Single-molecule mass measurements reveal distinct effects of sodium and potassium on mini-spidroin assembly

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Kosmotropic salts induce phase separation of spidroins, however, their exact role in assembly is not clear. Here, we investigate how sodium and potassium phosphate affect spidroin interactions via the single-molecule method mass photometry. We observed that spidroin oligomerization occurs at low nanomolar protein concentrations. Potassium ions were found to stabilize a compact conformation of individual spidroins and slow down pH-induced β-sheet aggregation, consistent with its more kosmotropic nature. Microfluidic MP showed that pre-assembly of the protein through salt-induced phase separation reduced the number and size of oligomeric intermediates that form upon acidification. Together, the findings suggest that spidroins have an inherent ability to self-assemble, blurring the line between one- and two-phase status. Subtle differences in ion composition are sufficient to change spidroin stability and assembly, potentially contributing to silk spinning in vivo by balancing storage stability with rapid fiber formation. Biological sciences/Biophysics/Single-molecule biophysics Biological sciences/Biotechnology/Biomaterials/Biomaterials – proteins Liquid-liquid phase separation single-molecule analysis microfluidics silk fiber formation Hofmeister series Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Spider silk is an extraordinary biomaterial due to its high tensile strength, extensibility, and biocompatibility. The primary protein components of spider silk are spidroins, highly dynamic proteins with a long, mostly disordered repeat region flanked by non-repetitive C- and N-terminal domains ( Fig. 1 a ) 1 , 2 . Spidroins undergo a complex and highly regulated assembly process during silk fiber formation. They are stored in the silk gland as a highly concentrated, soluble dope 3 , 4 . During spinning, the dope is extruded through a narrowing duct while experiencing shear forces and a pH drop from pH > 7 to ca. pH 5 ( Fig. 1 b ) 4 , 5 . Acidification induces antiparallel dimerization of the N-terminal domain (NT) which cross-links the spidroins 6 – 8 . The C-terminal domain (CT) is a constitutive dimer which is in some spidroins linked by a disulfide bond 9 . Each subunit contains a conserved salt bridge that is broken at low pH to initiate β-sheet aggregation 5 , 9 . Using engineered mini-spidroins composed of the terminal domains as well as a repetitive domain with two repeats (NT2RepCT), it was shown that the pH-induced structural changes in the NT and CT domains trigger the conversion of the soluble protein into solid fibers 10 , 11 . Another contributing factor in spidroin assembly is the presence of various ions in the silk gland. The sac of the gland contains high amounts of sodium and chloride (192 and 164 mM, respectively), which are exchanged for potassium and phosphate in the spinning duct ( Fig. 1 b ) 5 , 12 . Native major ampullate spidroins as well as recombinant mini-spidroins can undergo liquid-liquid phase separation (LLPS) in vitro when exposed to high concentrations (> 250 mM) of phosphate 13 – 16 . Phase separation is driven by low-affinity interactions between polyalanines as well as aromatic and basic residues in the repeat domains, giving rise to liquid-like macroscopic droplets 14 , 15 , 17 . LLPS has a pronounced effect on the aggregation behaviour of NT2RepCT, shifting from being stable at neutral pH to spontaneously converting into β-sheet-rich aggregates within hours 14 , 18 . This destabilizing effect is mediated by the CT domain, which contains an N-terminal disordered extension capable of LLPS 9 , 14 . Inducing LLPS of the extended CT domain strongly accelerates the conversion to β-sheet at low pH, whereas removal of the extension renders CT insensitive to LLPS conditions 14 . NMR studies have furthermore shown that ions affect the structure of the repeat domain, with chaotropic ions favoring a random coil conformation, whereas kosmotropic ions promote turn and helix formation 19 . Together, the observations suggest that kosmotropic ions tune LLPS-like interactions as part of the spinning process, although their exact concentrations in the gland have not been established 20 . While the kosmotropic nature of phosphate has been shown to be important for spidroin LLPS, the roles of sodium and potassium are less established. Both have similar Hofmeister properties, with potassium being only slightly more kosmotropic than sodium ( Fig. 1 b ) , and potassium and sodium phosphate buffers are similarly efficient at inducing spidroin LLPS in vitro 13 , 15 . The fact that the spider removes sodium and increases the potassium content of the dope during spinning thus raises the question whether both may affect spidroins differently 12 . Here, we employed mass photometry (MP) to unravel how different ion and pH regimes affect the interactions of the NT2RepCT mini-spidroin at the single-molecule level. We find that low concentrations of potassium, but not sodium, suppress oligomerization by stabilizing spidroin dimers in the dilute phase. Microfluidic rapid-mixing MP reveals that exposure of spidroins to potassium phosphate prior to pH-induced assembly reduces oligomeric intermediates compared to sodium phosphate. Our findings show that mini-spidroins interact at nanomolar concentrations, and that these interactions are modulated by sodium and potassium, which may help to control the formation of spidroin clusters. Results Potassium affects LLPS-induced aggregation of NT2RepCT YF To study the combined effects of phosphate salt and pH, we used a variant of NT2RepCT, where two tyrosines in each repeat were replaced with phenylalanine (NT2RepCT YF ) 21 . NT2RepCT YF readily undergoes droplet formation when exposed to 500 mM sodium or potassium phosphate (Fig. 1 c) 14 . As a first step, we investigated how the combinations of high and low phosphate buffer concentrations and high and low pH affect spidroin aggregation, using the β-sheet-sensitive fluorescent dye Thioflavin T (ThT). Exposure of NT2RepCT YF to pH 8 in the presence of 20 mM NaP i or KP i resulted in no change in ThT fluorescence. Lowering the pH to 5 caused a rapid increase in fluorescence, as expected for aggregation triggered by the NT and CT domains ( Fig. 1 d ) 5 . Under LLPS conditions (500 mM NaP i or KP i ) 14 , 15 , we observed a strong increase in fluorescence at pH 8 ( Fig. 1 e ) , with KP i being slightly slower than NaP i . These findings are in good agreement with the reported slow conversion of droplets into β-sheet spherical aggregates 18 . We then tested lowering the pH in addition to inducing LLPS by exposing the spidroins to a combination of 500 mM phosphate buffer and pH 5. We observed a more rapid fluorescence increase than at pH 8, suggesting that pH-induced aggregation supersedes aggregation at high phosphate concentration. Interestingly, 500 mM NaP i led to a greater fluorescence increase than KP i . This finding suggests that at high salt concentrations, sodium promotes spidroin aggregation more efficiently than potassium. Potassium reduces oligomer formation at nanomolar protein concentrations To investigate whether NaP i and KP i ions affect the interactions of mini-spidroins under assembly conditions, we turned to mass photometry (MP), which measures the molecular weight of proteins in solution based on their light scattering properties 22 . Importantly, MP typically operates at protein concentrations in the low nanomolar range, well below the threshold for NT2RepCT YF spinning or LLPS 10 , 15 , 23 . Therefore, it enables us to monitor how assembly states are impacted by high and low pH, as well as by low and high salt concentrations, without bias that could arise from protein partitioning into dilute and dense phases. For MP measurements, a 10 µM stock of NT2RepCT YF in MQ was diluted to 400 nM in different buffers, equilibrated for a minimum of 2 minutes, and subsequently loaded into the mass photometer sample chamber at a final concentration of 20 nM ( Fig. 2 a ) . In 20 mM NaP i at pH 8 ( Fig. 2 b, pink) , we observed NT2RepCT YF dimers (66 kDa) as the dominant species. We also detected oligomers of dimers, which could be resolved up to the 462 kDa 14-mer ( Fig. 2 b, pink insert) . When the protein was diluted in 20 mM NaP i at pH 5, signal intensity decreased by ca. 50%. This finding indicates loss of protein from the solution, which is consistent with pH-induced aggregation. Despite the reduced signal, spidroin oligomers up to a decamer (330 kDa) could again be observed, with the ratios between dimers and oligomers largely unchanged ( Fig. 2 b, purple) . When