The bacteriocin enterocin EJ97s retains its antibacterial effect upon interaction with human serum albumin

The bacteriocin enterocin EJ97s retains its antibacterial effect upon interaction with human serum albumin | 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 The bacteriocin enterocin EJ97s retains its antibacterial effect upon interaction with human serum albumin Ingvild Reinseth, Krister Gjestvang Grønlien, Thomas Førland Oftedal, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8041595/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 Enterocin EJ97s is a bacteriocin with potent activity against vancomycin-resistant Enterococcus faecalis . Since EntEJ97 is active in the presence of blood and has been shown to circulate at therapeutic concentrations without causing damage in a murine model, it is considered a promising candidate for treatment of E. faecalis infections. The aim of this study was to investigate the interaction of EntEJ97s with human serum albumin (HSA) as a step in the pre-clinical assessment of this bacteriocin. The binding association between EntEJ97s and HSA was investigated with fluorescence, nano differential scanning calorimetry (nano DSC) and nano isothermal titration calorimetry (nano ITC). The results show low to moderate binding affinity between EntEJ97s and HSA, with a stoichiometry of less than one EntEJ97s per HSA molecule. Nano ITC competition experiments with competitive ligands indicated that the peptide binds to both the Sudlow I site and the Sudlow II site, the two main drug binding sites on HSA. Molecular modelling efforts corroborated the findings of a binding in the vicinity of these sites. The antibacterial effect of the peptide was preserved in the presence of physiological and experimental concentrations of HSA, as indicated by minimum inhibitory concentration experiments, and spot-on-lawn assays. These findings accentuate that the properties EntEJ97s has in association with HSA are favourable for intravenous treatment. Applied & Industrial Microbiology bacteriocins antimicrobial peptides albumin binding nano differential scanning calorimetry molecular modelling nano isothermal titration calorimetry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Human serum albumin (HSA) is the most abundant plasma protein, comprising ~ 60% of the total plasma protein content. Consequently it has a significant impact on the constituents of the blood, where the physiological concentration of HSA is 0.6 mM ( 1 ). The main functions of HSA are maintenance of osmotic blood pressure, preservation of blood pH and binding and transport of various molecules ( 2 – 5 ). The HSA protein (66.5 kDa), composed of 585 amino acids, is a globular protein consisting of three domains (I, II and III) connected by flexible hinges and stabilized by disulfide bridges ( 2 , 5 , 6 ). Each of the three domains contains two subunits, labelled A and B. The two main drug binding sites are located in subdomains IIA and IIIA ( 7 , 8 ). These two sites are named Sudlow site I and II, respectively, and have different preferences for binding ligands ( 9 ). Sudlow site I selectively binds heterocyclic compounds such as warfarin or 4-hydroxycoumarine ( 8 , 10 ), while Sudlow site II prefers aromatic compounds such as ibuprofen ( 8 , 11 , 12 ). In spite of these preferences some compounds bind to several binding pockets in HSA. This is exemplified by the non-steroidal anti-inflammatory compounds naproxen and diflunisal which bind to two and three pockets on HSA, respectively ( 13 ). HSA plays a large role in transport of endogenous and exogenous compounds, carrying fatty acids, hormones, bilirubin, and other small molecules, as well as peptides ( 14 – 17 ). The interaction of various compounds with HSA is often reversible and can contribute to extending the half-life of the compounds in blood. In fact, recent research has explored the use of fusion to an albumin-binding domain as a strategy to associate therapeutic compounds with HSA ( 18 – 20 ). Conversely, interaction with HSA can also decrease the effectiveness of a drug; an example of this is the antibiotic oritavancin. The presence of HSA increases the minimum inhibitory concentration (MIC) of oritavancin against different strains of enterococci two- to eightfold ( 21 ). Increased MIC values due to HSA were also found for teicoplanin and daptomycin, while the MIC value of vancomycin and linezolid was unaltered ( 21 , 22 ). It has also been shown that binding to HSA is beneficial for strongly lipophilic compounds as it increases the solubility in plasma ( 9 , 23 ). However, if the binding affinity is too high, the interaction may be a disadvantage due to the necessity of an augmented dose ( 9 , 17 ). In addition, it must be considered whether the interaction disturbs the activity of the compound in question. Bacteriocins are bacterially produced antimicrobial peptides. While their ecological roles is linked to competition for niche and nutrients, these peptides hold promise for use in treatment of various infections ( 24 , 25 ). One of the major concerns of developing bacteriocins into viable alternatives to antibiotics is their delivery method, as oral delivery would leave them susceptible to proteolytic degradation ( 26 ). Several strategies for delivery have been suggested, such as nanoparticles, gels, as well as systemic delivery through intravenous infusion ( 27 , 28 ). Intravenous infusion bypasses many delivery hurdles, but the administered compound will interact with the blood constituents, such as HSA ( 29 – 31 ). Previously it has been shown that the activity of an antimicrobial peptide was reduced in the presence of bovine serum albumin (BSA) ( 32 ). Another study corroborated these results, indicating a 10-fold increase in MIC in the presence of BSA ( 33 ). Thus, any binding interaction with HSA can have significance in determining whether a bacteriocin can have a role in treatment of systemic infections, such as those caused by vancomycin resistant enterococci (VRE) ( 34 ). Antibiotic resistant bacteria are a major concern due to the limited development of new antibiotics and the absence of viable alternatives to antibiotics ( 35 ). VRE are of particular concern due to the remarkable resilience of these bacteria, being able to survive on surfaces for months, and withstand lysozyme, heat, alcohol, and salt ( 36 – 41 ). In addition, these bacteria are extremely proficient at exchanging genetic material often due to absence of CRISPR-Cas genes, making spread of resistance genes rapid and efficient ( 42 ). Bacteriocins represent an alternative treatment strategy against infections caused by antibiotic resistant pathogens, such as VRE ( 43 ). In terms of infection treatment, bacteriocins possess several advantageous properties. They often have narrow spectrum of activity, thereby specifically killing the pathogen while preserving the commensal microbiota ( 44 , 45 ).Furthermore, since bacteriocins mostly have different cellular targets and mechanisms of action compared to antibiotics, the risk of cross-resistance is low ( 46 , 47 ). Enterocin EJ97s (EntEJ97s), is a leaderless class II bacteriocin belonging to the so-called LsbB family of bacteriocins. EntEJ97s has potent activity against Enterococcus faecalis and lower, but still notable, activity against Enterococcus faecium ( 48 , 49 ). In aqueous solution, EntEJ97s exhibits a primarily unfolded confirmation, characterized by an absence of well-defined secondary structures. However, within this unfolded state, intermittent helical or turn-like structures emerge, suggesting dynamic fluctuations in the peptide conformation ( 50 ). The exact mechanism of action of EntEJ97s is not fully characterized, but it is known that it targets the transmembrane protease RseP as a receptor in the cell membrane of susceptible cells ( 51 ). RseP functions in regulated intramembrane proteolysis, where it performs the second cleavage of an anti-σ factor, resulting in the release of a σ factor that activates expression of genes involved in bacterial stress response ( 52 , 53 ). This study aimed to explore the interaction between EntEJ97s and HSA, and to determine if such interaction affected the antibacterial effect of the bacteriocin. The interactions were studied using a multifaceted approach. Fluorescence spectroscopy, nano differential scanning calorimetry (nano DSC) and nano isothermal titration calorimetry (nano ITC) were used to gain insights into the binding kinetics of EntEJ97s and HSA. To further support the experimental results, molecular modelling was performed. The antibacterial effect of the EntEJ97s in the presence of experimental and physiological concentrations of HSA was also investigated. Combining these approaches provided a comprehensive overview of the binding properties of HSA and EntEJ97s, which is essential for further development of EntEJ97s and similar bacteriocins, in treatment of systemic enterococcal infections. Methods Ligands and reagents The amino acid sequence of the peptide EntEJ97s is MIKKFPNPYTLAAKLTTYEINWYKQQYGRYPWERPVA ( 49 ) giving it a weight of ~ 4566 Da. All synthetic peptides were purchased from Pepmic co., Ltd., China with ≥ 99% purity. Lyophilized HSA (≥ 99% globulin, fatty acid free) was purchased from Sigma (A3782, Darmstadt, Germany). The reference ligands 4-hydroxycoumarine, dansylglycine, and ibuprofen were purchased from Sigma. Steady state fluorescence Steady state fluorescence measurements were performed on a Photon Technology International modular fluorescence system (London, Ontario, Canada) with Model 101 monochromator with f/4 0.2-m Czerny-Turner configuration. The instrument was equipped with a red-sensitive photomultiplier. The excitation source was a 75 W xenon lamp. The emission and excitation spectra were automatically corrected for, both the lamp spectral radiance and the detector quantum efficiency, by means of the acquisition software (FeliX32, PTI). The excitation and emission monochromator band passes were set at 2 nm. The excitation wavelength was 295 nm, and the emission was recorded in the range 300–500 nm. The measurements were performed in quartz cuvettes with 1 x 1 cm cell path at 25 ± 0.1°C (n = 3). The blanks consisted of EntEJ97s in Milli-Q water at similar concentration as the individual samples and were subtracted from the sample emission spectrum. Fluorescence lifetimes were recorded on an Easy Life V system (Optical Building Blocks Corp., Birmingham, New Jersey, US) equipped with a nanosecond pulsed LED excitation source at 297 nm, slit width 1.5 nm. Post-acquisition data handling was performed by means of the Easy Life V software. The lifetime was calculated from an average of 10 scans and the experiment was repeated on three different samples. A stock solution of 2 x 10 − 5 M HSA and a series of HSA / EntEJ97s assay solutions were prepared for the HSA fluorescence quenching experiments. The concentration of HSA was kept constant at 2 x 10 − 5 M while the concentration of EntEJ97s varied in the range of 1.5 x 10 − 5 – 6.0 x 10 − 5 M. The samples were incubated for 30 min before measurement. The overlapping of the excitation wavelength with the EntEJ97s absorption spectrum required correction for inner-filter effect according to Eq. 1 (54): F cor = F obs x 10 (Aex − Aem/ 2) ( 1 ) where F cor is the corrected fluorescence value, F obs is the measured fluorescence value, A