1\u0026ndash;40)(H6A, H13A, H14A) mutant peptide, abbreviated as Aβ\u003csub\u003eNoHis\u003c/sub\u003e, and uniformly \u003csup\u003e15\u003c/sup\u003eN-labeled Aβ\u003csub\u003e40\u003c/sub\u003e peptide were also purchased from AlexoTech AB. All Aβ peptide variants were stored at -80\u0026deg;C until use, when they were dissolved to monomeric form in 10 mM NaOH. The fresh solutions were sonicated for 5 mins in an ice-bath to dissolve possible pre-formed aggregates. Then, phosphate buffer or 2-(N-Morpholino)ethanesulfonic acid hydrate (MES) buffer was added. All preparation steps were performed on ice. The peptide concentrations were first estimated from the weight of the dry powder, and then more accurately determined by NanoDrop measurements of the solutions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Transmission electron microscopy (TEM) imaging\u003c/h2\u003e \u003cp\u003eNegative staining TEM images were recorded for 10 \u0026micro;M Aβ\u003csub\u003e40\u003c/sub\u003e peptide in 20 mM MES buffer, pH 7.3, that had been incubated for 20 hours on a thermo shaker operating at 37 \u003csup\u003eo\u003c/sup\u003eC and 300 rpm, together with either 0 \u0026micro;M, 0.5 \u0026micro;M, 1 \u0026micro;M, 3 \u0026micro;M, or 10 \u0026micro;M of Hg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. Then, 5 \u0026micro;L of each incubated sample were put on copper grids of 200 \u0026micro;m mesh size. The grids were covered with Pioloform film upon which a carbon layer had first been deposited and then glow-discharged using a Leica EM ACE600 carbon coater (Leica Microsystems, Germany). The Aβ\u003csub\u003e40\u003c/sub\u003e samples were absorbed to the grids for 5 mins, rinsed with Milli-Q water two times, and then stained for 2 mins with 2% aqueous solution of uranyl acetate. The excess stain was removed with filter paper, and then the samples were left to air-dry. A digital Orius SC1000 camera was used to record TEM images in a FEI Tecnai G2 Spirit electron microscope (FEI, The Netherlands) operating at 120 kV accelerating voltage.\u003c/p\u003e \u003cp\u003eTo investigate the effects of both monovalent Hg(I) and divalent Hg(II) on Aβ aggregation, all samples were prepared and incubated in both oxidizing and reducing conditions, respectively. Standard oxidizing conditions yield the Hg(II) form, which typically exists as free Hg\u003csup\u003e2+\u003c/sup\u003e ions. A reducing condition, roughly corresponding to the chemical environment inside the cell cytosol, was obtained by adding 1 mM of the reducing agent TCEP to the samples. This yields monovalent Hg(I), which typically exists as diatomic Hg\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e ions (Neisler and Pitzer, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Argent et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Fluorescence spectroscopy\u003c/h2\u003e \u003cp\u003eFluorescence measurements were carried out with a Jobin Yvon Horiba Fluorolog 3 fluorescence spectrometer (Longjumeau, France). Emission intensities at 306 nm (excitation 276 nm) were recorded at room temperature for samples of 20 \u0026micro;M Aβ peptide (Aβ\u003csub\u003e40\u003c/sub\u003e, Aβ\u003csub\u003eNoHis\u003c/sub\u003e, or Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e) in 20mM MES buffer, pH 5.1 or 7.3, using a quartz cuvette with 4mm path length and a total volume of 600 \u0026micro;L. For Aβ\u003csub\u003e40\u003c/sub\u003e at pH 7.3, measurements were also conducted in the presence of 50 mM SDS (sodium dodecyl sulfate) surfactant. Small aliquots of Hg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e were titrated to the samples (less than 3% of total volume added) using stock solutions of 2 mM, 3 mM, and 10 mM Hg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, respectively. The measured tyrosine fluorescence intensities were then plotted against the concentration of Hg ions. Dissociation constants (K\u003csub\u003eD\u003c/sub\u003e) for the Aβ\u0026middot;Hg complexes were calculated by fitting the data curves to Eq.\u0026nbsp;1, the Morrison equation (Kuzmic, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e):\u003c/p\u003e \u003cp\u003eEq.\u0026nbsp;1:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{I}={\\text{I}}_{0}+\\frac{{\\text{I}}_{{\\infty\\:}}-{\\text{I}}_{0}}{2\u0026middot;\\left[\\text{A}{\\beta\\:}\\right]}\u0026middot;\\left(\\left({\\text{K}}_{\\text{D}}+\\left[\\text{H}\\text{g}\\right]+\\left[\\text{A}{\\beta\\:}\\right]\\right)-\\sqrt{{\\left({\\text{K}}_{\\text{D}}+\\left[\\text{H}\\text{g}\\right]+\\left[\\text{A}{\\beta\\:}\\right]\\right)}^{2}-4\u0026middot;\\left[\\text{H}\\text{g}\\right]\u0026middot;\\left[\\text{A}{\\beta\\:}\\right]}\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eHere, I\u003csub\u003e0\u003c/sub\u003e is the initial fluorescence intensity with no added Hg ions, I\u003csub\u003e\u0026infin;\u003c/sub\u003e is the steady-state intensity at the end of the titration, [Hg] is the concentration of added Hg ions, K\u003csub\u003eD\u003c/sub\u003e is the dissociation constant, and [Aβ] is the peptide concentration. This version of the equation assumes that the peptide has a single binding site for Hg ions. Adding a linear term for the fluorescence quenching effect of free Hg ions appeared unnecessary (Lindgren et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In this study no corrections were made for possible interactions between the buffer and the Hg ions. The derived dissociation constants should therefore be considered as apparent, i.e. K\u003csub\u003eD\u003c/sub\u003e\u003csup\u003eApp\u003c/sup\u003e. However, the buffer effects are likely small, as MES is a Good buffer devised to have minimal interactions with metal ions and other cations (Good et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). For each condition three separate samples were prepared and measured, allowing mean