the experiment was repeated with KP i , only minor oligomerization of dimers was observed at high pH, both in terms of signal intensity and oligomer size ( Fig. 2 b, light blue insert). Lowering the pH to 5 resulted in an increase in NT2RepCT YF dimer signal and a near-complete loss of oligomers. Upon raising the salt concentration to 500 mM, no other assembly states than dimers were observed, both at low and high pH, as well as with NaP i and KP i (Figure S1 ) . We conclude that low amounts of potassium reduce the formation of NT2RepCT YF oligomers both at low and high pH compared to sodium. High sodium or potassium phosphate concentrations, on the other hand, generally favor the dimeric state of NT2RepCT YF under MP conditions. To gain more insights into how low concentrations of sodium and potassium affect NT2RepCT YF conformations, we turned to native ion mobility mass spectrometry (IM-MS). Here, electrospray ionization (ESI) is used to transfer intact protein complexes from solution to the gas phase and the confirmation of the ions is analyzed by measuring the time it takes them to traverse a gas-filled drift cell 24 . Although ESI relies on volatile buffers to produce ions, it tolerates low millimolar concentrations of non-volatile salts 25 . This feature of nMS has been used to study the effect of salts in the Hofmeister series on the conformations and stabilities of protein complexes 26 . We therefore recorded positive ionization mode mass spectra of 10 µM NT2RepCT YF in 100 mM ammonium acetate, which is commonly used to stabilize protein complexes during ESI. The solution was supplemented with either 10 mM sodium acetate or potassium acetate, as phosphate buffers cause signal suppression. We then compared the arrival times of the 12 + charge state of the monomer as well as the 10 + charge state of the dimer. The resulting arrival time distributions show that under otherwise identical conditions, the protein retains a more compact conformation in the presence of potassium compared to sodium, both in its monomeric and dimeric state, as well as with bound sodium or potassium ions (Figure S2) . These data indicate that protein oligomerization observed by MP coincides with a less compact conformation as seen by IM-MS. Potassium and sodium affect self-association of the repeat domains To gain insight into the contributions of the different domains of NT2RepCT YF to oligomerization, we performed the same MP experiments with NT2Rep, which lacks the CT domain ( Fig. 3 a ) , and the isolated NT domain ( Fig. 3 b ). Both proteins are too small to be detected as monomers (NT2Rep: 22 kDa, NT: 15 kDa) but can be detected in their dimeric states, since MP has a lower mass limit of approximately 30 kDa 23 . While not informing about the monomer/dimer ratio, the data reveal that a fraction of the protein is dimeric even at high pH. In 20 mM NaP i , pH 8, the dimer peak for NT2Rep showed a broad higher-mass shoulder which was poorly resolved, suggesting the presence of oligomers that range in size up to ca. 10 monomers ( Fig. 3 a, pink histogram). At pH 5, the intensity of the dimer peak increased a bit, and the shoulder peak persisted, but could be better resolved into tetramers, hexamers, and octamers ( Fig. 3 a, purple histogram). We speculate that the increased resolution is due to the oligomers being composed mainly of dimers (44 kDa). In 20 mM KP i we observed exclusively dimers and no higher oligomers ( Fig. 3 a, light blue and dark blue histograms) . When repeating the same experiment with NT, only dimers were detected ( Fig. 3 b ) . The NT dimer (30 kDa) is at the lower detection limit of the instrument and was not used for quantification. The histograms showed no notable differences between NaP i and KP i and no higher NT oligomers. MP of NT2Rep or NT in 500 mM NaP i or KP i showed an increase in dimers at pH 5 but otherwise no differences between conditions (Figure S3) . The finding that NT2Rep, but not NT, forms oligomers in 20 mM NaP i indicates that self-association is mediated by the repeat domains. NT2RepCT YF contains four repeat domains per dimer, which may account for the higher signal intensity of the oligomers formed by the full-length mini-spidroin ( Figs. 3 a, 2 b ) . Together, the data imply that at low ion and protein concentrations, KP i reduces oligomerization of repeat domains by promoting a compact conformation. Inducing LLPS suppresses NT2RepCTassembly intermediates A consideration when using MP to study spidroin assembly is the requirement to work in dilute solutions 23 , 27 . As shown above, KP i reduces oligomerization of NT2RepCT YF at nanomolar protein concentrations. At micromolar protein concentrations, on the other hand, KP i promotes protein interactions and LLPS, which is a common feature of several spider and silkworm fibroins 14 , 15 , 28 . We therefore asked whether we could adapt MP to be able to study the effects of KP i on NT2RepCT YF assembly under LLPS conditions while circumventing the need for micromolar protein concentrations required for LLPS 29 . We therefore turned to the MassFluidix system (Refeyn, Oxford, UK), which was designed to facilitate analysis of more concentrated solutions by including a rapid dilution step prior to data acquisition 27 . Briefly, the protein solution in buffer A is diluted in buffer B, followed by a 37 millisecond mixing time, and then measured in an MP flow cell 27 . We adapted this system for analysis of spidroin assembly by preparing 10 µM of NT2RepCT YF in LLPS- or non-LLPS buffer at pH 8 and diluting either solution 1000-fold in 20 mM NaP i at pH 5 to trigger assembly ( Fig. 4 a ) . Since NT dimerizes on the low-millisecond timescale 30 , we hypothesized that this setup could capture polymerization intermediates of NT2RepCT YF . First, we analysed NT2RepCT YF under non-LLPS conditions using 20 mM NaP i pH 8, as buffer A and B. The resulting histograms have lower signal intensity than those obtained at steady-state conditions ( Fig. 2 ) , and show mostly dimers with a minor fraction of higher oligomers ( Fig. 4 b, pink histogram). Next, we rapidly lowered the pH by using 20 mM NaP i pH 5, as buffer B. Strikingly, we observed a wide distribution of oligomeric states, ranging up to the megadalton range, whose intensity decayed exponentially with increasing molecular weight ( Fig. 4 b, purple histogram). The oligomeric states could not be resolved. Since low pH destabilizes the non-covalent CT dimer, and trimers and pentamers of mini-spidroins have been observed by nMS at pH 5 31 , the poor resolution might stem from the presence of even and odd oligomer stoichiometries. As these oligomers are not present in MP histograms recorded under steady-state conditions ( Fig. 2 ) , they likely represent assembly intermediates that form within milliseconds and subsequently escape the detection window by becoming too large, too polydisperse, or both. Next, we induced LLPS prior to diluting the protein for MP measurements, mimicking the proposed events in the gland. 20 When 10 µM of NT2RepCT YF in 500 mM KP i , pH 8 (buffer A, see Fig. 1 c) was diluted in 20 mM NaP i , pH 8 (buffer B), the MP histogram showed no pronounced difference compared the data obtained in 20 mM NaP i only ( Fig. 4 b, light blue histogram) . When the pH was rapidly lowered to 5, we observed no oligomerization, but instead a pronounced reduction in signal intensity ( Fig. 4 b, dark blue histogram) . These data contrast the observation made for non-LLPS conditions and allow us to delineate a role for LLPS during spidroin assembly: In 20 mM NaP i, pH 8, most spidroins are in the dilute phase. Lowering the pH to 5 causes polymerization via the NT domains, and we can observe a wide range of oligomeric states immediately afterwards. In 500 mM KP i , the proteins form liquid droplets that are held together by weak interactions between the repeat domains. When the pH is lowered to 5, these pre-assembled proteins immediately polymerize into oligomers that are too large for the detection window of the mass photometer. Assuming that NT dimerization is faster than droplet dissolution in the mixing capillary, low pH crosslinks the spidroins while they are closer in space, which we speculate gives rise to larger assemblies in a shorter time frame. Discussion Reproducing spinning conditions and monitoring their effects on spidroins at the molecular level is a major challenge in the experimental investigation of spider silk. Here, we have employed MP to investigate the effects of sodium and potassium phosphate, which are present in the silk gland, on the oligomeric state of the NT2RepCT YF mini-spidroin. MP is a single-molecule technique that can trace individual protein interactions, but requires nanomolar