ex and A em the measured absorbance value at the excitation and emission wavelengths, respectively. Absorption spectra were recorded between 190 and 700 nm on a Shimadzu UV-2101 PC UV-Vis scanning spectrophotometer using a quartz cuvette with 1 cm cell path. The fluorescence quenching process was evaluated by application of the Stern-Volmer equation (Eq. 2, (54)): F 0 / F cor = 1 + k q τ 0 [Q] = 1 + K sv [Q] ( 2 ) where F 0 and F cor are the corrected HSA fluorescence intensities in the absence and presence of EntEJ97s, respectively, [Q] is the known EntEJ97s concentration, k q is the bimolecular quenching constant, τ 0 is the fluorescence lifetime of the unquenched HSA fluorophore, and K sv is the Stern-Volmer quenching constant. The data were presented as a plot of F 0 / F cor versus [Q]. The number of binding sites (n) per HSA molecule and the observed binding constant (K a ) can be calculated from the fluorescence data using Eq. 3 (55): lg (F 0 – F cor / F cor ) = lg K a + n lg[Q] (Eq. 3) where F 0 and F cor are the fluorescence values applied in the Stern-Volmer equation (i.e., the corrected HSA fluorescence intensities in the absence and presence of EntEJ97s, respectively), [Q] is the molar concentration of free EntEJ97s and K a is the observed binding constant. A plot of lg (F 0 – F cor / F cor ) versus lg [Q] resulted in a straight line (R 2 ≥ 0.94). Nano differential scanning calorimetry (nano DSC) The experiments were performed in triplicate using a nano DSC instrument (TA® instruments, New Castle, DE, USA). A range of EntEJ97s concentrations were used (0–1 mM), while the HSA concentration was kept constant at 30 µM. The samples were prepared in Milli-Q water. The samples were incubated for 30 minutes at 37°C and all working solutions were degassed with a vacuum degasser (TA® instruments). The measured temperature interval was between 20°C and 90°C, with a scanning rate of 1°C min − 1 , at a pressure of 3 atm. Baseline scans of Milli-Q water were recorded and subtracted from the sample scans. Data was analysed using NanoAnalyze software from TA® Instruments, v3.12.5 (Waters LLC, New Castle, DE, US), fitting the data to the two-state scaled model. To compensate for baseline variations between the initial and final states, the calorimetric data was corrected with a sigmoidal baseline in the NanoAnalyze software. The temperature (T) and the enthalpy change (ΔH) of each transition were evaluated. Nano isothermal titration calorimetry (nano ITC) The experiments were performed in triplicate on a nano Isothermal Titration Calorimeter (TA® instruments, New Castle, DE, USA), with a cell volume of 200 µL. All samples were prepared in Milli-Q water. The final concentration of HSA was 0.8 mM. The reference cell was filled with 300 µL of Milli-Q water and changed every second day or after two titrations. All working solutions were degassed with a vacuum degasser (TA® instruments). The peptide was titrated into Milli-Q water in duplicate as a control of the signal caused by the dilution. The concentration of the peptide EntEJ97s was always 2.0 mM. The competition ligands were incubated with HSA for 30 minutes at 25°C in a 1:1 molar ratio. The active cell volume was 190 µL and the syringe volume was 50 µL. 50 consecutive 1 µL injections were carried out with 500 s spacing between injections. The stirring rate was set at 200 rpm and the temperature was kept at 25°C. A 300-second baseline was established and assessed before commencement of the experiment. The nano ITC data was analysed using the NanoAnalyze software from TA® Instruments, v3.12.5 (Waters LLC, New Castle, DE, US). To generate the binding isotherm the independent model from the program was used, with adjustments to accommodate a larger ΔH in order to fit the data. The first injection of all experiments was omitted in the final analysis. Modelling of protein-protein interactions Rigid-body protein-protein docking studies were performed using FTDock and scored using pyDock ( 56 , 57 ). A three-dimensional structure of EntEJ97s was obtained using AlphaFold2 v2.3.2 while the structure for human serum albumin was obtained from the RCSB protein data bank (PDB 1AO6). A total of 10,000 docking poses (complexes) were predicted and scored by pyDock, the 100 best-scoring complexes were kept for further analysis. To obtain representative clusters from the ensemble of best-scoring complexes, a cluster analysis was performed using Biomol2Clust ( 58 ). Molecular interactions between EntEJ97s and HSA were predicted using The Residue Interaction Network Generator v4 (RING) and the web-based 3D structure viewer and structure analysis tool iCn3D ( 59 , 60 ). Antibacterial activity HSA (30 µM or 600µM) was weighed out in Milli-Q water together with EntEJ97s (0.025, 0.1 or 0.3 mM) and incubated at 37°C for 30 minutes with shaking at 100 rpm. The minimum inhibitory concentration (MIC) of the antimicrobials with HSA was determined as the minimum concentration that inhibited growth by at least 50% compared to control by OD 600 (without added antimicrobial, but with HSA) in 200 µL culture. The antimicrobial assay was performed with a microtiter assay as previously described ( 61 ). A standard spot-on-lawn assay was set up with indicator strains ( E. faecalis Symbioflor and E. faecalis MMH 594), and 10 µL of the EntEJ97s / HSA mixture. The antibacterial activity experiments were repeated two times. Results Steady-state fluorescence shows that EntEJ97s bind to HSA To get initial insights into the potential interaction between EntEJ97s and HSA, analysis of changes in the intrinsic HSA fluorescence in the presence of EntEJ97s was analyzed. The fluorescence quenching was evaluated by the Stern-Volmer equation. There was a linear dependency of F / F cor upon the concentration of EntEJ97 in the concentration range examined (R 2 ≥ 0.90), suggesting a direct interaction between HSA and EntEJ97s. A representative Stern-Volmer plot is shown in Fig. 1 . To evaluate the stability of the interaction, the bimolecular quenching constant k q and the Stern-Volmer quenching constant K sv were determined. The values for k q and K sv of 1.35 ± 0.50 x 10 13 M − 1 s − 1 and 6.53 ± 2.69 x 10 3 M − 1 , respectively (n = 3) (Table 1 ), suggest formation of a static complex between HSA and EntEJ97s. The lifetime of the HSA fluorophore in the absence of EntEJ97s was found to be 5.62 ± 0.47 ns (n = 3). Table 1 Parameters for EntEJ97s and HSA association extracted from fluorescence and isothermal titration calorimetry (ITC) experiments. Parameter Fluorescence ITC K sv (M − 1 ) 6.53 ± 2.69 x 10 3 k q (M − 1 s − 1 ) 1.35 ± 0.50 x 10 13 n 0.78 0.44 K a (M − 1 ) 7.10 ± 0.49 x 10 2 7.56 x 10 3 -TΔS (kcal/mol) 51.14 ΔH° (kcal/mol) -56.43 ΔG° (kcal/mol) -5.29 ΔS° (kcal/mol) -0.17 The number of binding sites and the binding constant (K a ) were calculated to be 0.8 and 7.10 ± 0.49 x 10 2 (Table 1 ), respectively. The fluorescence emission maximum of HSA remained constant at ≈ 338 nm in all samples. In the UV-absorption spectrum (Fig. 2 ), the peak at 278 nm also remained unchanged while there was a major change in the absorption spectrum in the range 190–235 nm. The absorption in this range was nearly eliminated in the presence of EJ97 but a new maximum occurred at 235 nm (Fig. 2 ). Nano differential scanning calorimetry indicates that EntEJ97s interacts with several domains of HSA As mentioned above, the structure of HSA is subdivided into three domains: domain I (residues 1-195); II (196–383); and III (384–585), where each domain is formed by two subdomains (A and B). Each domain of HSA denatures at specific temperatures. Nano DSC, in which changes in the heat flow associated with the thermal denaturation of HSA is monitored, was used to determine how EntEJ97s interacted with three domains of HSA and whether interactions alter the thermal stability of the protein. We hypothesized that the data obtained from the nano DSC measurements of HSA can be resolved into three transitions, one for each of the three domains of this protein. In addition, predenaturation transitions were observed at lower measured temperatures. Table 2 presents the obtained melting temperatures (T) and enthalpy changes (ΔH) for HSA in the presence of the peptide EntEJ97s at different concentrations. Major changes in T and ΔH (transitions) of HSA were observed at low concentrations of EntEJ97s (≤ 0.025 mM) compared to situation where not HSA is present. The largest changes occurred in domain I and II. At increasing concentrations of HSA, no transitions were observed for domain III ≥ 0.05 mM and by domain II at concentrations ≥ 0.6 mM. Domain I retained transitions except for the highest concentration of the peptide (1.0 mM). The lack of transitions suggests that the domain is completely stabilized or that aggregation or precipitation occurs. Two predenaturation peaks appear in the concentration range 0.025–0.7 mM of EntEJ97s. Table 2 Results of nano-DSC measurements for human serum albumin at a constant concentration of 30 µM with varying concentrations of EntEJ97s. The data includes the melting temperature (T) and the enthalpy change for each condition (ΔH), n = 3. Sample T (°C) / ΔH (kJ/mol) HSA (µM) EntEJ97s (mM) Predenat-uration Predenaturation Domain I Domain II Domain III 30 0 48.88 / 557.3 - 62.62 / 669.77 66.51 / 457.37 72.40 / 612.03 30 0.01 49.20 / 639.93 - 65.79 / 576.80 68.57 / 735.23 72.05 / 693.44 30 0.025 48.64 / 951.63 51.40 / 912.83 65.14 / 819.23 68.37 / 717.90 72.42 / 644.33 30 0.05 49.86 / 788.83 53.03 / 1091.67 63.00 / 1124.33 65.96 / 803.50 - 30 0.1 51.60 / 691.40 54.62 / 1087.07 64.21 / 1001.93 66.86 / 703.00 - 30 0.3 51.43 / 870.50 54.55 / 854.17 66.76 / 687.43 70.95 / 672.17 - 30 0.5 50.41 / 693.43 53.79 / 997.33 66.01 / 641.97 70.41 / 630.47 - 30 0.6 47.24 / 830.53 50.45 / 926.57 64.42 / 454.23 - - 30 0.7 48.90 / 601.90 - 62.25 / 469.20 - - 30 1.0 - - - - - Nano isothermal titration calorimetry indicates that EntEJ97s interferes with both Sudlow site I and II Within HSA, two primary drug-binding sites are generally recognized; Sudlow site I located in subdomain IIA (also known as the warfarin-azapropazone site) and Sudlow site II site located in subdomain IIIA (also known as the indole-benzodiazepine site). Nano ITC measurements were performed to further investigate the thermodynamic properties of the interaction between EntEJ97s and HSA. First, EntEJ97s was titrated into 0.8 mM HSA in a non-competitive nano ITC experiment. Table 1 presents the thermodynamic data from this experiment, including stoichiometry (n = 0.44), and the association constant (K a ) of 7.56 x 10 3 M − 1 . Figure 3 shows the nano ITC binding data of EntEJ97s titrated into HSA. Together, this further supports the interaction between HSA and EntEJ97s and suggests that there is less than one EntEJ97s molecule bound per HSA. Next, competitive nano ITC experiments were performed using ligands with specific affinity to Sudlow site I or II in HSA; 4 – hydroxycoumarine (hcm, Sudlow site I ligand), dansylglycine (dgly, Sudlow site II ligand) and ibuprofen (ibu, Sudlow site II ligand, weak Sudlow site I ligand) (Fig. 4 ) to decipher whether the interaction interferes with any of the drug binding sites. These competitive experiments indicated that EntEJ97s competed with both drug-binding site I and II reference ligands. This was demonstrated by a decrease in the heat signals when the peptide