K\u003csub\u003eD\u003c/sub\u003e\u003csup\u003eApp\u003c/sup\u003e values to be calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Nuclear magnetic resonance (NMR) spectroscopy\u003c/h2\u003e \u003cp\u003eNuclear magnetic resonance (NMR) spectroscopy measurements were performed on a 700 MHz Bruker Avance spectrometer equipped with a cryoprobe. Two-dimensional \u003csup\u003e1\u003c/sup\u003eH,\u003csup\u003e15\u003c/sup\u003eN-HSQC spectra were recorded at 5\u0026deg;C for 92 \u0026micro;M monomeric \u003csup\u003e15\u003c/sup\u003eN-labelled Aβ\u003csub\u003e40\u003c/sub\u003e peptide in 20 mM MES buffer at pH 5.1, (90/10 H\u003csub\u003e2\u003c/sub\u003eO/D\u003csub\u003e2\u003c/sub\u003eO), before and after addition of 92 \u0026micro;M Hg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. The Topspin v.3.6.2 software was used to process the NMR spectra, and the crosspeaks were assigned according to previously published data at both neutral pH (Danielsson et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Yamaguchi et al., \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Roche et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and at acidic pH (Ghalebani et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 CD spectroscopy\u003c/h2\u003e \u003cp\u003eCircular dichroism (CD) spectra were recorded between 195 nm and 250 nm, with 0.5 nm step size, using a Chirascan CD instrument (Applied Photophysics Ltd., U.K.) operating at 20\u0026deg;C. A quartz cuvette with 2 mm pathlength was used to hold 600 \u0026micro;L of either 10 \u0026micro;M Aβ\u003csub\u003e40\u003c/sub\u003e peptide or 10 \u0026micro;M Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e peptide in 20 mM phosphate buffer, pH 7.3. Both samples also contained 50 mM SDS surfactant. As this concentration is much higher than the critical micelle concentration (cmc) of SDS, which in pure water at 25\u0026deg;C is 8.2 mM (Dominguez et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), the SDS forms micelles that constitute a simple but efficient membrane model (\u0026Ouml;sterlund et al., \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). As the SDS micelle concentration is much higher than the Aβ concentration, there should be no more than one Aβ peptide in each micelle.\u003c/p\u003e \u003cp\u003eTo both Aβ variants, i.e. Aβ\u003csub\u003e40\u003c/sub\u003e and Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e, the dissolved mercury salt Hg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e was added in steps of 16 \u0026micro;M, 56 \u0026micro;M, 156 \u0026micro;M and 256 \u0026micro;M. The recorded spectra were processed with the Chirascan Pro-Data v.4.4.1 software (Applied Photophysics Ltd., U.K.), including smoothing with an eight points Savitzky-Golay smoothing filter.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Transmission electron microscopy (TEM) imaging\u003c/h2\u003e \u003cp\u003eTEM images were recorded to study the morphologies of aggregates of 10 \u0026micro;M Aβ\u003csub\u003e40\u003c/sub\u003e peptide, incubated for 20 hours together with different concentrations of Hg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e. The incubations were carried out either in MES buffer only (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), or together with 1 mM of the reducing agent TCEP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnder standard oxidizing conditions, i.e. without TCEP present, the aggregated control sample of Aβ\u003csub\u003e40\u003c/sub\u003e peptide without added mercury displays slender fibrils that are several microns long, together with smaller aggregate particles that might be protofibrils (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This is in line with previous \u003cem\u003ein vitro\u003c/em\u003e studies of the size and shape of self-aggregated Aβ\u003csub\u003e40\u003c/sub\u003e fibrils (Wallin et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), which appear to grow out from globular protofibrillar assemblies (Luo et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). A similar fibril morphology is observed for the Aβ\u003csub\u003e40\u003c/sub\u003e samples incubated with 0.5 \u0026micro;M Hg(II) ions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The Aβ\u003csub\u003e40\u003c/sub\u003e sample with 1 \u0026micro;M Hg(II) ions is somewhat different, displaying a mixture of slender fibrils, small aggregate particles, and larger amorphous aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The Aβ\u003csub\u003e40\u003c/sub\u003e samples with 3 \u0026micro;M and 10 \u0026micro;M Hg(II) contain no fibrils, but only small and large aggregate particles (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). The sample with 10 \u0026micro;M Hg(II) is dominated by large aggregated particles, roughly half a micron across (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). This shows that Hg(II) ions promote formation of large amorphous aggregates rather than slender fibrils. The effect is clearly concentration-dependent, and the formation of long fibrils is completely inhibited already at sub-stoichiometric mercury concentrations, i.e. with 3 \u0026micro;M Hg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e to 10 \u0026micro;M Aβ\u003csub\u003e40\u003c/sub\u003e peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor the samples incubated under reducing conditions, i.e. with 1 mM TCEP present, the Aβ\u003csub\u003e40\u003c/sub\u003e control sample without added mercury again displays numerous amyloid fibrils, several microns long, together with small aggregate particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The Aβ\u003csub\u003e40\u003c/sub\u003e samples incubated together with 0.5 \u0026micro;M, 1 \u0026micro;M, and 3 \u0026micro;M Hg(I) display similar combinations of amyloid fibrils and small amorphous aggregates (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). At 