protein concentrations that do not represent native or biomimetic spinning conditions 32 . However, using microfluidics to rapidly switch solution conditions and protein concentrations, we were able to directly observe how ions and pH synergistically affect spidroin assembly. The data from MP indicate different effects of sodium and potassium on NT2RepCT YF that are consistent with their positions in the Hofmeister series. Potassium ions, which are slightly kosmotropic, reduce self-association of the repeat domains and stabilize NT2RepCT YF dimers. The slightly chaotropic sodium ions destabilize the protein and enable intermolecular interactions between the repeat domains. Both cations are close to the middle of the Hofmeister range, yet the fact that we observe effects at low ion concentrations suggests that the partially structured repeat domain may be sensitive to such subtle differences or engage in ion-specific interactions. Native spidroins contain hundreds of repeats, which may render them more susceptible to minor changes in ion composition. Here, the potassium-induced compaction of individual spidroins that we observe at nanomolar protein concentrations likely stems from increased intramolecular interactions in the repeat domain. At higher protein concentrations, potassium shifts from promoting intramolecular to intermolecular contacts. In line with this interpretation, Numata and colleagues have reported that kosmotropic ions induce oligomerization of the repeat domain at millimolar peptide concentrations 19 . Potassium is likely to have a pronounced effect on phase transitions in native spidroins, considering their high number of repeats and extreme concentration in the gland. Recombinant mini-spidroins require high phosphate concentrations to undergo LLPS 13 – 15 . Importantly, spidroin droplets formed at high salt are metastable and undergo spontaneous aggregation, which would be detrimental in native silk dope 18 . We speculate that much lower amounts of kosmotropic ions would be required to promote LLPS-like interactions in the silk gland where there is a higher concentration of repeat domains. MassFluidix MP, where interference from high amounts of KP i is mitigated by rapid dilution, provides an indication how LLPS could contribute to silk assembly. We find that pre-assembly of NT2RepCT YF into macroscopic droplets reduces oligomeric intermediates when polymerization is triggered by lowering the pH. Considering the sensitivity to potassium that we observe with mini-spidroins in dilute solutions, we speculate that even a small shift in the potassium content of the gland may move the spidroins in their phase space to facilitate more efficient spinning. Linder and co-workers have recently observed by analytical ultracentrifugation that NT2RepCT forms clusters that transition to aggregates as a function of protein concentration, temperature, or pH 33 . Their findings show that the protein occupies a two-phase regime even below the threshold for LLPS. The MP data now reveal that the tendency to oligomerize extends to the low nanomolar range, further blurring the distinction between single proteins, clusters, and droplets. Rather than presenting a phase diagram with a defined solubility line, spidroins are inherently bound to self-assemble via their repeat regions, which can be fine-tuned by ions. This behaviour is markedly different from other phase-separating proteins. For example, MP has shown that LLPS of α-Synuclein does not proceed via small oligomers but is driven by the formation of megadalton clusters at micromolar protein concentrations 29 . We speculate that the ability to assemble spidroins with minimal intermediates helps spiders to achieve their high spinning speeds. Materials and Methods Protein sequences Protein expression and purification All proteins were expressed and purified as previously described, however sonication (6 min, 2 s on, 8 s off, 30%) was used for cell disruption 5,34 . NT2repCT YF was dialyzed into deionized water while NT2rep and NT were kept in 20 mM Tris, pH 8. After concentrating (NT to 10 mM, NT2rep and NT2repCT YF to 200 – 300 μM) using Amicon spin filters (Millipore), all proteins were stored at -20°C. Microscopy Bright-field microscopy images of 10 μM NT2repCT YF droplets in 20 mM or 500 mM KP i , pH 8, were acquired using a Nikon Eclipse Ti series inverted microscope (Nikon) equipped with a Crest X-light V2 series confocal unit (Nikon) using a Plan Apo 40x objective (Nikon) and a Zyla sCMOS camera (Andor). Thioflavin T Assay Aggregation kinetics were monitored by measuring the ThT fluorescence (excitation: 445-8 nm, emission: 488-15 nm) using a CLARIOstar microplate reader (BMG labtech) with the gain set to 2000. In black half-area 96-well polystyrene microplates with a transparent bottom (Corning), 40 uL 10 μM ThT and 25 μM NT2RepCT YF were mixed in the different buffers (20 mM NaP i pH 8, 20 mM NaP i pH 5, 20 mM KP i pH 8, 20 mM KP i pH 5, 500 mM NaP i pH 8, 500 mM NaP i pH 5, 500 mM KP i pH 8, 500 mM KP i pH 5) and the plate sealed with transparent cover film to avoid evaporation. All measurements were conducted at 30°C without agitation. Mass photometry All mass photometry experiments were carried out at room temperature using the Refeyn Two MP instrument (Refeyn) in the normal measurement mode with regular image size. Proteins were pre-diluted first to 10 μM in 20 mM NaP i pH8 and from that further diluted (400 nM) into the desired buffer. A final concentration of 20 nM (NT2RepCT YF , NT-2rep) or 100 nM (NT) was then used on the mass photometry slide. Each measurement was recorded for 1 min (3000 frames) using the AcquireMP (2024 R1.1) software. Histograms were created in DiscoverMP (v2024 R1) with β-amylase (56, 112 and 224 kDa) or bovine serum albumin (66 and 132 kDa) in 20 mM NaP i pH 8 as mass calibrants. Data were analyzed using DiscoverMP software (Refeyn). Unbinding peak intensities were significantly lower than the binding peak intensities in all instances . Mass Fluidix Mass Fluidix-MP measurements were carried out using 1st generation microfluidic chips (Refeyn). 10 μM NT2RepCT YF in 20 mM NaPi, pH 8, or in 500 mM KPi, pH 8, were pumped via the sample inlet. 20 mM NaPi, pH 8 or 20 mM NaPi, pH 5, were supplied via the buffer inlet. Inlet tubes were washed with MQ and primed with buffer before and between runs. Nitrogen pressure was used to control the sample and buffer flow. Pressure and related flow rates were adjusted manually using the OxyGEN software. Flow rates were adjusted to 1 μL/min and 999 μL/min for sample and buffer, respectively, resulting in 1500 - 3000 binding events/min. Histograms were created in DiscoverMP (v2024 R1) and β-amylase (56, 112 and 224 kDa) or bovine serum albumin (66 and 132 kDa) was used as mass calibrant. Native ion mobility mass spectrometry NT2RepCT YF was buffer-exchanged into 100 mM ammonium acetate, or into 100 mM ammonium acetate supplemented with 10 mM sodium acetate (NaAc) or 10 mM potassium acetate (KAc), pH 8 using BioSpin6 columns (BioRad, CA, USA). Samples were loaded into nESI capillaries (Thermo Fisher Scientific Inc., MA, USA). Mass spectra were recorded in positive ionization mode on a Waters Synapt G1 traveling-wave IM mass spectrometer (MS Vision, The Netherlands). The capillary voltage was maintained at 1,5 kV and the sample cone was 100 V. The source temperature was +30°C. The trap and transfer collision energies were 50 V. The trap gas was argon at a flow rate of 1.5 mL/min, the IMS gas was nitrogen at a flow rate of 18.5 mL/min. The ion mobility-mass spectrometry settings were: IMS wave height 10 V and IMS wave velocity 350 m/s. Data were analyzed using MassLynx 4.1 (Waters, UK). Declarations Acknowledgements ML is supported by a KI faculty-funded Career Position, a Cancerfonden Project grant (22-2023 Pj), a VR Project Grant (2024-04483), a KAW Project grant (2022.0032), and a Consolidator Grant from the Swedish Society for Medical Research (SSMF). AR is supported by the Swedish Research Council (2024-02919), FORMAS (2023-01313), Knut and Alice Wallenberg Foundation (grant 2023.0331) and Olle Engkvists Stiftelse (233-0334). Competing Interest Statement The authors declare no competing interests. Data Availability Statement All data are available from the corresponding authors upon reasonable request. References Hu, X.; Vasanthavada, K.; Kohler, K.; McNary, S.; Moore, A. M. F.; Vierra, C. A. Molecular Mechanisms of Spider Silk. Cell. Mol. 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Engineered Spider Silk Proteins for Biomimetic Spinning of Fibers with Toughness Equal to Dragline Silks. Adv. Funct. Mater. 