was titrated into HSA incubated with hcm, dgly or ibu at a 1:1 molar ratio. Molecular docking and interaction analysis To corroborate the results obtained from the fluorescence quenching and competitive binding assays, molecular docking was performed between EntEJ97s and HSA using FTDock to identify probable binding sites. The molecular docking analysis revealed three distinct clusters representing potential binding sites of EntEJ97s to HSA. The best predicted binding site of EntEJ97s was to regions of subdomains IB (residue 108–196) and IIA (residue 197–297), with the C-terminus located at the Sudlow site I entrance towards the drug-binding site (Fig. 5 ). In this complex, EntEJ97s was predicted to interact with HSA via hydrogen-bonding, π-stacking, and π-cation pairs (Table 3 ). The two other potential binding sites (complex 2 and 3, Table 3 ) involved only hydrogen-bonding and π-cation interactions. Among these, the second ranked complex involved interactions with residues in domain III, where the Sudlow site II is located. Table 3 Docking results. Molecular interactions (bonds) between HSA and EntEJ97s in the three predicted complexes. Residue interactions are from HSA (first residue) to EntEJ97s. Complex/rank H-bond pairs π-stacking pairs π-cation pairs 1 Arg 218 -Ala 37 Glu 292 -Arg 29 Phe 156 -Tyr 27 His 288 -Tyr 27 Arg 160 -Tyr 27 Lys 181 -Trp 22 2 Glu 376 -Tyr 9 Arg 410 -Tyr 18 Lys 351 -Phe 5 Arg 410 -Tyr 18 Lys 414 -Tyr 18 3 Ser 273 -Trp 22 Ser 273 -Tyr 18 Lys 225 -Tyr 18 Antimicrobial activity of EntEJ97s is retained in the presence of HSA A previous work has shown that the association of a short cationic antimicrobial peptide with serum albumin reduced the ability of the peptide to kill bacteria 10-fold ( 33 ). With the intention of investigating whether the same is true for the association between EntEJ97s and HSA, a standard MIC assay (Table 4 ) and a spot-on-lawn assay (Fig. 6 ), were set up. The MIC concentrations were the same when HSA was present or absent, with one exception (30 µM HSA and 0.3 mM EntEJ97s, Table 4 ). To corroborate these findings, the spot-on-lawn assay was performed and demonstrated equivalent findings (Fig. 6 ); the inhibition zones of the peptide are comparable independent of the presence of HSA. As expected, the inhibition zones are larger for the higher concentrations of bacteriocin, but there is no inhibitory effect of HSA alone (Fig. 6 ). Table 4 Estimated MIC 50 of EntEJ97s (in µg/ml) against E. faecalis MMH594 with and without experimental (30µM) and physiological (600µM) concentrations of HSA. EntEJs (mM) Without HSA HSA (30 µM) HSA (600 µM) 0.025 0.34–0.69 0.69 0.34 0.1 0.36 0.36 0.36 0.3 0.13–0.27 0.27 0.27 Discussion Many bacteriocins have been proposed as alternatives for treatment of infection, but very few have reached clinical trials. The difficulty of oral delivery caused by the proteolysis of the peptide in the digestive system, could contribute to this lack of progress. A way around this predicament is to deliver the peptide intravenously, but this implicates interaction with plasma proteins such as HSA. The current work presents the results from binding analysis between bacteriocin EntEJ97s and HSA using steady-state fluorescence, nano DSC and nano ITC. In addition, the results are compared to predictions from molecular modelling. Together, the results indicate that EntEJ97s interacts with HSA, albeit with low affinity. Importantly, however, the interactions between HSA and EntEJ97s, do not seem to negatively impact the antimicrobial effect of the peptide. Initially, steady state fluorescence was used to demonstrate that EntEJ97s interacts with HSA. The intrinsic fluorescence of HSA can originate from three amino acids: Trp, Tyr and Phe where Trp emission is dominant. HSA contains only one Trp unit, Trp-214, which is located in the hydrophobic subdomain IIA (Sudlow site I). The Trp fluorophore can be specifically excited at 295 nm leading to an emission spectrum with maximum around 320–325 nm if deeply embedded in the hydrophobic environment, and around 340 nm when exposed to the hydrophilic surroundings. The latter was representative for our data with an observed maximum at 338 nm (Fig. 2 ), indicating that the Trp residue and HSA is in its native folded state. The emission wavelength remained constant independent of EntEJ97s concentration, suggesting only minor changes in the polarity of the microenvironment surrounding the HSA fluorophore with the concentrations tested. The fluorescence intensity of HSA showed a linear decrease with increasing concentration of EntEJ97s (Fig. 1 ), which implies an interaction between the two molecules. The decrease in fluorescence intensity (i.e. quenching) can be due to different mechanisms. Collisional (dynamic) quenching is a result of the deactivation of the excited state fluorophore upon contact with some other molecule (i.e. quencher) during the lifetime of the excited state. Static quenching is a result of the formation of a non-fluorescent complex, known as a ground state complex, between the quencher and the fluorophore. Both processes can be expressed by the Stern-Volmer equation; with dynamic quenching, the bimolecular quenching constant (k q ) is below 2 x 10 10 M − 1 s − 1 in aqueous solution ( 54 ). The calculated value of k q in the present work,1.35 ± 0.50 x 10 13 M − 1 s − 1 , is three orders of magnitudes higher than k q in dynamic quenching meaning that the quenching process resulted from static, ground state complex formation between EntEJ97s and HSA. Such complex formation will in many cases lead to perturbation of the absorption spectrum of the fluorophore. The absorption spectra of HSA include absorption between 190–235 nm, with a maximum at about 208 nm, which can be ascribed to the overall three-dimensional conformation of the protein ( 62 ), while the observed peak at 278 nm represents the aromatic amino acids (i.e., Trp, Tyr, and Phe). The gradual elimination of the absorption below 235 nm in the presence of EntEJ97s (Fig. 2 ) indicated that binding of EntEJ97s to HSA induced a change in the protein structure, further supporting ground state complex formation between HSA and EntEJ97s. The binding affinity between HSA and EntEJ97s and the stoichiometry was also estimated from the steady state fluorescence data. In case of static quenching, the Stern-Volmer quenching constant (K sv ) can be interpreted as the association constant (K a ) for the complex formation between ligand and protein, assumed that K sv [F 0 ] < < 1 where [F 0 ] is the initial concentration of fluorophore ( 63 ). In the present work, K sv [F 0 ] was in the range 0.1–0.4, and the calculated K sv therefore represented a rough estimate of the complex association constant. Calculation of K a from Eq. 3 resulted in a slightly lower value (Table 1 ). It has previously been reported that such calculations should be based on a ratio of [drug] / [HSA] ranging from 1 to 10 ( 64 ). This criterion was fulfilled in the current study, except for the lowest EntEJ97s concentration (ratio 0.75). A binding constant in the range 1–15 x 10 4 M − 1 is considered as moderate binding ( 55 ). The K a and K sv values obtained in the present work were, 7.1 x 10 2 and 6.5 x 10 3 M − 1 , respectively, suggesting a weak and reversible interaction between quencher (EntEJ97s) and fluorophore (HSA). This is supported by the K a determined from ITC data, which was in the same range (7.56 x 10 3 M − 1 ). The binding stoichiometry (n) between HSA and EntEJ97s was also estimated from both the fluorescence and nano ITC data, and found to be 0.78 and 0.44, respectively. Thus, both studies suggest less than one EntEJ97s molecules bound per HSA under the given experimental conditions (Table 1 ). This further points to a rather weak drug-protein interaction but can also be due to multiple binding sites and/or a receptor molecule that is partly unfolded ( 65 ). These results are consistent with previous investigations of antimicrobial peptides and HSA, reporting a stoichiometry less than 1:1 ( 17 ). The discrepancy between some of the values determined by fluorescence and nano ITC (e.g., association constant, binding stoichiometry) could be due to several factors. For instance, the fluorescence method represents steady-state conditions while nano ITC represent dynamic processes. Fluorescence describes the final state after equilibrium between the two measured components is achieved, while nano ITC describes the heat absorbed or released during binding events in real time. Also, the methods of analyzing the data are different; while complex model fitting are used for nano ITC data, simpler models such as Stern-Volmer, are used for fluorescence data. Molecules in non-covalent interactions can be governed by Van der Waals interactions, hydrophobic forces, and electrostatic forces. The thermodynamic parameters can be interpreted to determine what type of forces govern the interaction in question. The thermodynamic parameters determined in the nano ITC experiments were ΔH° = -56.43 kcal/mol, ΔG° = -5.292 kcal/mol, ΔS° = − 0.17 kcal/mol (Table 1 ). When Van der Waals and hydrogen bonding causes interactions, ΔH° < 0 and ΔS° 0, and, lastly, if hydrophobic interactions play a major role, ΔH° >0 and ΔS° >0 ( 62 ). Our nano ITC data thus suggest that the interaction between EntEJ97s and HSA is governed by Van der Waals and hydrogen bond interactions (Table 1 , Fig. 3 ). Additionally, the negative ΔH° and ΔG° indicate that the interaction is enthalpy driven (negative ΔH°) and that the binding process is spontaneous (negative ΔG°). The molecular docking analysis supports these findings, as it revealed that hydrogen bond interactions as well as π-stacking and π-cation pairs can occur (Table 3 ). Few studies have been performed on interactions between antimicrobial peptides and HSA, but one other study investigating several cationic antimicrobial peptides also found low binding affinities in nano ITC experiments ( 17 ). However, the negative ΔS° and the positive ΔH° shown for these peptides indicate that electrostatic forces govern the interaction ( 17 , 62 ). Additionally, unlike EntEJ97s described here, the peptides from the previous study lost effect in the presence of physiological HSA concentrations, indicating a different condition of binding ( 33 ). The results presented here also provide insights into the site(s) of interaction between HSA and EntEJ97s. Our nano DSC data indicates that at the lower concentrations (0.01 and 0.025 mM), the largest changes in thermal stability of the protein occur in domain I and II, as supported by the molecular docking analysis (Table 2 ). However, at higher concentrations, the transition of domain III vanishes, indicating there is no thermally induced unfolding of this domain of the protein. At 0.6 mM EntEJ97s the transition of domain II also disappears, and at 1.0 mM concentration no transitions were found (Table 2 ). Such lack of transitions suggest either that the complex is completely stable or that there are aggregation/precipitations that have hindered the nano DSC measurements. However, our current data does not allow us to distinguish between these two options. Therefore, the HSA:EntEJ97s interaction at high concentrations of EntEJ97s should be further investigated . To further investigate how EntEJ97s binds to HSA, nano ITC experiments were performed with the competitive