10 \u0026micro;M added Hg(I) the Aβ\u003csub\u003e40\u003c/sub\u003e peptide no longer forms slender fibrils, but rather amorphous aggregates in various sizes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). This shows that Hg(I) has a similar, albeit weaker, concentration-dependent effect on Aβ\u003csub\u003e40\u003c/sub\u003e aggregation as Hg(II). For example, complete inhibition of Aβ\u003csub\u003e40\u003c/sub\u003e fibrillation is achieved by Hg(I) at stoichiometric concentrations (1:1 Hg(I):Aβ\u003csub\u003e40\u003c/sub\u003e ratio; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), but by Hg(II) already at sub-stoichiometric concentrations (3:10 Hg(II):Aβ\u003csub\u003e40\u003c/sub\u003e ratio; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Fluorescence measurements of Hg(II) binding affinity\u003c/h2\u003e \u003cp\u003eHg ions have previously been shown to be able to quench the intrinsic fluorescence of Tyr residues (Berntsson et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), similar to e.g. Ag(I), Cu(II), and UO\u003csub\u003e2\u003c/sub\u003e(II) ions (Alies et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lindgren et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tiiman et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Berntsson et al., 2023a; Lakela et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The effect of added Hg(II) on the fluorescence of Tyr10, the only natural fluorophore in Aβ peptides, was therefore used to evaluate binding affinities for Hg(II)\u0026middot;Aβ complexes in different conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), using three replicates for each Aβ variant and/or condition,\u003c/p\u003e \u003cp\u003eFor titrations with Hg(II) at pH 7.3, the fluorescence intensity curves can be fitted to Eq.\u0026nbsp;1 with good accuracy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This shows that the binding interactions are dominated by a single binding site. As seen in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Hg(II) displays the strongest binding to the Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e variant (3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.1 \u0026micro;M), weakest binding to Aβ\u003csub\u003eNoHis\u003c/sub\u003e (60\u0026thinsp;\u0026plusmn;\u0026thinsp;18 \u0026micro;M), and intermediate binding affinity to wild-type Aβ\u003csub\u003e40\u003c/sub\u003e (28\u0026thinsp;\u0026plusmn;\u0026thinsp;8 \u0026micro;M). The apparent dissociation constant for the Hg(II)\u0026middot;Aβ\u003csub\u003e40\u003c/sub\u003e complex in the presence of SDS micelles is 20\u0026thinsp;\u0026plusmn;\u0026thinsp;9 \u0026micro;M, which is very similar to the 28\u0026thinsp;\u0026plusmn;\u0026thinsp;8 \u0026micro;M value observed in buffer only (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although Aβ peptides are known to interact with SDS micelles (Jarvet et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; \u0026Ouml;sterlund et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2018\u003c/span\u003e)., which are a very simple membrane model, these interactions apparently have no significant effect on the Hg(II) binding affinity.\u003c/p\u003e \u003cp\u003eAt pH 5.1 the overall fluorescence quenching effect is smaller, possibly suggesting generally weaker binding at acidic pH (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Due to this smaller effect, the K\u003csub\u003eD\u003c/sub\u003e\u003csup\u003eApp\u003c/sup\u003e values derived at pH 5.1 have larger errors than those at pH 7.3 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The Hg(II) affinity values at pH 5.1 are 45\u0026thinsp;\u0026plusmn;\u0026thinsp;31 \u0026micro;M for Aβ\u003csub\u003e40\u003c/sub\u003e, 8\u0026thinsp;\u0026plusmn;\u0026thinsp;5 \u0026micro;M for Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e, and 11\u0026thinsp;\u0026plusmn;\u0026thinsp;5 \u0026micro;M for Aβ\u003csub\u003eNoHis\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Both Aβ\u003csub\u003e40\u003c/sub\u003e and Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e display weaker binding to Hg(II) at pH 5.1 than at pH 7.3. However, for neither peptide the difference is statistically significant, mainly due to the larger errors at pH 5.1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For Aβ\u003csub\u003eNoHis\u003c/sub\u003e, the Hg(II) binding is instead somewhat stronger at lower pH (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe most reasonable explanation for weaker Hg(II) binding to Aβ\u003csub\u003e40\u003c/sub\u003e and Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e at acidic pH is protonation of His residues in Aβ, as this would disfavour binding of cations. This implies that His6, His13 and/or His14 are involved as binding ligands, which is supported by Aβ\u003csub\u003eNoHis\u003c/sub\u003e displaying weaker Hg(II) binding than Aβ\u003csub\u003e40\u003c/sub\u003e and Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e at neutral pH (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Also, as there are no His residues that can be protonated in Aβ\u003csub\u003eNoHis\u003c/sub\u003e, this peptide variant does not show weaker Hg(II) binding at acidic pH (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBinding titrations were also carried out with Hg(I), i.e. under reducing conditions obtained by addition of 1 mM TCEP to the samples. These titrations were not successful, as they did not produce meaningful binding curves (data not shown).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eApparent dissociation constants (K\u003csub\u003eD\u003c/sub\u003e\u003csup\u003eApp\u003c/sup\u003e) in \u0026micro;M for Aβ\u003cb\u003e\u0026middot;\u003c/b\u003eHg(II) complexes, measured at different conditions and with different Aβ peptide variants. Three separate samples were prepared and measured for each condition, labelled 1, 2 and 3 in the table. The K\u003csub\u003eD\u003c/sub\u003e\u003csup\u003eApp\u003c/sup\u003e values were obtained by fitting Eq.