2022 , 32 (23), 2200986. https://doi.org/10.1002/adfm.202200986. Additional Declarations There is NO Competing Interest. Supplementary Files OsterholzetalSupplementaryinformation.docx SI Figures 1-3 Cite Share Download PDF Status: Published Journal Publication published 02 Jan, 2026 Read the published version in Communications Materials → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-7639041","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":522026437,"identity":"b69cb9d0-25e5-4606-9414-a2ec5e7e96e2","order_by":0,"name":"Michael 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08:20:03","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":129899,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7639041/v1/3db295a2a39a70df248ec46f.html"},{"id":93207392,"identity":"217d48ae-c6cb-40bc-9dd5-47da9a5e08b8","added_by":"auto","created_at":"2025-10-10 08:20:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":223743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSpidroin- and silk gland architecture and salt- and pH-induced aggregation of NT2RepCT\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eYF\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e. (a) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eStructure of the NT2RepCT mini-spidroin. The NT domain (blue) undergoes dimerization below pH 6. The repeat domain (green) composed of two poly-alanine segments and disordered linkers drives LLPS in the presence of NaP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e or KP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e. The CT domain (yellow) is a constitutive dimer and undergoes β-sheet aggregation when exposed to acidic pH or high NaP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e or KP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e concentrations. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(b)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Architecture of the silk gland. The spinning dope is stored in the sac at pH 7-8, but undergoes acidification to pH 5, as well as increased phosphate and potassium concentrations, during passage through the spinning duct. The Hofmeister series for cations is shown below. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(c)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e Light microscopy of NT2RepCT\u003c/em\u003e\u003csup\u003e\u003cem\u003eYF\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e shows droplet formation in the presence of 500 mM KP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(d)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e ThT fluorescence shows that pH-induced aggregation of NT2RepCT\u003c/em\u003e\u003csup\u003e\u003cem\u003eYF\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e is near-identical in 20 mM\u0026nbsp; KP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e and 20 mM NaP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(e)\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e ThT assays of NT2RepCT\u003c/em\u003e\u003csup\u003e\u003cem\u003eYF\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e in 500 mM phosphate buffer show pronounced aggregation at pH 8 and rapid aggregation accompanied by reduced ThT fluorescence at pH 5. At pH 5 and 500 mM phosphate buffer, sodium increases aggregation compared to potassium. Lines and shaded areas indicate the average and standard deviation, respectively, of three repeats.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7639041/v1/6617d5ab58d04599a23f23bc.png"},{"id":93208908,"identity":"1c526d1f-ba07-4b4a-b08b-60a8f6859d0e","added_by":"auto","created_at":"2025-10-10 08:36:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":145344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eLow concentrations of potassium suppress NT2RepCT\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eYF\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e oligomerization. (a) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eExperimental strategy for steady-state MP. NT2RepCT\u003c/em\u003e\u003csup\u003e\u003cem\u003eYF\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e is diluted 500 times in NaP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e or KP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e buffer prior to MP measurements. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(b) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eMP histograms show oligomers of dimers up to 16-mers in NaP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e at pH 8 (pink histogram). The signal intensity decreases in NaP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e at pH 5 (purple histogram). In the presence of KP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e, fewer oligomers are detected at pH 8 (light blue histogram), and none at pH 5 (dark blue histogram). Theoretical molecular weights and oligomeric states are indicated by red dashed lines.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7639041/v1/c29d4d388ccb3db0054b2e79.png"},{"id":93207393,"identity":"39baee7d-bb36-4b99-b6c3-db2b49184e1a","added_by":"auto","created_at":"2025-10-10 08:20:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":123891,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eNT2Rep, but not NT, oligomerizes in a potassium-sensitive manner. (a) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eMP histograms of NT2Rep at pH 8 (top row) or 5 (bottom row) show poorly resolved oligomers in the presence of 20 mM NaP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e. The oligomers are slightly better resolved at pH 5 than at pH 8. No oligomers are detected in KP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(b) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eHistograms of the NT domain dimer show no major differences between NaP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e. and KP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e buffers. Theoretical molecular weights and oligomeric states are indicated by red dashed lines.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7639041/v1/0acc166d260f512e635ef70f.png"},{"id":93207395,"identity":"f337e18a-370c-4650-a777-f4de365cabed","added_by":"auto","created_at":"2025-10-10 08:20:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":146253,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMicrofluidic MP captures the effect of LLPS on NT2RepCT\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003eYF\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e assembly intermediates. (a) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eArchitecture of the MassFluidix system. A 10 μM solution of NT2RepCT\u003c/em\u003e\u003csup\u003e\u003cem\u003eYF\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e in non-LLPS or LLPS buffer (buffer A) is rapidly diluted 1000-fold in 20 mM NaP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003cem\u003e buffer at pH 8 or pH 5 (buffer B), and measured in an MP flow cell following 37 ms mixing time. \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e(b) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eMP histograms of\u0026nbsp; NT2RepCT\u003c/em\u003e\u003csup\u003e\u003cem\u003eYF\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e diluted from non-LLPS buffer (pink and purple histograms) show that lowering the pH to 5 induces broad oligomerization. When NT2RepCT\u003c/em\u003e\u003csup\u003e\u003cem\u003eYF\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e is diluted from LLPS-buffer (light blue and dark blue histograms), lowering the pH results in loss of signal and no detectable oligomers. Theoretical molecular weights and oligomeric states are indicated by red dashed lines.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7639041/v1/98c5776a9f55099df6d73ede.png"},{"id":101839444,"identity":"85d0845f-87bb-4915-8afa-99ef92dcfc3b","added_by":"auto","created_at":"2026-02-04 08:22:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1586773,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7639041/v1/e0ab5a37-8a57-4af9-adb7-e924f2624eba.pdf"},{"id":93207402,"identity":"8212c480-a4ac-48d7-a114-641e82791856","added_by":"auto","created_at":"2025-10-10 08:20:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":706786,"visible":true,"origin":"","legend":"SI Figures 1-3","description":"","filename":"OsterholzetalSupplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7639041/v1/7a8df5866ae8800a30b03b13.