ligands 4 – hydroxycoumarine (hcm, Sudlow site I ligand), dansylglycine (dgly, Sudlow site II ligand) and ibuprofen (ibu, Sudlow site II ligand, weak Sudlow site I ligand). All these experiments indicated reduced enthalpy changes, supporting the presence of competitive interactions at both binding sites (Fig. 4 ). The molecular docking experiment supported that EntEJ97s could bind both in the vicinity of Sudlow sites I and II, with the best predicted binding close to site I. Together, the results therefore suggest that EntEJ97s may bind several sites on HSA, with preferential interaction close Sudlow site I. Previous in vitro studies showed a somewhat decreased antibacterial effect of EntEJ97s in blood and more negatively affected in blood compared to plasma ( 66 ). Ex vivo the peptides were able to kill bacteria as effectively in blood as in media, and in vivo the peptide circulated in systemic circulation in therapeutic concentrations ( 67 ). This correlates well with the findings from the current work, that the low affinity binding to HSA does not cause a decrease in antimicrobial effect at either of the concentrations of HSA tested (Fig. 6 , Table 4 ). The findings are contrary to findings with other antimicrobial peptides, which have demonstrated decreased antibacterial efficacy due to HSA-binding, ( 17 ), however, these are completely different peptides with different sizes compared to EntEJ97s, which likely influence the results. Taken together, our results indicate that there is an interaction with low affinity between EntEJ97s and HSA, most likely involving both of the most important binding sites; Sudlow site I and II. However, this interaction does not seem to interfere with the antibacterial effect of EntEJ97s. The potent effect of EntEJ97s in the presence of HSA is promising for developing EntEJ97s into an intravenous treatment against infections, such as those caused by VRE. Declarations Funding The work was supported by a grant from the Research Council of Norway (FRIMEDBIO grant #275190). Conflict of Interest The authors declare that they have no conflicts of interest. Data Availability Statement All data are incorporated into the article and its online supplementary material. Author Contributions IR designed and performed the experiments, analysed the data, and wrote the manuscript. HHT performed the fluorescence experiments, wrote the manuscript, designed experiments, analysed the data and revised the manuscript. TO performed the modelling experiments and wrote the manuscript. KGG, MK and HC designed experiments, analysed the data, and revised the manuscript. References V. Mishra, R. J. Heath, Structural and Biochemical Features of Human Serum Albumin Essential for Eukaryotic Cell Culture. Int J Mol Sci 22 (2021). X. M. He, D. C. Carter, Atomic structure and chemistry of human serum albumin. Nature 358 , 209-215 (1992). N. Abdollahpour, V. Soheili, M. 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08:57:28","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":71029,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8041595/v1/c5d8a72941e832f529109957.png"},{"id":95525762,"identity":"39b7aba9-2d39-4777-9b38-b41e19567056","added_by":"auto","created_at":"2025-11-10 10:05:39","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":125860,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8041595/v1/17b15812dcadf32bdb17ecd9.png"},{"id":95367736,"identity":"ec0ab5f5-6e46-4da6-8b75-e73b8324ae00","added_by":"auto","created_at":"2025-11-07 08:57:29","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":136278,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8041595/v1/15e238ee0ed1bd1b6400211b.png"},{"id":95526165,"identity":"3f24fcab-a681-403d-af27-fb7277d35a38","added_by":"auto","created_at":"2025-11-10 10:06:26","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144586,"visible":true,"origin":"","legend":"","description":"","filename":"rs80415950structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8041595/v1/57507ebaad9336c47c37937f.xml"},{"id":95367738,"identity":"1d4bcf5c-0277-42dc-900e-19007c681f68","added_by":"auto","created_at":"2025-11-07 08:57:29","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":153362,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8041595/v1/4451d017ad446cffa2ef6466.html"},{"id":95367716,"identity":"6c5008dd-29f9-4500-be1c-e7a0e45c09ef","added_by":"auto","created_at":"2025-11-07 08:57:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":37974,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA representative Stern-Volmer plot of HSA fluorescence quenched by increasing concentration of EntEJ97.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8041595/v1/374cdaee6fef2c8718f1a8ff.png"},{"id":95367717,"identity":"7f92efd5-c1ad-4c4d-9fdf-239af88a269b","added_by":"auto","created_at":"2025-11-07 08:57:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAbsorption spectrum of HSA in Milli-Q water with and without EntEJ97.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8041595/v1/bdb5cee8ade2db1334127f55.png"},{"id":95525625,"identity":"84b717da-3a92-4b18-9bf5-196b48247bf6","added_by":"auto","created_at":"2025-11-10 10:05:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":71622,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNano ITC binding data of 2.0 mM EntEJ97s titrated into 0.8mM HSA. A. \u003c/strong\u003eRaw data as µcal/sec is plotted against time in seconds. The control experiment (EntEJ97s in Milli-Q water) is shown in red. \u003cstrong\u003eB.\u003c/strong\u003eIntegrated heats fitted through the independent model. Control experiment shown in open squares. Data collected with NanoITC (TA® instruments) and analyzed in NanoAnalyze (TA® instruments). Figures made with GraphPad Prism (V 10.1.2).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8041595/v1/74d51949e90bc4618fe9d798.png"},{"id":95367724,"identity":"972616f5-f68d-425d-9230-1f6d30c0ef3b","added_by":"auto","created_at":"2025-11-07 08:57:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":118552,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNano ITC binding data of 2.0mM EntEJ97s in HSA with competing ligands. A. \u003c/strong\u003eCompetitive experiment of HSA incubated in a 1:1 molar ratio with drug site I reference ligand 4-Hydroxycoumarine. \u003cstrong\u003eB. \u003c/strong\u003eCompetitive experiment of HSA incubated in a 1:1 molar ratio with drug site II ligand dansyglycine. \u003cstrong\u003eC. \u003c/strong\u003eCompetitive experiment of HSA incubated in a 1:1 molar ratio with drug site II, and weak site II ligand ibuprofen. The first injections are missing possibly due to air in the syringe. \u003cstrong\u003eD. \u003c/strong\u003eCompetitive experiment of HSA incubated in a 1:1 molar ratio with dansylglycine and ibuprofen. In the upper panel the raw data as µcal/sec is plotted against time in seconds. In the lower panel the integrated heat in kcal/mol is plotted against injection number. Data collected with NanoITC (TA® instruments) and analysed in NanoAnalyze (TA® instruments). Figures made with GraphPad Prism (V 10.1.2).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8041595/v1/88f7636e9918999b1ad18db8.png"},{"id":95367720,"identity":"cdd19602-d245-4b09-98b0-3ddcc5d0b6ca","added_by":"auto","created_at":"2025-11-07 08:57:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":251953,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHighest-scoring HSA-EntEJ97s complex obtained by docking analysis\u003c/strong\u003e. Colored spheres show the drug binding sites Sudlow I (teal) and Sudlow II (green). Binding sites were determined from structures of HSA in complex with warfarin (PDB ID 2BXD) and diazepam (PDB ID 2BXF).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8041595/v1/ce16462f51240e777536c70a.png"},{"id":95525252,"identity":"4dd71f62-09ef-4f42-ba52-8fd15e108399","added_by":"auto","created_at":"2025-11-10 10:04:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":112354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibacterial effect of EntEJ97s in the presence of experimental and physiological concentrations of HSA. \u003c/strong\u003e10 µL of EntEJ97s was spotted on indicator bacteria (here represented by \u003cem\u003eE. faecalis \u003c/em\u003eMMH594), in three different concentrations as indicated. Only HSA was added as a control (0 mM).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8041595/v1/8e88cc23602455fe9f057a6a.png"},{"id":95530822,"identity":"7cb35fbe-f69f-46dc-8c68-41a4dfbc5c0e","added_by":"auto","created_at":"2025-11-10 10:22:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1894011,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8041595/v1/f7e29bd3-60ef-4521-8485-f759de1e3307.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eThe bacteriocin enterocin EJ97s retains its antibacterial effect upon interaction with human serum albumin\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman serum albumin (HSA) is the most abundant plasma protein, comprising\u0026thinsp;~\u0026thinsp;60% of the total plasma protein content. Consequently it has a significant impact on the constituents of the blood, where the physiological concentration of HSA is 0.6 mM (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The main functions of HSA are maintenance of osmotic blood pressure, preservation of blood pH and binding and transport of various molecules (\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The HSA protein (66.5 kDa), composed of 585 amino acids, is a globular protein consisting of three domains (I, II and III) connected by flexible hinges and stabilized by disulfide bridges (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Each of the three domains contains two subunits, labelled A and B. The two main drug binding sites are located in subdomains IIA and IIIA (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). These two sites are named Sudlow site I and II, respectively, and have different preferences for binding ligands (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Sudlow site I selectively binds heterocyclic compounds such as warfarin or 4-hydroxycoumarine (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), while Sudlow site II prefers aromatic compounds such as ibuprofen (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In spite of these preferences some compounds bind to several binding pockets in HSA. This is exemplified by the non-steroidal anti-inflammatory compounds naproxen and diflunisal which bind to two and three pockets on HSA, respectively (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHSA plays a large role in transport of endogenous and exogenous compounds, carrying fatty acids, hormones, bilirubin, and other small molecules, as well as peptides (\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The interaction of various compounds with HSA is often reversible and can contribute to extending the half-life of the compounds in blood. In fact, recent research has explored the use of fusion to an albumin-binding domain as a strategy to associate therapeutic compounds with HSA (\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Conversely, interaction with HSA can also decrease the effectiveness of a drug; an example of this is the antibiotic oritavancin. The presence of HSA increases the minimum inhibitory concentration (MIC) of oritavancin against different strains of enterococci two- to eightfold (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Increased MIC values due to HSA were also found for teicoplanin and daptomycin, while the MIC value of vancomycin and linezolid was unaltered (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). It has also been shown that binding to HSA is beneficial for strongly lipophilic compounds as it increases the solubility in plasma (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). However, if the binding affinity is too high, the interaction may be a disadvantage due to the necessity of an augmented dose (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). In addition, it must be considered whether the interaction disturbs the activity of the compound in question.