\u0026nbsp;1 to the binding curves shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean K\u003csub\u003eD\u003c/sub\u003e\u003csup\u003eApp\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAβ\u003c/b\u003e\u003csub\u003e\u003cb\u003e40\u003c/b\u003e\u003c/sub\u003e \u003cb\u003epH 7.3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e38.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAβ\u003c/b\u003e\u003csub\u003e\u003cb\u003e40\u003c/b\u003e\u003c/sub\u003e \u003cb\u003epH 5.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e87.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e32.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e45\u0026thinsp;\u0026plusmn;\u0026thinsp;31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAβ\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u0026minus;40\u003c/b\u003e\u003c/sub\u003e \u003cb\u003epH 7.3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4\u0026nbsp;\u0026plusmn; 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAβ\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u0026minus;40\u003c/b\u003e\u003c/sub\u003e \u003cb\u003epH 5.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e8\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAβ\u003c/b\u003e\u003csub\u003e\u003cb\u003eNoHis\u003c/b\u003e\u003c/sub\u003e \u003cb\u003epH 7.3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e70.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e74.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e60\u0026thinsp;\u0026plusmn;\u0026thinsp;18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAβ\u003c/b\u003e\u003csub\u003e\u003cb\u003eNoHis\u003c/b\u003e\u003c/sub\u003e \u003cb\u003epH 5.1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e11\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAβ\u003c/b\u003e\u003csub\u003e\u003cb\u003e40\u003c/b\u003e\u003c/sub\u003e \u003cb\u003epH 7.3\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e+\u0026thinsp;50 mM SDS\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 NMR spectroscopy\u003c/h2\u003e \u003cp\u003eHigh-resolution NMR experiments were performed to investigate possible residue-specific molecular interactions between Hg(II) and monomeric Aβ\u003csub\u003e40\u003c/sub\u003e peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The experiments were conducted at pH 5.1, where histidine residues are protonated. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA shows 2D \u003csup\u003e1\u003c/sup\u003eH,\u003csup\u003e15\u003c/sup\u003eN-HSQC spectra for the amide cross-peak region of the Aβ\u003csub\u003e40\u003c/sub\u003e peptide, recorded before and after addition of Hg(II) in a 1:1 Hg(II):Aβ\u003csub\u003e40\u003c/sub\u003e ratio. Although there is a general loss of signal intensity for all residues after added Hg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, the signal loss is most pronounced in the N-terminal segment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This shows that there are specific binding interactions between Hg(II) and certain N-terminal residues. Because Hg(II) is diamagnetic with a 5d\u003csup\u003e10\u003c/sup\u003e electronic configuration, the increased loss of cross-peak intensity for these amino acids is not caused by paramagnetic effects. Instead, it likely results from intermediate chemical exchange on the NMR time-scale (Farrar and Becker, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1971\u003c/span\u003e), between the Hg(II)\u003cb\u003e\u0026middot;\u003c/b\u003eAβ\u003csub\u003e40\u003c/sub\u003e complex and free Aβ\u003csub\u003e40\u003c/sub\u003e peptide. This could be similar to the effects previously observed when Aβ peptide interacts with diamagnetic Zn(II) ions (Danielsson et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Fawzi et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wallin et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Circular dichroism (CD) spectroscopy\u003c/h2\u003e \u003cp\u003eCD spectroscopy was used to monitor possible effects of Hg(II) on the secondary structure of monomeric Aβ\u003csub\u003e40\u003c/sub\u003e and Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e peptides, investigated in a membrane-mimicking environment consisting of SDS micelles. In this environment and without added Hg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, both peptides display CD spectra characteristic for α-helix structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), i.e. with typical minima around 222 and 208 nm (W\u0026auml;rml\u0026auml;nder et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The spectra of the two peptides are however not identical, as they differ in the ratios between the CD intensities at 208 and 222 nm (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This shows that the two peptide variants have slightly different α-helix conformations in micelles, which is consistent with earlier studies showing Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e in SDS micelles to have a higher 222/208 ratio than Aβ\u003csub\u003e40\u003c/sub\u003e (Berntsson et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). Furthermore, the overall α-helix CD signal is stronger for Aβ\u003csub\u003e40\u003c/sub\u003e (-6300 at 208 nm) than for Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e (-3900 at 208 nm), which is in line with earlier measurements of these two peptides (Berntsson et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAddition of Hg(II) induces similar concentration-dependent structural transitions in both peptide variants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). For the Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e peptide, the start and end spectra have similar characteristic minima around 208 