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Single-molecule mass measurements reveal distinct effects of sodium and potassium on mini-spidroin assembly","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpider silk is an extraordinary biomaterial due to its high tensile strength, extensibility, and biocompatibility. The primary protein components of spider silk are spidroins, highly dynamic proteins with a long, mostly disordered repeat region flanked by non-repetitive C- and N-terminal domains \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Spidroins undergo a complex and highly regulated assembly process during silk fiber formation. They are stored in the silk gland as a highly concentrated, soluble dope\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. During spinning, the dope is extruded through a narrowing duct while experiencing shear forces and a pH drop from pH\u0026thinsp;\u0026gt;\u0026thinsp;7 to \u003cem\u003eca.\u003c/em\u003e pH 5 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Acidification induces antiparallel dimerization of the N-terminal domain (NT) which cross-links the spidroins\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The C-terminal domain (CT) is a constitutive dimer which is in some spidroins linked by a disulfide bond\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Each subunit contains a conserved salt bridge that is broken at low pH to initiate β-sheet aggregation\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Using engineered mini-spidroins composed of the terminal domains as well as a repetitive domain with two repeats (NT2RepCT), it was shown that the pH-induced structural changes in the NT and CT domains trigger the conversion of the soluble protein into solid fibers\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAnother contributing factor in spidroin assembly is the presence of various ions in the silk gland. The sac of the gland contains high amounts of sodium and chloride (192 and 164 mM, respectively), which are exchanged for potassium and phosphate in the spinning duct \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Native major ampullate spidroins as well as recombinant mini-spidroins can undergo liquid-liquid phase separation (LLPS) \u003cem\u003ein vitro\u003c/em\u003e when exposed to high concentrations (\u0026gt;\u0026thinsp;250 mM) of phosphate\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Phase separation is driven by low-affinity interactions between polyalanines as well as aromatic and basic residues in the repeat domains, giving rise to liquid-like macroscopic droplets\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. LLPS has a pronounced effect on the aggregation behaviour of NT2RepCT, shifting from being stable at neutral pH to spontaneously converting into β-sheet-rich aggregates within hours\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. This destabilizing effect is mediated by the CT domain, which contains an N-terminal disordered extension capable of LLPS\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Inducing LLPS of the extended CT domain strongly accelerates the conversion to β-sheet at low pH, whereas removal of the extension renders CT insensitive to LLPS conditions\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. NMR studies have furthermore shown that ions affect the structure of the repeat domain, with chaotropic ions favoring a random coil conformation, whereas kosmotropic ions promote turn and helix formation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Together, the observations suggest that kosmotropic ions tune LLPS-like interactions as part of the spinning process, although their exact concentrations in the gland have not been established\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWhile the kosmotropic nature of phosphate has been shown to be important for spidroin LLPS, the roles of sodium and potassium are less established. Both have similar Hofmeister properties, with potassium being only slightly more kosmotropic than sodium \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, and potassium and sodium phosphate buffers are similarly efficient at inducing spidroin LLPS \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The fact that the spider removes sodium and increases the potassium content of the dope during spinning thus raises the question whether both may affect spidroins differently\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eHere, we employed mass photometry (MP) to unravel how different ion and pH regimes affect the interactions of the NT2RepCT mini-spidroin at the single-molecule level. We find that low concentrations of potassium, but not sodium, suppress oligomerization by stabilizing spidroin dimers in the dilute phase. Microfluidic rapid-mixing MP reveals that exposure of spidroins to potassium phosphate prior to pH-induced assembly reduces oligomeric intermediates compared to sodium phosphate. Our findings show that mini-spidroins interact at nanomolar concentrations, and that these interactions are modulated by sodium and potassium, which may help to control the formation of spidroin clusters.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePotassium affects LLPS-induced aggregation of NT2RepCT\u003csup\u003eYF\u003c/sup\u003e\u003c/h2\u003e\u003cp\u003eTo study the combined effects of phosphate salt and pH, we used a variant of NT2RepCT, where two tyrosines in each repeat were replaced with phenylalanine (NT2RepCT\u003csup\u003eYF\u003c/sup\u003e)\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. NT2RepCT\u003csup\u003eYF\u003c/sup\u003e readily undergoes droplet formation when exposed to 500 mM sodium or potassium phosphate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. As a first step, we investigated how the combinations of high and low phosphate buffer concentrations and high and low pH affect spidroin aggregation, using the β-sheet-sensitive fluorescent dye Thioflavin T (ThT). Exposure of NT2RepCT\u003csup\u003eYF\u003c/sup\u003e to pH 8 in the presence of 20 mM NaP\u003csub\u003ei\u003c/sub\u003e or KP\u003csub\u003ei\u003c/sub\u003e resulted in no change in ThT fluorescence. Lowering the pH to 5 caused a rapid increase in fluorescence, as expected for aggregation triggered by the NT and CT domains \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Under LLPS conditions (500 mM NaP\u003csub\u003ei\u003c/sub\u003e or KP\u003csub\u003ei\u003c/sub\u003e)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, we observed a strong increase in fluorescence at pH 8 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e, with KP\u003csub\u003ei\u003c/sub\u003e being slightly slower than NaP\u003csub\u003ei\u003c/sub\u003e. These findings are in good agreement with the reported slow conversion of droplets into β-sheet spherical aggregates\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. We then tested lowering the pH in addition to inducing LLPS by exposing the spidroins to a combination of 500 mM phosphate buffer and pH 5. We observed a more rapid fluorescence increase than at pH 8, suggesting that pH-induced aggregation supersedes aggregation at high phosphate concentration. Interestingly, 500 mM NaP\u003csub\u003ei\u003c/sub\u003e led to a greater fluorescence increase than KP\u003csub\u003ei\u003c/sub\u003e. This finding suggests that at high salt concentrations, sodium promotes spidroin aggregation more efficiently than potassium.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePotassium reduces oligomer formation at nanomolar protein concentrations\u003c/h3\u003e\n\u003cp\u003eTo investigate whether NaP\u003csub\u003ei\u003c/sub\u003e and KP\u003csub\u003ei\u003c/sub\u003e ions affect the interactions of mini-spidroins under assembly conditions, we turned to mass photometry (MP), which measures the molecular weight of proteins in solution based on their light scattering properties\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Importantly, MP typically operates at protein concentrations in the low nanomolar range, well below the threshold for NT2RepCT\u003csup\u003eYF\u003c/sup\u003e spinning or LLPS\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Therefore, it enables us to monitor how assembly states are impacted by high and low pH, as well as by low and high salt concentrations, without bias that could arise from protein partitioning into dilute and dense phases. For MP measurements, a 10 \u0026micro;M stock of NT2RepCT\u003csup\u003eYF\u003c/sup\u003e in MQ was diluted to 400 nM in different buffers, equilibrated for a minimum of 2 minutes, and subsequently loaded into the mass photometer sample chamber at a final concentration of 20 nM \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. In 20 mM NaP\u003csub\u003ei\u003c/sub\u003e at pH 8 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cb\u003epink)\u003c/b\u003e, we observed NT2RepCT\u003csup\u003eYF\u003c/sup\u003e dimers (66 kDa) as the dominant species. We also detected oligomers of dimers, which could be resolved up to the 462 kDa 14-mer \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cb\u003epink insert)\u003c/b\u003e. When the protein was diluted in 20 mM NaP\u003csub\u003ei\u003c/sub\u003e at pH 5, signal intensity decreased by \u003cem\u003eca.