\u003c/p\u003e\u003cp\u003eBacteriocins are bacterially produced antimicrobial peptides. While their ecological roles is linked to competition for niche and nutrients, these peptides hold promise for use in treatment of various infections (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). One of the major concerns of developing bacteriocins into viable alternatives to antibiotics is their delivery method, as oral delivery would leave them susceptible to proteolytic degradation (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Several strategies for delivery have been suggested, such as nanoparticles, gels, as well as systemic delivery through intravenous infusion (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Intravenous infusion bypasses many delivery hurdles, but the administered compound will interact with the blood constituents, such as HSA (\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Previously it has been shown that the activity of an antimicrobial peptide was reduced in the presence of bovine serum albumin (BSA) (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Another study corroborated these results, indicating a 10-fold increase in MIC in the presence of BSA (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Thus, any binding interaction with HSA can have significance in determining whether a bacteriocin can have a role in treatment of systemic infections, such as those caused by vancomycin resistant enterococci (VRE) (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAntibiotic resistant bacteria are a major concern due to the limited development of new antibiotics and the absence of viable alternatives to antibiotics (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). VRE are of particular concern due to the remarkable resilience of these bacteria, being able to survive on surfaces for months, and withstand lysozyme, heat, alcohol, and salt (\u003cspan additionalcitationids=\"CR37 CR38 CR39 CR40\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). In addition, these bacteria are extremely proficient at exchanging genetic material often due to absence of CRISPR-Cas genes, making spread of resistance genes rapid and efficient (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Bacteriocins represent an alternative treatment strategy against infections caused by antibiotic resistant pathogens, such as VRE (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). In terms of infection treatment, bacteriocins possess several advantageous properties. They often have narrow spectrum of activity, thereby specifically killing the pathogen while preserving the commensal microbiota (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).Furthermore, since bacteriocins mostly have different cellular targets and mechanisms of action compared to antibiotics, the risk of cross-resistance is low (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Enterocin EJ97s (EntEJ97s), is a leaderless class II bacteriocin belonging to the so-called LsbB family of bacteriocins. EntEJ97s has potent activity against \u003cem\u003eEnterococcus faecalis\u003c/em\u003e and lower, but still notable, activity against \u003cem\u003eEnterococcus faecium\u003c/em\u003e (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). In aqueous solution, EntEJ97s exhibits a primarily unfolded confirmation, characterized by an absence of well-defined secondary structures. However, within this unfolded state, intermittent helical or turn-like structures emerge, suggesting dynamic fluctuations in the peptide conformation (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). The exact mechanism of action of EntEJ97s is not fully characterized, but it is known that it targets the transmembrane protease RseP as a receptor in the cell membrane of susceptible cells (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). RseP functions in regulated intramembrane proteolysis, where it performs the second cleavage of an anti-σ factor, resulting in the release of a σ factor that activates expression of genes involved in bacterial stress response (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study aimed to explore the interaction between EntEJ97s and HSA, and to determine if such interaction affected the antibacterial effect of the bacteriocin. The interactions were studied using a multifaceted approach. Fluorescence spectroscopy, nano differential scanning calorimetry (nano DSC) and nano isothermal titration calorimetry (nano ITC) were used to gain insights into the binding kinetics of EntEJ97s and HSA. To further support the experimental results, molecular modelling was performed. The antibacterial effect of the EntEJ97s in the presence of experimental and physiological concentrations of HSA was also investigated. Combining these approaches provided a comprehensive overview of the binding properties of HSA and EntEJ97s, which is essential for further development of EntEJ97s and similar bacteriocins, in treatment of systemic enterococcal infections.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eLigands and reagents\u003c/h2\u003e\u003cp\u003eThe amino acid sequence of the peptide EntEJ97s is MIKKFPNPYTLAAKLTTYEINWYKQQYGRYPWERPVA (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) giving it a weight of ~\u0026thinsp;4566 Da. All synthetic peptides were purchased from Pepmic co., Ltd., China with \u0026ge;\u0026thinsp;99% purity. Lyophilized HSA (\u0026ge;\u0026thinsp;99% globulin, fatty acid free) was purchased from Sigma (A3782, Darmstadt, Germany). The reference ligands 4-hydroxycoumarine, dansylglycine, and ibuprofen were purchased from Sigma.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSteady state fluorescence\u003c/h3\u003e\n\u003cp\u003eSteady state fluorescence measurements were performed on a Photon Technology International modular fluorescence system (London, Ontario, Canada) with Model 101 monochromator with f/4 0.2-m Czerny-Turner configuration. The instrument was equipped with a red-sensitive photomultiplier. The excitation source was a 75 W xenon lamp. The emission and excitation spectra were automatically corrected for, both the lamp spectral radiance and the detector quantum efficiency, by means of the acquisition software (FeliX32, PTI). The excitation and emission monochromator band passes were set at 2 nm. The excitation wavelength was 295 nm, and the emission was recorded in the range 300\u0026ndash;500 nm. The measurements were performed in quartz cuvettes with 1 x 1 cm cell path at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u0026deg;C (n\u0026thinsp;=\u0026thinsp;3). The blanks consisted of EntEJ97s in Milli-Q water at similar concentration as the individual samples and were subtracted from the sample emission spectrum. Fluorescence lifetimes were recorded on an Easy Life V system (Optical Building Blocks Corp., Birmingham, New Jersey, US) equipped with a nanosecond pulsed LED excitation source at 297 nm, slit width 1.5 nm. Post-acquisition data handling was performed by means of the Easy Life V software. The lifetime was calculated from an average of 10 scans and the experiment was repeated on three different samples.\u003c/p\u003e\u003cp\u003eA stock solution of 2 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M HSA and a series of HSA / EntEJ97s assay solutions were prepared for the HSA fluorescence quenching experiments. The concentration of HSA was kept constant at 2 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M while the concentration of EntEJ97s varied in the range of 1.5 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e \u0026ndash; 6.0 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e M. The samples were incubated for 30 min before measurement.\u003c/p\u003e\u003cp\u003eThe overlapping of the excitation wavelength with the EntEJ97s absorption spectrum required correction for inner-filter effect according to Eq.\u0026nbsp;1 (54):\u003c/p\u003e\u003cp\u003eF\u003csub\u003ecor\u003c/sub\u003e = F\u003csub\u003eobs\u003c/sub\u003e x 10 \u003csup\u003e(Aex \u0026minus; Aem/ 2)\u003c/sup\u003e (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\u003cp\u003ewhere F\u003csub\u003ecor\u003c/sub\u003e is the corrected fluorescence value, F\u003csub\u003eobs\u003c/sub\u003e is the measured fluorescence value, A\u003csub\u003eex\u003c/sub\u003e and A\u003csub\u003eem\u003c/sub\u003e the measured absorbance value at the excitation and emission wavelengths, respectively. Absorption spectra were recorded between 190 and 700 nm on a Shimadzu UV-2101 PC UV-Vis scanning spectrophotometer using a quartz cuvette with 1 cm cell path.\u003c/p\u003e\u003cp\u003eThe fluorescence quenching process was evaluated by application of the Stern-Volmer equation (Eq.\u0026nbsp;2, (54)):\u003c/p\u003e\u003cp\u003eF\u003csub\u003e0\u003c/sub\u003e / F\u003csub\u003ecor\u003c/sub\u003e = 1 + k\u003csub\u003eq\u003c/sub\u003eτ\u003csub\u003e0\u003c/sub\u003e [Q]\u0026thinsp;=\u0026thinsp;1\u0026thinsp;+\u0026thinsp;K\u003csub\u003esv\u003c/sub\u003e [Q] (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\u003cp\u003ewhere F\u003csub\u003e0\u003c/sub\u003e and F\u003csub\u003ecor\u003c/sub\u003e are the corrected HSA fluorescence intensities in the absence and presence of EntEJ97s, respectively, [Q] is the known EntEJ97s concentration, k\u003csub\u003eq\u003c/sub\u003e is the bimolecular quenching constant, τ\u003csub\u003e0\u003c/sub\u003e is the fluorescence lifetime of the unquenched HSA fluorophore, and K\u003csub\u003esv\u003c/sub\u003e is the Stern-Volmer quenching constant. The data were presented as a plot of F\u003csub\u003e0\u003c/sub\u003e / F\u003csub\u003ecor\u003c/sub\u003e versus [Q].\u003c/p\u003e\u003cp\u003eThe number of binding sites (n) per HSA molecule and the observed binding constant (K\u003csub\u003ea\u003c/sub\u003e) can be calculated from the fluorescence data using Eq.\u0026nbsp;3 (55):\u003c/p\u003e\u003cp\u003elg (F\u003csub\u003e0\u003c/sub\u003e \u0026ndash; F\u003csub\u003ecor\u003c/sub\u003e / F\u003csub\u003ecor\u003c/sub\u003e)\u0026thinsp;=\u0026thinsp;lg K\u003csub\u003ea\u003c/sub\u003e + n lg[Q] (Eq.\u0026nbsp;3)\u003c/p\u003e\u003cp\u003ewhere F\u003csub\u003e0\u003c/sub\u003e and F\u003csub\u003ecor\u003c/sub\u003e are the fluorescence values applied in the Stern-Volmer equation (i.e., the corrected HSA fluorescence intensities in the absence and presence of EntEJ97s, respectively), [Q] is the molar concentration of free EntEJ97s and K\u003csub\u003ea\u003c/sub\u003e is the observed binding constant. A plot of lg (F\u003csub\u003e0\u003c/sub\u003e \u0026ndash; F\u003csub\u003ecor\u003c/sub\u003e / F\u003csub\u003ecor\u003c/sub\u003e) versus lg [Q] resulted in a straight line (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.94).