and 222 nm, and there is an isodichroic point around 200 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This show that the transition is from one well-defined α-helical conformation to another. The main difference between the start and end spectra is the 222/208 ratio, which increases from 0.67 without Hg(II) to 0.84 with 256 \u0026micro;M added Hg(II) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Such spectral changes are known to correspond to changes in helix supercoiling, i.e. when two or more α-helices form coiled coils via hydrophobic interactions (Lau et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e1992b\u003c/span\u003e; \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003ea\u003c/span\u003e). In such structures, a 222/208 ratio close to 1 reflects large amounts of superhelicity (Barbar and Nyarko, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor the Aβ\u003csub\u003e40\u003c/sub\u003e peptide, addition of Hg(II) induces a structural transition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) that is similar but not identical to the one observed for the Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). That is, for Aβ\u003csub\u003e40\u003c/sub\u003e there is no clear isodichroic point (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), and the 222/208 ratios remain around 0.5 during the titration (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It is therefore less clear how bound Hg(II) ions modulate the α-helical structure of wildtype Aβ\u003csub\u003e40\u003c/sub\u003e peptide.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCD signal intensities at 208 nm and 222 nm for the Aβ\u003csub\u003e40\u003c/sub\u003e and Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e peptide variants, as a function of added Hg(II). The values are derived from the CD spectra shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWavelength (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 \u0026micro;M Hg(II)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 \u0026micro;M Hg(II)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56\u0026micro;M Hg(II)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e156 \u0026micro;M Hg(II)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e256 \u0026micro;M Hg(II)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eAβ\u003c/b\u003e\u003csub\u003e\u003cb\u003e40\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-6332\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-6510\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6123\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-4965\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-4055\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3406\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3447\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-2865\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-2522\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e222/208 ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.538\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.538\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.529\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.577\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eAβ\u003c/b\u003e\u003csub\u003e\u003cb\u003e4\u0026minus;40\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-3974\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3351\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2827\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-2357\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-2100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2679\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2446\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-1851\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-1772\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e222/208 ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eHarmful metal exposure has for a long time been suspected to contribute to neurodegenerative diseases (Pamphlett and Kum Jew, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Koski et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Squitti et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; \u0026Aring;str\u0026ouml;m and Roos, \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Babic Leko et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Pamphlett and Bishop, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Roos and W\u0026auml;rml\u0026auml;nder, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Althobaiti, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Thus, both endogenous metals such as Cu, Fe and Zn, and numerous exogenous metals have been investigated in relation to AD, either by measuring metal concentrations in blood and brains of AD patients (Xu et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Babic Leko et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Scolari Grotto and Glaser, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), or by \u003cem\u003ein vitro\u003c/em\u003e studies of metal ion interactions with disease-related molecules such as Aβ and tau (Faller and Hureau, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; W\u0026auml;rml\u0026auml;nder et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ahmadi et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lermyte et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; W\u0026auml;rml\u0026auml;nder et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Di Natale et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur TEM images show that both Hg(I) and Hg(II) affect Aβ\u003csub\u003e40\u003c/sub\u003e peptide aggregation, by inhibiting fibril formation and instead promoting formation of large amorphous clump-like aggregates (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The effect is somewhat stronger for