\u003c/em\u003e 50%. This finding indicates loss of protein from the solution, which is consistent with pH-induced aggregation. Despite the reduced signal, spidroin oligomers up to a decamer (330 kDa) could again be observed, with the ratios between dimers and oligomers largely unchanged \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cb\u003epurple)\u003c/b\u003e. When the experiment was repeated with KP\u003csub\u003ei\u003c/sub\u003e, only minor oligomerization of dimers was observed at high pH, both in terms of signal intensity and oligomer size \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cb\u003elight blue insert).\u003c/b\u003e Lowering the pH to 5 resulted in an increase in NT2RepCT\u003csup\u003eYF\u003c/sup\u003e dimer signal and a near-complete loss of oligomers. Upon raising the salt concentration to 500 mM, no other assembly states than dimers were observed, both at low and high pH, as well as with NaP\u003csub\u003ei\u003c/sub\u003e and KP\u003csub\u003ei\u003c/sub\u003e \u003cb\u003e(Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e. We conclude that low amounts of potassium reduce the formation of NT2RepCT\u003csup\u003eYF\u003c/sup\u003e oligomers both at low and high pH compared to sodium. High sodium or potassium phosphate concentrations, on the other hand, generally favor the dimeric state of NT2RepCT\u003csup\u003eYF\u003c/sup\u003e under MP conditions.\u003c/p\u003e\u003cp\u003eTo gain more insights into how low concentrations of sodium and potassium affect NT2RepCT\u003csup\u003eYF\u003c/sup\u003e conformations, we turned to native ion mobility mass spectrometry (IM-MS). Here, electrospray ionization (ESI) is used to transfer intact protein complexes from solution to the gas phase and the confirmation of the ions is analyzed by measuring the time it takes them to traverse a gas-filled drift cell\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Although ESI relies on volatile buffers to produce ions, it tolerates low millimolar concentrations of non-volatile salts\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. This feature of nMS has been used to study the effect of salts in the Hofmeister series on the conformations and stabilities of protein complexes\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. We therefore recorded positive ionization mode mass spectra of 10 \u0026micro;M NT2RepCT\u003csup\u003eYF\u003c/sup\u003e in 100 mM ammonium acetate, which is commonly used to stabilize protein complexes during ESI. The solution was supplemented with either 10 mM sodium acetate or potassium acetate, as phosphate buffers cause signal suppression. We then compared the arrival times of the 12\u0026thinsp;+\u0026thinsp;charge state of the monomer as well as the 10\u0026thinsp;+\u0026thinsp;charge state of the dimer. The resulting arrival time distributions show that under otherwise identical conditions, the protein retains a more compact conformation in the presence of potassium compared to sodium, both in its monomeric and dimeric state, as well as with bound sodium or potassium ions \u003cb\u003e(Figure S2)\u003c/b\u003e. These data indicate that protein oligomerization observed by MP coincides with a less compact conformation as seen by IM-MS.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003ePotassium and sodium affect self-association of the repeat domains\u003c/h3\u003e\n\u003cp\u003eTo gain insight into the contributions of the different domains of NT2RepCT\u003csup\u003eYF\u003c/sup\u003e to oligomerization, we performed the same MP experiments with NT2Rep, which lacks the CT domain \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e, and the isolated NT domain \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e).\u003c/b\u003e Both proteins are too small to be detected as monomers (NT2Rep: 22 kDa, NT: 15 kDa) but can be detected in their dimeric states, since MP has a lower mass limit of approximately 30 kDa\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. While not informing about the monomer/dimer ratio, the data reveal that a fraction of the protein is dimeric even at high pH. In 20 mM NaP\u003csub\u003ei\u003c/sub\u003e, pH 8, the dimer peak for NT2Rep showed a broad higher-mass shoulder which was poorly resolved, suggesting the presence of oligomers that range in size up to ca. 10 monomers \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cb\u003epink histogram).\u003c/b\u003e At pH 5, the intensity of the dimer peak increased a bit, and the shoulder peak persisted, but could be better resolved into tetramers, hexamers, and octamers \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cb\u003epurple histogram).\u003c/b\u003e We speculate that the increased resolution is due to the oligomers being composed mainly of dimers (44 kDa). In 20 mM KP\u003csub\u003ei\u003c/sub\u003e we observed exclusively dimers and no higher oligomers \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cb\u003elight blue and dark blue histograms)\u003c/b\u003e. When repeating the same experiment with NT, only dimers were detected \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. The NT dimer (30 kDa) is at the lower detection limit of the instrument and was not used for quantification. The histograms showed no notable differences between NaP\u003csub\u003ei\u003c/sub\u003e and KP\u003csub\u003ei\u003c/sub\u003e and no higher NT oligomers. MP of NT2Rep or NT in 500 mM NaP\u003csub\u003ei\u003c/sub\u003e or KP\u003csub\u003ei\u003c/sub\u003e showed an increase in dimers at pH 5 but otherwise no differences between conditions \u003cb\u003e(Figure S3)\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eThe finding that NT2Rep, but not NT, forms oligomers in 20 mM NaP\u003csub\u003ei\u003c/sub\u003e indicates that self-association is mediated by the repeat domains. NT2RepCT\u003csup\u003eYF\u003c/sup\u003e contains four repeat domains per dimer, which may account for the higher signal intensity of the oligomers formed by the full-length mini-spidroin \u003cb\u003e(\u003c/b\u003eFigs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Together, the data imply that at low ion and protein concentrations, KP\u003csub\u003ei\u003c/sub\u003e reduces oligomerization of repeat domains by promoting a compact conformation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eInducing LLPS suppresses NT2RepCTassembly intermediates\u003c/h3\u003e\n\u003cp\u003eA consideration when using MP to study spidroin assembly is the requirement to work in dilute solutions\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. As shown above, KP\u003csub\u003ei\u003c/sub\u003e reduces oligomerization of NT2RepCT\u003csup\u003eYF\u003c/sup\u003e at nanomolar protein concentrations. At micromolar protein concentrations, on the other hand, KP\u003csub\u003ei\u003c/sub\u003e promotes protein interactions and LLPS, which is a common feature of several spider and silkworm fibroins\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. We therefore asked whether we could adapt MP to be able to study the effects of KP\u003csub\u003ei\u003c/sub\u003e on NT2RepCT\u003csup\u003eYF\u003c/sup\u003e assembly under LLPS conditions while circumventing the need for micromolar protein concentrations required for LLPS\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We therefore turned to the MassFluidix system (Refeyn, Oxford, UK), which was designed to facilitate analysis of more concentrated solutions by including a rapid dilution step prior to data acquisition\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Briefly, the protein solution in buffer A is diluted in buffer B, followed by a 37 millisecond mixing time, and then measured in an MP flow cell\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. We adapted this system for analysis of spidroin assembly by preparing 10 \u0026micro;M of NT2RepCT\u003csup\u003eYF\u003c/sup\u003e in LLPS- or non-LLPS buffer at pH 8 and diluting either solution 1000-fold in 20 mM NaP\u003csub\u003ei\u003c/sub\u003e at pH 5 to trigger assembly \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. Since NT dimerizes on the low-millisecond timescale\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, we hypothesized that this setup could capture polymerization intermediates of NT2RepCT\u003csup\u003eYF\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFirst, we analysed NT2RepCT\u003csup\u003eYF\u003c/sup\u003e under non-LLPS conditions using 20 mM NaP\u003csub\u003ei\u003c/sub\u003e pH 8, as buffer A and B. The resulting histograms have lower signal intensity than those obtained at steady-state conditions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, and show mostly dimers with a minor fraction of higher oligomers \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, \u003cb\u003epink histogram).