\u003c/p\u003e\n\u003ch3\u003eNano differential scanning calorimetry (nano DSC)\u003c/h3\u003e\n\u003cp\u003eThe experiments were performed in triplicate using a nano DSC instrument (TA\u0026reg; instruments, New Castle, DE, USA). A range of EntEJ97s concentrations were used (0\u0026ndash;1 mM), while the HSA concentration was kept constant at 30 \u0026micro;M. The samples were prepared in Milli-Q water. The samples were incubated for 30 minutes at 37\u0026deg;C and all working solutions were degassed with a vacuum degasser (TA\u0026reg; instruments). The measured temperature interval was between 20\u0026deg;C and 90\u0026deg;C, with a scanning rate of 1\u0026deg;C min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, at a pressure of 3 atm. Baseline scans of Milli-Q water were recorded and subtracted from the sample scans. Data was analysed using NanoAnalyze software from TA\u0026reg; Instruments, v3.12.5 (Waters LLC, New Castle, DE, US), fitting the data to the two-state scaled model. To compensate for baseline variations between the initial and final states, the calorimetric data was corrected with a sigmoidal baseline in the NanoAnalyze software. The temperature (T) and the enthalpy change (ΔH) of each transition were evaluated.\u003c/p\u003e\n\u003ch3\u003eNano isothermal titration calorimetry (nano ITC)\u003c/h3\u003e\n\u003cp\u003eThe experiments were performed in triplicate on a nano Isothermal Titration Calorimeter (TA\u0026reg; instruments, New Castle, DE, USA), with a cell volume of 200 \u0026micro;L. All samples were prepared in Milli-Q water. The final concentration of HSA was 0.8 mM. The reference cell was filled with 300 \u0026micro;L of Milli-Q water and changed every second day or after two titrations. All working solutions were degassed with a vacuum degasser (TA\u0026reg; instruments). The peptide was titrated into Milli-Q water in duplicate as a control of the signal caused by the dilution. The concentration of the peptide EntEJ97s was always 2.0 mM. The competition ligands were incubated with HSA for 30 minutes at 25\u0026deg;C in a 1:1 molar ratio. The active cell volume was 190 \u0026micro;L and the syringe volume was 50 \u0026micro;L. 50 consecutive 1 \u0026micro;L injections were carried out with 500 s spacing between injections. The stirring rate was set at 200 rpm and the temperature was kept at 25\u0026deg;C. A 300-second baseline was established and assessed before commencement of the experiment.\u003c/p\u003e\u003cp\u003eThe nano ITC data was analysed using the NanoAnalyze software from TA\u0026reg; Instruments, v3.12.5 (Waters LLC, New Castle, DE, US). To generate the binding isotherm the independent model from the program was used, with adjustments to accommodate a larger ΔH in order to fit the data. The first injection of all experiments was omitted in the final analysis.\u003c/p\u003e\n\u003ch3\u003eModelling of protein-protein interactions\u003c/h3\u003e\n\u003cp\u003eRigid-body protein-protein docking studies were performed using FTDock and scored using pyDock (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). A three-dimensional structure of EntEJ97s was obtained using AlphaFold2 v2.3.2 while the structure for human serum albumin was obtained from the RCSB protein data bank (PDB 1AO6). A total of 10,000 docking poses (complexes) were predicted and scored by pyDock, the 100 best-scoring complexes were kept for further analysis. To obtain representative clusters from the ensemble of best-scoring complexes, a cluster analysis was performed using Biomol2Clust (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Molecular interactions between EntEJ97s and HSA were predicted using The Residue Interaction Network Generator v4 (RING) and the web-based 3D structure viewer and structure analysis tool iCn3D (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAntibacterial activity\u003c/h2\u003e\u003cp\u003eHSA (30 \u0026micro;M or 600\u0026micro;M) was weighed out in Milli-Q water together with EntEJ97s (0.025, 0.1 or 0.3 mM) and incubated at 37\u0026deg;C for 30 minutes with shaking at 100 rpm. The minimum inhibitory concentration (MIC) of the antimicrobials with HSA was determined as the minimum concentration that inhibited growth by at least 50% compared to control by OD\u003csub\u003e600\u003c/sub\u003e (without added antimicrobial, but with HSA) in 200 \u0026micro;L culture. The antimicrobial assay was performed with a microtiter assay as previously described (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). A standard spot-on-lawn assay was set up with indicator strains (\u003cem\u003eE. faecalis\u003c/em\u003e Symbioflor and \u003cem\u003eE. faecalis\u003c/em\u003e MMH 594), and 10 \u0026micro;L of the EntEJ97s / HSA mixture. The antibacterial activity experiments were repeated two times.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eSteady-state fluorescence shows that EntEJ97s bind to HSA\u003c/h2\u003e\u003cp\u003eTo get initial insights into the potential interaction between EntEJ97s and HSA, analysis of changes in the intrinsic HSA fluorescence in the presence of EntEJ97s was analyzed. The fluorescence quenching was evaluated by the Stern-Volmer equation. There was a linear dependency of F / F\u003csub\u003ecor\u003c/sub\u003e upon the concentration of EntEJ97 in the concentration range examined (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.90), suggesting a direct interaction between HSA and EntEJ97s. A representative Stern-Volmer plot is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eTo evaluate the stability of the interaction, the bimolecular quenching constant k\u003csub\u003eq\u003c/sub\u003e and the Stern-Volmer quenching constant K\u003csub\u003esv\u003c/sub\u003e were determined. The values for k\u003csub\u003eq\u003c/sub\u003e and K\u003csub\u003esv\u003c/sub\u003e of 1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 x 10\u003csup\u003e13\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 6.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69 x 10\u003csup\u003e3\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively (n\u0026thinsp;=\u0026thinsp;3) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), suggest formation of a static complex between HSA and EntEJ97s. The lifetime of the HSA fluorophore in the absence of EntEJ97s was found to be 5.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 ns (n\u0026thinsp;=\u0026thinsp;3).\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\u003eParameters for EntEJ97s and HSA association extracted from fluorescence and isothermal titration calorimetry (ITC) experiments.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFluorescence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eITC\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\u003eK\u003c/b\u003e\u003csub\u003e\u003cb\u003esv\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(M\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.69 x 10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ek\u003c/b\u003e\u003csub\u003e\u003cb\u003eq\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(M\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e \u003cb\u003es\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 x 10\u003csup\u003e13\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003en\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eK\u003c/b\u003e\u003csub\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(M\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u0026thinsp;1\u003c/b\u003e\u003c/sup\u003e\u003cb\u003e)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 x 10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.56 x 10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e-TΔS (kcal/mol)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e51.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eΔH\u0026deg; (kcal/mol)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-56.43\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eΔG\u0026deg; (kcal/mol)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-5.29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eΔS\u0026deg; (kcal/mol)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-0.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe number of binding sites and the binding constant (K\u003csub\u003ea\u003c/sub\u003e) were calculated to be 0.8 and 7.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 x 10\u003csup\u003e2\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), respectively.\u003c/p\u003e\u003cp\u003eThe fluorescence emission maximum of HSA remained constant at \u0026asymp;\u0026thinsp;338 nm in all samples. In the UV-absorption spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the peak at 278 nm also remained unchanged while there was a major change in the absorption spectrum in the range 190\u0026ndash;235 nm. The absorption in this range was nearly eliminated in the presence of EJ97 but a new maximum occurred at 235 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eNano differential scanning calorimetry indicates that EntEJ97s interacts with several domains of HSA\u003c/h2\u003e\u003cp\u003eAs mentioned above, the structure of HSA is subdivided into three domains: domain I (residues 1-195); II (196\u0026ndash;383); and III (384\u0026ndash;585), where each domain is formed by two subdomains (A and B). Each domain of HSA denatures at specific temperatures. Nano DSC, in which changes in the heat flow associated with the thermal denaturation of HSA is monitored, was used to determine how EntEJ97s interacted with three domains of HSA and whether interactions alter the thermal stability of the protein. We hypothesized that the data obtained from the nano DSC measurements of HSA can be resolved into three transitions, one for each of the three domains of this protein. In addition, predenaturation transitions were observed at lower measured temperatures. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e presents the obtained melting temperatures (T) and enthalpy changes (ΔH) for HSA in the presence of the peptide EntEJ97s at different concentrations. Major changes in T and ΔH (transitions) of HSA were observed at low concentrations of EntEJ97s (\u0026le;\u0026thinsp;0.025 mM) compared to situation where not HSA is present. The largest changes occurred in domain I and II. At increasing concentrations of HSA, no transitions were observed for domain III\u0026thinsp;\u0026ge;\u0026thinsp;0.05 mM and by domain II at concentrations\u0026thinsp;\u0026ge;\u0026thinsp;0.6 mM. Domain I retained transitions except for the highest concentration of the peptide (1.0 mM). The lack of transitions suggests that the domain is completely stabilized or that aggregation or precipitation occurs. Two predenaturation peaks appear in the concentration range 0.025\u0026ndash;0.7 mM of EntEJ97s.\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\u003eResults of nano-DSC measurements for human serum albumin at a constant concentration of 30 \u0026micro;M with varying concentrations of EntEJ97s. The data includes the melting temperature (T) and the enthalpy change for each condition (ΔH), n\u0026thinsp;=\u0026thinsp;3.