Hg(II), which completely inhibits fibril formation already at sub-stoichiometric concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), while Hg(I) inhibits fibrillation at stoichiometric amounts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). These results support earlier studies on the effect of Hg(II) on Aβ fibrillation (Wallin et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Similar effects on Aβ aggregation have also been observed for ions of the heavy metals Ag, Cd, Pb and U (Wallin et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wallin et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Berntsson et al., 2023a; Lakela et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). As Hg has been found to induce harmful aggregation of other proteins such as γ-crystallins (Dominguez-Calva et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and ovalbumin (Mathew et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), it is possible that one toxic mechanism of mercury, and perhaps of heavy metals in general, could be to induce misfolding and aggregation of proteins and peptides (Sharma et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Tamas et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt therefore appears very likely that the aggregation-modulating properties of Hg(I) and Hg(II) would affect the structure and toxicity of Aβ oligomers, which are considered a main toxic species in AD neuropathology (Glabe, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Ghosh et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, due to the intrinsic Hg toxicity, it is difficult to compare the toxicities of Aβ aggregates formed in the presence or absence of Hg. It has still not been clarified if harmful Aβ aggregation occurs intracellularly or extracellularly in AD brains. Because the Hg(II) form exists in oxidizing environments, i.e. extracellularly, and the Hg(I) form exists in reducing environments, i.e. intracellularly, the TEM images in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e prove that Hg ions can interfere with Aβ aggregation both inside and outside neural cells. As discussed above in section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e, Hg(II) usually occurs as a free Hg\u003csup\u003e2+\u003c/sup\u003e ion, while Hg(I) typically occurs as the diatomic Hg\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2+\u003c/sup\u003e ion (Neisler and Pitzer, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Argent et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe fluorescence quenching experiments show that Hg(II) binds Aβ\u003csub\u003e40\u003c/sub\u003e peptide with apparent dissociation constants in the micromolar range (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). At neutral pH, Hg(II) binds wildtype Aβ\u003csub\u003e40\u003c/sub\u003e peptide with an apparent dissociation constant of 28\u0026thinsp;\u0026plusmn;\u0026thinsp;8 \u0026micro;M. Because the values in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e have not been corrected for buffer effects, the true affinity values should be somewhat lower (Alies et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The Hg(II) binding affinity is similar to Aβ\u003csub\u003e40\u003c/sub\u003e binding to Ag(I), Ni(II) and Zn(II) (Danielsson et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Berntsson et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e; Lakela et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), stronger than binding to Li(I), Mn(II) and uranyl ions (Wallin et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Berntsson et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Berntsson et al., 2023a), but weaker than binding to Cu(II) (Alies et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lindgren et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe somewhat stronger binding of Hg(II) to Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e than to wildtype Aβ\u003csub\u003e40\u003c/sub\u003e (4\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026micro;M vs. 28\u0026thinsp;\u0026plusmn;\u0026thinsp;8 \u0026micro;M; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) suggests that Hg(II) could display strong binding to ATCUN (amino-terminal Cu,Ni-binding) motifs. These motifs consist of an N-terminal tripeptide with a His residue at position three, i.e. X-Z-His, and are known to strongly bind metal ions such as Cu(II) and Ni(II) (Stefaniak et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lefevre et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Noorm\u0026auml;gi et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Truncating the first three residues in Aβ\u003csub\u003e40\u003c/sub\u003e produces an ATCUN motif, as His6 then becomes residue number three. Because Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e and other N-terminally truncated peptide variants are abundant in both healthy and AD brains (Wirths et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Stefaniak et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), it has been argued that the observed stronger Cu(II) binding to Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e peptide could be of importance in AD progression (Stefaniak and Bal, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Stefaniak et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lefevre et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Cu(II) and Ni(II) have the electron configurations [Ar]3d\u003csup\u003e9\u003c/sup\u003e and [Ar]3d\u003csup\u003e8\u003c/sup\u003e, respectively, and usually prefer square planar or tetrahedral binding configurations with a coordination number of four (Flowers et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Hg(II) has the electron configuration [Xe]4f\u003csup\u003e14\u003c/sup\u003e5d\u003csup\u003e10\u003c/sup\u003e, and can adopt a wide range of coordination geometries, such as octahedral, T-shaped, square planar, linear, and tetrahedral (Argent et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Khavasi and Azhdari Tehrani, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Khavasi and Mir Mohammad Sadegh, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Thus, it is conceivable that Hg(II) might benefit from the advantageous binding properties of ATCUN binding sites, just like Cu(II) and Ni(II).