\u003c/b\u003e Next, we rapidly lowered the pH by using 20 mM NaP\u003csub\u003ei\u003c/sub\u003e pH 5, as buffer B. Strikingly, we observed a wide distribution of oligomeric states, ranging up to the megadalton range, whose intensity decayed exponentially with increasing molecular weight \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, \u003cb\u003epurple histogram).\u003c/b\u003e The oligomeric states could not be resolved. Since low pH destabilizes the non-covalent CT dimer, and trimers and pentamers of mini-spidroins have been observed by nMS at pH 5\u003csup\u003e31\u003c/sup\u003e, the poor resolution might stem from the presence of even and odd oligomer stoichiometries. As these oligomers are not present in MP histograms recorded under steady-state conditions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, they likely represent assembly intermediates that form within milliseconds and subsequently escape the detection window by becoming too large, too polydisperse, or both.\u003c/p\u003e\u003cp\u003eNext, we induced LLPS prior to diluting the protein for MP measurements, mimicking the proposed events in the gland.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e When 10 \u0026micro;M of NT2RepCT\u003csup\u003eYF\u003c/sup\u003e in 500 mM KP\u003csub\u003ei\u003c/sub\u003e, pH 8 (buffer A, see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) was diluted in 20 mM NaP\u003csub\u003ei\u003c/sub\u003e, pH 8 (buffer B), the MP histogram showed no pronounced difference compared the data obtained in 20 mM NaP\u003csub\u003ei\u003c/sub\u003e only \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, \u003cb\u003elight blue histogram)\u003c/b\u003e. When the pH was rapidly lowered to 5, we observed no oligomerization, but instead a pronounced reduction in signal intensity \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, \u003cb\u003edark blue histogram)\u003c/b\u003e. These data contrast the observation made for non-LLPS conditions and allow us to delineate a role for LLPS during spidroin assembly: In 20 mM NaP\u003csub\u003ei,\u003c/sub\u003e pH 8, most spidroins are in the dilute phase. Lowering the pH to 5 causes polymerization \u003cem\u003evia\u003c/em\u003e the NT domains, and we can observe a wide range of oligomeric states immediately afterwards. In 500 mM KP\u003csub\u003ei\u003c/sub\u003e, the proteins form liquid droplets that are held together by weak interactions between the repeat domains. When the pH is lowered to 5, these pre-assembled proteins immediately polymerize into oligomers that are too large for the detection window of the mass photometer. Assuming that NT dimerization is faster than droplet dissolution in the mixing capillary, low pH crosslinks the spidroins while they are closer in space, which we speculate gives rise to larger assemblies in a shorter time frame.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eReproducing spinning conditions and monitoring their effects on spidroins at the molecular level is a major challenge in the experimental investigation of spider silk. Here, we have employed MP to investigate the effects of sodium and potassium phosphate, which are present in the silk gland, on the oligomeric state of the NT2RepCT\u003csup\u003eYF\u003c/sup\u003e mini-spidroin. MP is a single-molecule technique that can trace individual protein interactions, but requires nanomolar protein concentrations that do not represent native or biomimetic spinning conditions\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, using microfluidics to rapidly switch solution conditions and protein concentrations, we were able to directly observe how ions and pH synergistically affect spidroin assembly.\u003c/p\u003e\u003cp\u003eThe data from MP indicate different effects of sodium and potassium on NT2RepCT\u003csup\u003eYF\u003c/sup\u003e that are consistent with their positions in the Hofmeister series. Potassium ions, which are slightly kosmotropic, reduce self-association of the repeat domains and stabilize NT2RepCT\u003csup\u003eYF\u003c/sup\u003e dimers. The slightly chaotropic sodium ions destabilize the protein and enable intermolecular interactions between the repeat domains. Both cations are close to the middle of the Hofmeister range, yet the fact that we observe effects at low ion concentrations suggests that the partially structured repeat domain may be sensitive to such subtle differences or engage in ion-specific interactions. Native spidroins contain hundreds of repeats, which may render them more susceptible to minor changes in ion composition. Here, the potassium-induced compaction of individual spidroins that we observe at nanomolar protein concentrations likely stems from increased intramolecular interactions in the repeat domain. At higher protein concentrations, potassium shifts from promoting intramolecular to intermolecular contacts. In line with this interpretation, Numata and colleagues have reported that kosmotropic ions induce oligomerization of the repeat domain at millimolar peptide concentrations\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePotassium is likely to have a pronounced effect on phase transitions in native spidroins, considering their high number of repeats and extreme concentration in the gland. Recombinant mini-spidroins require high phosphate concentrations to undergo LLPS\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Importantly, spidroin droplets formed at high salt are metastable and undergo spontaneous aggregation, which would be detrimental in native silk dope\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. We speculate that much lower amounts of kosmotropic ions would be required to promote LLPS-like interactions in the silk gland where there is a higher concentration of repeat domains. MassFluidix MP, where interference from high amounts of KP\u003csub\u003ei\u003c/sub\u003e is mitigated by rapid dilution, provides an indication how LLPS could contribute to silk assembly. We find that pre-assembly of NT2RepCT\u003csup\u003eYF\u003c/sup\u003e into macroscopic droplets reduces oligomeric intermediates when polymerization is triggered by lowering the pH. Considering the sensitivity to potassium that we observe with mini-spidroins in dilute solutions, we speculate that even a small shift in the potassium content of the gland may move the spidroins in their phase space to facilitate more efficient spinning.\u003c/p\u003e\u003cp\u003eLinder and co-workers have recently observed by analytical ultracentrifugation that NT2RepCT forms clusters that transition to aggregates as a function of protein concentration, temperature, or pH\u003csup\u003e33\u003c/sup\u003e. Their findings show that the protein occupies a two-phase regime even below the threshold for LLPS. The MP data now reveal that the tendency to oligomerize extends to the low nanomolar range, further blurring the distinction between single proteins, clusters, and droplets. Rather than presenting a phase diagram with a defined solubility line, spidroins are inherently bound to self-assemble via their repeat regions, which can be fine-tuned by ions. This behaviour is markedly different from other phase-separating proteins. For example, MP has shown that LLPS of α-Synuclein does not proceed via small oligomers but is driven by the formation of megadalton clusters at micromolar protein concentrations\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We speculate that the ability to assemble spidroins with minimal intermediates helps spiders to achieve their high spinning speeds.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eProtein sequences\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eProtein expression and purification\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll proteins were expressed and purified as previously described, however sonication (6 min, 2 s on, 8 s off, 30%) was used for cell disruption\u003csup\u003e5,34\u003c/sup\u003e. NT2repCT\u003csup\u003eYF\u003c/sup\u003e was dialyzed into deionized water while NT2rep and NT were kept in 20 mM Tris, pH 8. After concentrating (NT to 10 mM, NT2rep and NT2repCT\u003csup\u003eYF\u003c/sup\u003e to 200 \u0026ndash; 300 \u0026mu;M) using Amicon spin filters (Millipore), all proteins were stored at -20\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMicroscopy\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBright-field microscopy images of 10 \u0026mu;M NT2repCT\u003csup\u003eYF\u003c/sup\u003e droplets in 20 mM or 500 mM KP\u003csub\u003ei\u003c/sub\u003e, pH 8, were acquired using a Nikon Eclipse Ti series inverted microscope (Nikon) equipped with a Crest X-light V2 series confocal unit (Nikon) using a Plan Apo 40x objective (Nikon) and a Zyla sCMOS camera (Andor).