\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\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e\u003cp\u003eT (\u0026deg;C) / ΔH (kJ/mol)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eHSA (\u0026micro;M)\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eEntEJ97s (mM)\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ePredenat-uration\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ePredenaturation\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eDomain I\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eDomain II\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eDomain III\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e48.88 / 557.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e62.62 / 669.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e66.51 / 457.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e72.40 / 612.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e49.20 / 639.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e65.79 / 576.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e68.57 / 735.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e72.05 / 693.44\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.025\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e48.64 / 951.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e51.40 / 912.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e65.14 / 819.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e68.37 / 717.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e72.42 / 644.33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e49.86 / 788.83\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e53.03 / 1091.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e63.00 / 1124.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e65.96 / 803.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e51.60 / 691.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54.62 / 1087.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e64.21 / 1001.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e66.86 / 703.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e51.43 / 870.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54.55 / 854.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e66.76 / 687.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e70.95 / 672.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50.41 / 693.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e53.79 / 997.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e66.01 / 641.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e70.41 / 630.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47.24 / 830.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50.45 / 926.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e64.42 / 454.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e48.90 / 601.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e62.25 / 469.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\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=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eNano isothermal titration calorimetry indicates that EntEJ97s interferes with both Sudlow site I and II\u003c/h2\u003e\u003cp\u003eWithin HSA, two primary drug-binding sites are generally recognized; Sudlow site I located in subdomain IIA (also known as the warfarin-azapropazone site) and Sudlow site II site located in subdomain IIIA (also known as the indole-benzodiazepine site). Nano ITC measurements were performed to further investigate the thermodynamic properties of the interaction between EntEJ97s and HSA. First, EntEJ97s was titrated into 0.8 mM HSA in a non-competitive nano ITC experiment. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the thermodynamic data from this experiment, including stoichiometry (n\u0026thinsp;=\u0026thinsp;0.44), and the association constant (K\u003csub\u003ea\u003c/sub\u003e) of 7.56 x 10\u003csup\u003e3\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows the nano ITC binding data of EntEJ97s titrated into HSA. Together, this further supports the interaction between HSA and EntEJ97s and suggests that there is less than one EntEJ97s molecule bound per HSA.\u003c/p\u003e\u003cp\u003eNext, competitive nano ITC experiments were performed using ligands with specific affinity to Sudlow site I or II in HSA; 4 \u0026ndash; hydroxycoumarine (hcm, Sudlow site I ligand), dansylglycine (dgly, Sudlow site II ligand) and ibuprofen (ibu, Sudlow site II ligand, weak Sudlow site I ligand) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e) to decipher whether the interaction interferes with any of the drug binding sites. These competitive experiments indicated that EntEJ97s competed with both drug-binding site I and II reference ligands. This was demonstrated by a decrease in the heat signals when the peptide was titrated into HSA incubated with hcm, dgly or ibu at a 1:1 molar ratio.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eMolecular docking and interaction analysis\u003c/h2\u003e\u003cp\u003eTo corroborate the results obtained from the fluorescence quenching and competitive binding assays, molecular docking was performed between EntEJ97s and HSA using FTDock to identify probable binding sites. The molecular docking analysis revealed three distinct clusters representing potential binding sites of EntEJ97s to HSA.\u003c/p\u003e\u003cp\u003eThe best predicted binding site of EntEJ97s was to regions of subdomains IB (residue 108\u0026ndash;196) and IIA (residue 197\u0026ndash;297), with the C-terminus located at the Sudlow site I entrance towards the drug-binding site (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In this complex, EntEJ97s was predicted to interact with HSA via hydrogen-bonding, π-stacking, and π-cation pairs (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The two other potential binding sites (complex 2 and 3, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) involved only hydrogen-bonding and π-cation interactions. Among these, the second ranked complex involved interactions with residues in domain III, where the Sudlow site II is located.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDocking results. Molecular interactions (bonds) between HSA and EntEJ97s in the three predicted complexes. Residue interactions are from HSA (first residue) to EntEJ97s.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eComplex/rank\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eH-bond pairs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eπ-stacking pairs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eπ-cation pairs\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eArg\u003csub\u003e218\u003c/sub\u003e-Ala\u003csub\u003e37\u003c/sub\u003e\u003c/p\u003e\u003cp\u003eGlu\u003csub\u003e292\u003c/sub\u003e-Arg\u003csub\u003e29\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePhe\u003csub\u003e156\u003c/sub\u003e-Tyr\u003csub\u003e27\u003c/sub\u003e\u003c/p\u003e\u003cp\u003eHis\u003csub\u003e288\u003c/sub\u003e-Tyr\u003csub\u003e27\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eArg\u003csub\u003e160\u003c/sub\u003e-Tyr\u003csub\u003e27\u003c/sub\u003e\u003c/p\u003e\u003cp\u003eLys\u003csub\u003e181\u003c/sub\u003e-Trp\u003csub\u003e22\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGlu\u003csub\u003e376\u003c/sub\u003e-Tyr\u003csub\u003e9\u003c/sub\u003e\u003c/p\u003e\u003cp\u003eArg\u003csub\u003e410\u003c/sub\u003e-Tyr\u003csub\u003e18\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLys\u003csub\u003e351\u003c/sub\u003e-Phe\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\u003cp\u003eArg\u003csub\u003e410\u003c/sub\u003e-Tyr\u003csub\u003e18\u003c/sub\u003e\u003c/p\u003e\u003cp\u003eLys\u003csub\u003e414\u003c/sub\u003e-Tyr\u003csub\u003e18\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSer\u003csub\u003e273\u003c/sub\u003e-Trp\u003csub\u003e22\u003c/sub\u003e\u003c/p\u003e\u003cp\u003eSer\u003csub\u003e273\u003c/sub\u003e-Tyr\u003csub\u003e18\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLys\u003csub\u003e225\u003c/sub\u003e-Tyr\u003csub\u003e18\u003c/sub\u003e\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=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eAntimicrobial activity of EntEJ97s is retained in the presence of HSA\u003c/h2\u003e\u003cp\u003eA previous work has shown that the association of a short cationic antimicrobial peptide with serum albumin reduced the ability of the peptide to kill bacteria 10-fold (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). With the intention of investigating whether the same is true for the association between EntEJ97s and HSA, a standard MIC assay (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and a spot-on-lawn assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003e), were set up. The MIC concentrations were the same when HSA was present or absent, with one exception (30 \u0026micro;M HSA and 0.3 mM EntEJ97s, Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). To corroborate these findings, the spot-on-lawn assay was performed and demonstrated equivalent findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003e); the inhibition zones of the peptide are comparable independent of the presence of HSA. As expected, the inhibition zones are larger for the higher concentrations of bacteriocin, but there is no inhibitory effect of HSA alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEstimated MIC\u003csub\u003e50\u003c/sub\u003e of EntEJ97s (in \u0026micro;g/ml) against \u003cem\u003eE. faecalis\u003c/em\u003e MMH594 with and without experimental (30\u0026micro;M) and physiological (600\u0026micro;M) concentrations of HSA.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEntEJs (mM)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWithout HSA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHSA (30 \u0026micro;M)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHSA (600 \u0026micro;M)\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\u003e0.025\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.34\u0026ndash;0.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e0.1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.36\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e0.3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.13\u0026ndash;0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.27\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":"Discussion","content":"\u003cp\u003eMany bacteriocins have been proposed as alternatives for treatment of infection, but very few have reached clinical trials. The difficulty of oral delivery caused by the proteolysis of the peptide in the digestive system, could contribute to this lack of progress. A way around this predicament is to deliver the peptide intravenously, but this implicates interaction with plasma proteins such as HSA. The current work presents the results from binding analysis between bacteriocin EntEJ97s and HSA using steady-state fluorescence, nano DSC and nano ITC. In addition, the results are compared to predictions from molecular modelling. Together, the results indicate that EntEJ97s interacts with HSA, albeit with low affinity. Importantly, however, the interactions between HSA and EntEJ97s, do not seem to negatively impact the antimicrobial effect of the peptide.\u003c/p\u003e\u003cp\u003eInitially, steady state fluorescence was used to demonstrate that EntEJ97s interacts with HSA.