\u003c/p\u003e \u003cp\u003eThe stronger binding to the ATCUN motif in Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e implies that His6 is involved as a binding ligand for Hg(II). Hg ions often bind to proteins via thiol or selenol groups (Suzuki et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Rooney, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Riccardi et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Kang et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which are not present in Aβ peptides. Instead, earlier studies have suggested that the three N-terminal His residues, i.e. His6, His13 and His4, could be involved in coordinating Hg(II) to Aβ (Wallin et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). This is supported by the weaker Hg(II) binding affinity to the Aβ\u003csub\u003eNoHis\u003c/sub\u003e mutant, i.e. 60\u0026thinsp;\u0026plusmn;\u0026thinsp;18 uM at pH 7.3, compared to the 28\u0026thinsp;\u0026plusmn;\u0026thinsp;8 \u0026micro;M observed for wildtype Aβ\u003csub\u003e40\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Additional evidence is provided by the weaker binding of Hg(II) to both Aβ\u003csub\u003e40\u003c/sub\u003e and Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e at acidic pH, compared to the affinities at neutral pH (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The most reasonable explanation for these results is protonation of the three Aβ His residues, which have pKa values around 6 (Gielnik et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Protonated binding ligands are of course less effective at binding cations. However, our NMR results show that also at acidic pH, Hg(II) still displays residue-specific binding to the N-terminal region up to His14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Earlier work has demonstrated that Cu(II) binding to Aβ peptides occurs via multiple binding configurations, which involve different combinations of His6, His13, His14 and anionic residues such as Asp1 and Glu11 (Faller and Hureau, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). It is quite possible that a similar ensamble of different binding configurations exists also for Hg(II) binding to Aβ\u003csub\u003e40\u003c/sub\u003e peptide, especially given the propensity of Hg(II) to adopt many different coordination geometries (Argent et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Khavasi and Azhdari Tehrani, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Khavasi and Mir Mohammad Sadegh, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe have so far not been able to obtain binding affinities for Hg(I) binding to Aβ. A recent study demonstrated weaker Hg(I) binding and stronger Hg(II) binding to the ApoE protein, where the binding sites appear to involve His residues (Berntsson et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This suggests that also Aβ peptides might display weaker binding to Hg(I) than to Hg(II), although this is something that should be further investigated in future studies. The ApoE protein study furthermore demonstrated almost identical Hg(II) binding affinities to the three protein variants ApoE2, ApoE3 and ApoE4 (Berntsson et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This result refuted the idea that the missing cysteines in ApoE4 would make it a worse Hg transporter than ApoE2 and ApoE3, which previously was considered the most likely explanation as to why ApoE4 is a risk factor in Hg exposure (Mutter et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Mutter et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Arrifano et al., 2018a; Arrifano et al., 2018b). Now, other explanations need to be considered and investigated.\u003c/p\u003e \u003cp\u003eThe binding affinity of the Hg(II)\u0026middot;Aβ\u003csub\u003e40\u003c/sub\u003e complex is not much changed when micelles of 50 mM SDS are added to the sample, i.e. from 28\u0026thinsp;\u0026plusmn;\u0026thinsp;8 \u0026micro;M to 20\u0026thinsp;\u0026plusmn;\u0026thinsp;9 \u0026micro;M (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The 50 mM of SDS molecules corresponds to slightly less than 1 mM of micelles, which is much higher than 10 \u0026micro;M Aβ peptide. Consequently, there is arguably no more than one Aβ peptide in each micelle. It is well established that the central and C-terminal hydrophobic Aβ segments insert themselves into SDS micelles, where they adopt α-helical conformations (Jarvet et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; \u0026Ouml;sterlund et al., \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The hydrophilic N-terminal Aβ segment is hanging outside the membrane surface, where it is free to e.g. bind metal ions. Thus, the current results are in line with previous studies showing that Aβ N-terminal metal binding is not much affected when the peptide binds to membranes or membrane mimetica (Lindgren et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). These results also suggest that Hg(II) binding should be very similar for Aβ\u003csub\u003e40\u003c/sub\u003e and the slightly longer Aβ\u003csub\u003e42\u003c/sub\u003e peptide, as the two additional hydrophobic C-terminal residues in Aβ\u003csub\u003e42\u003c/sub\u003e should not affect the N-terminal metal binding properties.