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThioflavin T Assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAggregation kinetics were monitored by measuring the ThT fluorescence (excitation: 445-8 nm, emission: 488-15 nm) using a CLARIOstar microplate reader (BMG labtech) with the gain set to 2000. In black half-area 96-well polystyrene microplates with a transparent bottom (Corning), 40 uL 10 \u0026mu;M ThT and 25 \u0026mu;M NT2RepCT\u003csup\u003eYF\u003c/sup\u003e were mixed in the different buffers (20 mM NaP\u003csub\u003ei\u003c/sub\u003e pH 8, 20 mM NaP\u003csub\u003ei\u003c/sub\u003e pH 5, 20 mM KP\u003csub\u003ei\u003c/sub\u003e pH 8, 20 mM KP\u003csub\u003ei\u003c/sub\u003e pH 5, 500 mM NaP\u003csub\u003ei\u003c/sub\u003e pH 8, 500 mM NaP\u003csub\u003ei\u003c/sub\u003e pH 5, 500 mM KP\u003csub\u003ei\u003c/sub\u003e pH 8, 500 mM KP\u003csub\u003ei\u003c/sub\u003e pH 5) and the plate sealed with transparent cover film to avoid evaporation. All measurements were conducted at 30\u0026deg;C without agitation.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMass photometry\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll mass photometry experiments were carried out at room temperature using the Refeyn Two\u003csup\u003eMP\u0026nbsp;\u003c/sup\u003einstrument (Refeyn) in the normal measurement mode with regular image size. Proteins were pre-diluted first to 10 \u0026mu;M in 20 mM NaP\u003csub\u003ei\u003c/sub\u003e pH8 and from that further diluted (400 nM) into the desired buffer. A final concentration of 20 nM (NT2RepCT\u003csup\u003eYF\u003c/sup\u003e, NT-2rep) or 100 nM (NT) was then used on the mass photometry slide. Each measurement was recorded for 1 min (3000 frames) using the AcquireMP (2024 R1.1) software. Histograms were created in DiscoverMP (v2024 R1) with \u0026beta;-amylase (56, 112 and 224\u0026thinsp;kDa) or bovine serum albumin (66 and 132 kDa) in 20 mM NaP\u003csub\u003ei\u003c/sub\u003e pH 8 as mass calibrants. Data were analyzed using DiscoverMP software (Refeyn). Unbinding peak intensities were significantly lower than the binding peak intensities in all instances\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMass Fluidix\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMass Fluidix-MP measurements were carried out using 1st generation microfluidic chips (Refeyn). 10 \u0026mu;M NT2RepCT\u003csup\u003eYF\u003c/sup\u003e in 20 mM NaPi, pH 8, or in 500 mM KPi, pH 8, were pumped \u003cem\u003evia\u003c/em\u003e the sample inlet. 20 mM NaPi, pH 8 or 20 mM NaPi, pH 5, were supplied \u003cem\u003evia\u003c/em\u003e the buffer inlet. Inlet tubes were washed with MQ and primed with buffer before and between runs. Nitrogen pressure was used to control the sample and buffer flow. Pressure and related flow rates were adjusted manually using the OxyGEN software. Flow rates were adjusted to 1 \u0026mu;L/min and 999 \u0026mu;L/min for sample and buffer, respectively, resulting in 1500 - 3000 binding events/min. Histograms were created in DiscoverMP (v2024 R1) and \u0026beta;-amylase (56, 112 and 224\u0026thinsp;kDa) or bovine serum albumin (66 and 132 kDa) was used as mass calibrant.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNative ion mobility mass spectrometry\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNT2RepCT\u003csup\u003eYF\u0026nbsp;\u003c/sup\u003ewas buffer-exchanged into 100 mM ammonium acetate, or into 100 mM ammonium acetate supplemented with 10 mM sodium acetate (NaAc) or 10 mM potassium acetate (KAc), pH 8 using BioSpin6 columns (BioRad, CA, USA). Samples were loaded into nESI capillaries (Thermo Fisher Scientific Inc., MA, USA). Mass spectra were recorded in positive ionization mode on a Waters Synapt G1 traveling-wave IM mass spectrometer (MS Vision, The Netherlands). The capillary voltage was maintained at 1,5 kV and the sample cone was 100 V. The source temperature was +30\u0026deg;C. The trap and transfer collision energies were 50 V. The trap gas was argon at a flow rate of 1.5 mL/min, the IMS gas was nitrogen at a flow rate of 18.5 mL/min. The ion mobility-mass spectrometry settings were: IMS wave height 10 V and IMS wave velocity 350 m/s. Data were analyzed using MassLynx 4.1 (Waters, UK).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eML is supported by a KI faculty-funded Career Position, a Cancerfonden Project grant (22-2023 Pj), a VR Project Grant (2024-04483), a KAW Project grant (2022.0032), and a Consolidator Grant from the Swedish Society for Medical Research (SSMF). AR is supported by the Swedish Research Council (2024-02919), FORMAS (2023-01313), Knut and Alice Wallenberg Foundation (grant 2023.0331) and Olle Engkvists Stiftelse (233-0334).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available from the corresponding authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHu, X.; Vasanthavada, K.; Kohler, K.; McNary, S.; Moore, A. M. F.; Vierra, C. A. Molecular Mechanisms of Spider Silk. \u003cem\u003eCell. Mol. Life Sci. \u003c/em\u003e\u003cstrong\u003e2006\u003c/strong\u003e, \u003cem\u003e63\u003c/em\u003e (17), 1986\u0026ndash;1999. https://doi.org/10.1007/s00018-006-6090-y.\u003c/li\u003e\n\u003cli\u003eJin, H.-J.; Kaplan, D. L. 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Mater. \u003c/em\u003e\u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e32\u003c/em\u003e (23), 2200986. https://doi.org/10.1002/adfm.202200986.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Liquid-liquid phase separation, single-molecule analysis, microfluidics, silk fiber formation, Hofmeister series","lastPublishedDoi":"10.21203/rs.3.rs-7639041/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7639041/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpider silk formation involves tightly regulated protein assembly influenced by pH and the presence of ions. Kosmotropic salts induce phase separation of spidroins, however, their exact role in assembly is not clear. Here, we investigate how sodium and potassium phosphate affect spidroin interactions via the single-molecule method mass photometry. We observed that spidroin oligomerization occurs at low nanomolar protein concentrations. Potassium ions were found to stabilize a compact conformation of individual spidroins and slow down pH-induced β-sheet aggregation, consistent with its more kosmotropic nature. Microfluidic MP showed that pre-assembly of the protein through salt-induced phase separation reduced the number and size of oligomeric intermediates that form upon acidification. Together, the findings suggest that spidroins have an inherent ability to self-assemble, blurring the line between one- and two-phase status. Subtle differences in ion composition are sufficient to change spidroin stability and assembly, potentially contributing to silk spinning\u003cem\u003e in vivo\u003c/em\u003e by balancing storage stability with rapid fiber formation.\u003c/p\u003e","manuscriptTitle":"Single-molecule mass measurements reveal distinct effects of sodium and potassium on mini-spidroin assembly","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-10 08:19:58","doi":"10.21203/rs.3.rs-7639041/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-materials","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsmat","sideBox":"Learn more about [Communications Materials](https://www.nature.com/commsmat/)","snPcode":"","submissionUrl":"","title":"Communications Materials","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"38eaec8c-f1bb-4b9f-b714-645a6820c9c4","owner":[],"postedDate":"October 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55479875,"name":"Biological sciences/Biophysics/Single-molecule biophysics"},{"id":55479876,"name":"Biological sciences/Biotechnology/Biomaterials/Biomaterials \u0026#x2013; proteins"}],"tags":[],"updatedAt":"2026-02-04T08:21:58+00:00","versionOfRecord":{"articleIdentity":"rs-7639041","link":"https://doi.org/10.1038/s43246-025-01051-3","journal":{"identity":"communications-materials","isVorOnly":false,"title":"Communications Materials"},"publishedOn":"2026-01-02 05:00:00","publishedOnDateReadable":"January 2nd, 2026"},"versionCreatedAt":"2025-10-10 08:19:58","video":"","vorDoi":"10.1038/s43246-025-01051-3","vorDoiUrl":"https://doi.org/10.1038/s43246-025-01051-3","workflowStages":[]},"version":"v1","identity":"rs-7639041","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7639041","identity":"rs-7639041","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}