\u003c/p\u003e \u003cp\u003eThe CD results show that addition of Hg(II) induces concentration-dependent changes in the α-helical structure of Aβ peptides positioned in SDS micelles (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). For Aβ\u003csub\u003e4\u0026minus;40\u003c/sub\u003e, the structural transition involves Hg(II)-induced formation of coil-coil interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), similar to previously observed effects of e.g. Cu(II) and Ni(II) ions (Tiiman et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Berntsson et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). For Aβ\u003csub\u003e40\u003c/sub\u003e, the nature of the structural changes are less clear, and may involve other secondary structures than α-helix (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The observed structural rearrangements of Aβ peptides in a membrane-mimicking environment could be biologically relevant, as membrane disruption is one likely toxic mechanism of Aβ oligomers (W\u0026auml;rml\u0026auml;nder et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Structural alterations are also a likely explanation for the previously reported observation that binding of Hg(II) reduces the propensity of Aβ\u003csub\u003e42\u003c/sub\u003e to form ion channels in membranes (Meleleo et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eHg(II) binds to the N-terminal segment of biologically relevant Aβ peptide variants. The Hg(II) binding affinity is in the low \u0026micro;M range, with the three N-terminal Aβ His residues involved as binding ligands. Hg(II) binding induces structural alterations in Aβ monomers positioned in membrane-mimicking SDS micelles. Equimolar amounts of either Hg(I) or Hg(II) inhibit normal Aβ fibrillation by instead directing the aggregation process towards formation of large amorphous aggregates. As Aβ aggregation is considered a major toxic mechanism in AD progression, the observed Hg-induced structural rearrangements are likely relevant for AD neuropathology. The capacity to induce misfolding and aggregation of proteins and peptides might be a general toxic mechanism of mercury.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003e \u003cb\u003eConflict of interest\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eAG, JJ, MP, and SW are shareholders in CellPept Sweden AB. Neither this company, nor the funding organizations, had any role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from the Estonian Research Council to MP (no. PRG1506) and to PP (no. PRG1289), from the Magnus Bergvall Foundation to SW, and from the Swedish Brain Foundation and the Swedish Natural Science Research Council to AG.\u003c/p\u003e\u003ch2\u003eData availability:\u003c/h2\u003e \u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbelein A, Abrahams JP, Danielsson J, Gr\u0026auml;slund A, Jarvet J, Luo J, Tiiman A, W\u0026auml;rml\u0026auml;nder SK (2014) The hairpin conformation of the amyloid beta peptide is an important structural motif along the aggregation pathway. J Biol Inorg Chem 19(4\u0026ndash;5):623\u0026ndash;634\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmadi S, Zhu S, Sharma R, Wilson DJ, Kraatz HB (2019) Interaction of metal ions with tau protein. The case for a metal-mediated tau aggregation. 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Biochim Biophys Acta Proteins Proteom 1867(5):492\u0026ndash;501\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Inorganic mercury, Alzheimer’s disease, Amyloid aggregation, Metal-protein binding, Neurodegeneration, Heavy metal toxicity, Spectroscopy, Transmission electron microscopy","lastPublishedDoi":"10.21203/rs.3.rs-5888115/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5888115/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMercury (Hg) exposure is a possible risk factor for Alzheimer´s disease (AD), and some studies have found higher Hg levels in AD patients. Yet, the evidence is inconclusive, and a mechanism linking Hg exposure to AD neuropathology remains to be found. The hallmark of AD brains is deposits of insoluble amyloid plaques consisting mainly of aggregated amyloid-β (Aβ) peptides.\u003c/p\u003e\n\u003cp\u003eHere, we use transmission electron microscopy (TEM) and biophysical spectroscopy techniques to study \u003cem\u003ein vitro\u003c/em\u003e interactions between inorganic Hg and the pathologically relevant Aβ(1-40) and Aβ(4-40) variants and the Aβ(1-40)(H6A, H13A, H14A) mutant. For the first time, the effect on Aβ aggregation of both Hg(I) and Hg(II) is compared.\u003c/p\u003e\n\u003cp\u003eHg(II) binds to Aβ(1-40) with an apparent binding affinity of 28±8 µM. The N-terminal His6, His13 and His14 residues are involved in binding coordination. Hg(II) binding induces structural alterations (coil-coil interactions) in Aβ monomers positioned in membrane-mimicking SDS micelles. Equimolar amounts of either Hg(I) or Hg(II) inhibit normal Aβ fibrillation by directing the aggregation process towards formation of large amorphous aggregates. All these structural rearrangements may be relevant for the harmful Aβ aggregation processes involved in AD brain pathology. Inducing protein misfolding and aggregation might be a general toxic mechanism of mercury.\u003c/p\u003e","manuscriptTitle":"Binding of Hg(I) and Hg(II) to amyloid-beta (Aβ) peptide variants: effects on structure and aggregation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-24 15:43:32","doi":"10.21203/rs.3.rs-5888115/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12215e6f-0b6a-4e92-9d85-77cc2dd3b8e7","owner":[],"postedDate":"January 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":43308398,"name":"Biophysics"},{"id":43308399,"name":"General Biochemistry"},{"id":43308400,"name":"Chemical Biology"}],"tags":[],"updatedAt":"2025-01-24T15:43:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-24 15:43:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5888115","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5888115","identity":"rs-5888115","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}