\u003c/p\u003e\u003cp\u003eThe intrinsic fluorescence of HSA can originate from three amino acids: Trp, Tyr and Phe where Trp emission is dominant. HSA contains only one Trp unit, Trp-214, which is located in the hydrophobic subdomain IIA (Sudlow site I). The Trp fluorophore can be specifically excited at 295 nm leading to an emission spectrum with maximum around 320\u0026ndash;325 nm if deeply embedded in the hydrophobic environment, and around 340 nm when exposed to the hydrophilic surroundings. The latter was representative for our data with an observed maximum at 338 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating that the Trp residue and HSA is in its native folded state. The emission wavelength remained constant independent of EntEJ97s concentration, suggesting only minor changes in the polarity of the microenvironment surrounding the HSA fluorophore with the concentrations tested. The fluorescence intensity of HSA showed a linear decrease with increasing concentration of EntEJ97s (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which implies an interaction between the two molecules. The decrease in fluorescence intensity (i.e. quenching) can be due to different mechanisms. Collisional (dynamic) quenching is a result of the deactivation of the excited state fluorophore upon contact with some other molecule (i.e. quencher) during the lifetime of the excited state. Static quenching is a result of the formation of a non-fluorescent complex, known as a ground state complex, between the quencher and the fluorophore. Both processes can be expressed by the Stern-Volmer equation; with dynamic quenching, the bimolecular quenching constant (k\u003csub\u003eq\u003c/sub\u003e) is below 2 x 10\u003csup\u003e10\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in aqueous solution (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). The calculated value of k\u003csub\u003eq\u003c/sub\u003e in the present work,1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 x 10\u003csup\u003e13\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, is three orders of magnitudes higher than k\u003csub\u003eq\u003c/sub\u003e in dynamic quenching meaning that the quenching process resulted from static, ground state complex formation between EntEJ97s and HSA. Such complex formation will in many cases lead to perturbation of the absorption spectrum of the fluorophore. The absorption spectra of HSA include absorption between 190\u0026ndash;235 nm, with a maximum at about 208 nm, which can be ascribed to the overall three-dimensional conformation of the protein (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e), while the observed peak at 278 nm represents the aromatic amino acids (i.e., Trp, Tyr, and Phe). The gradual elimination of the absorption below 235 nm in the presence of EntEJ97s (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e) indicated that binding of EntEJ97s to HSA induced a change in the protein structure, further supporting ground state complex formation between HSA and EntEJ97s.\u003c/p\u003e\u003cp\u003eThe binding affinity between HSA and EntEJ97s and the stoichiometry was also estimated from the steady state fluorescence data. In case of static quenching, the Stern-Volmer quenching constant (K\u003csub\u003esv\u003c/sub\u003e) can be interpreted as the association constant (K\u003csub\u003ea\u003c/sub\u003e) for the complex formation between ligand and protein, assumed that K\u003csub\u003esv\u003c/sub\u003e [F\u003csub\u003e0\u003c/sub\u003e]\u0026thinsp;\u0026lt;\u0026thinsp;\u0026lt;\u0026thinsp;1 where [F\u003csub\u003e0\u003c/sub\u003e] is the initial concentration of fluorophore (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). In the present work, K\u003csub\u003esv\u003c/sub\u003e [F\u003csub\u003e0\u003c/sub\u003e] was in the range 0.1\u0026ndash;0.4, and the calculated K\u003csub\u003esv\u003c/sub\u003e therefore represented a rough estimate of the complex association constant. Calculation of K\u003csub\u003ea\u003c/sub\u003e from Eq.\u0026nbsp;3 resulted in a slightly lower value (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It has previously been reported that such calculations should be based on a ratio of [drug] / [HSA] ranging from 1 to 10 (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). This criterion was fulfilled in the current study, except for the lowest EntEJ97s concentration (ratio 0.75). A binding constant in the range 1\u0026ndash;15 x 10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is considered as moderate binding (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). The K\u003csub\u003ea\u003c/sub\u003e and K\u003csub\u003esv\u003c/sub\u003e values obtained in the present work were, 7.1 x 10\u003csup\u003e2\u003c/sup\u003e and 6.5 x 10\u003csup\u003e3\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, suggesting a weak and reversible interaction between quencher (EntEJ97s) and fluorophore (HSA). This is supported by the K\u003csub\u003ea\u003c/sub\u003e determined from ITC data, which was in the same range (7.56 x 10\u003csup\u003e3\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003eThe binding stoichiometry (n) between HSA and EntEJ97s was also estimated from both the fluorescence and nano ITC data, and found to be 0.78 and 0.44, respectively. Thus, both studies suggest less than one EntEJ97s molecules bound per HSA under the given experimental conditions (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This further points to a rather weak drug-protein interaction but can also be due to multiple binding sites and/or a receptor molecule that is partly unfolded (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). These results are consistent with previous investigations of antimicrobial peptides and HSA, reporting a stoichiometry less than 1:1 (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). The discrepancy between some of the values determined by fluorescence and nano ITC (e.g., association constant, binding stoichiometry) could be due to several factors. For instance, the fluorescence method represents steady-state conditions while nano ITC represent dynamic processes. Fluorescence describes the final state after equilibrium between the two measured components is achieved, while nano ITC describes the heat absorbed or released during binding events in real time. Also, the methods of analyzing the data are different; while complex model fitting are used for nano ITC data, simpler models such as Stern-Volmer, are used for fluorescence data.\u003c/p\u003e\u003cp\u003eMolecules in non-covalent interactions can be governed by Van der Waals interactions, hydrophobic forces, and electrostatic forces. The thermodynamic parameters can be interpreted to determine what type of forces govern the interaction in question. The thermodynamic parameters determined in the nano ITC experiments were ΔH\u0026deg; = -56.43 kcal/mol, ΔG\u0026deg; = -5.292 kcal/mol, ΔS\u0026deg;\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.17 kcal/mol (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). When Van der Waals and hydrogen bonding causes interactions, ΔH\u0026deg; \u0026lt; 0 and ΔS\u0026deg; \u0026lt; 0. Electrostatic effect is the main force if ΔH\u0026deg; \u0026asymp; 0 and ΔS\u0026deg; \u0026gt;0, and, lastly, if hydrophobic interactions play a major role, ΔH\u0026deg; \u0026gt;0 and ΔS\u0026deg; \u0026gt;0 (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). Our nano ITC data thus suggest that the interaction between EntEJ97s and HSA is governed by Van der Waals and hydrogen bond interactions (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Additionally, the negative ΔH\u0026deg; and ΔG\u0026deg; indicate that the interaction is enthalpy driven (negative ΔH\u0026deg;) and that the binding process is spontaneous (negative ΔG\u0026deg;). The molecular docking analysis supports these findings, as it revealed that hydrogen bond interactions as well as π-stacking and π-cation pairs can occur (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Few studies have been performed on interactions between antimicrobial peptides and HSA, but one other study investigating several cationic antimicrobial peptides also found low binding affinities in nano ITC experiments (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). However, the negative ΔS\u0026deg; and the positive ΔH\u0026deg; shown for these peptides indicate that electrostatic forces govern the interaction (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). Additionally, unlike EntEJ97s described here, the peptides from the previous study lost effect in the presence of physiological HSA concentrations, indicating a different condition of binding (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe results presented here also provide insights into the site(s) of interaction between HSA and EntEJ97s. Our nano DSC data indicates that at the lower concentrations (0.01 and 0.025 mM), the largest changes in thermal stability of the protein occur in domain I and II, as supported by the molecular docking analysis (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, at higher concentrations, the transition of domain III vanishes, indicating there is no thermally induced unfolding of this domain of the protein. At 0.6 mM EntEJ97s the transition of domain II also disappears, and at 1.0 mM concentration no transitions were found (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Such lack of transitions suggest either that the complex is completely stable or that there are aggregation/precipitations that have hindered the nano DSC measurements. However, our current data does not allow us to distinguish between these two options. Therefore, the HSA:EntEJ97s interaction at high concentrations of EntEJ97s should be further investigated .\u003c/p\u003e\u003cp\u003eTo further investigate how EntEJ97s binds to HSA, nano ITC experiments were performed with the competitive ligands 4 \u0026ndash; hydroxycoumarine (hcm, Sudlow site I ligand), dansylglycine (dgly, Sudlow site II ligand) and ibuprofen (ibu, Sudlow site II ligand, weak Sudlow site I ligand). All these experiments indicated reduced enthalpy changes, supporting the presence of competitive interactions at both binding sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The molecular docking experiment supported that EntEJ97s could bind both in the vicinity of Sudlow sites I and II, with the best predicted binding close to site I. Together, the results therefore suggest that EntEJ97s may bind several sites on HSA, with preferential interaction close Sudlow site I.\u003c/p\u003e\u003cp\u003ePrevious \u003cem\u003ein vitro\u003c/em\u003e studies showed a somewhat decreased antibacterial effect of EntEJ97s in blood and more negatively affected in blood compared to plasma (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). \u003cem\u003eEx vivo\u003c/em\u003e the peptides were able to kill bacteria as effectively in blood as in media, and \u003cem\u003ein vivo\u003c/em\u003e the peptide circulated in systemic circulation in therapeutic concentrations (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). This correlates well with the findings from the current work, that the low affinity binding to HSA does not cause a decrease in antimicrobial effect at either of the concentrations of HSA tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The findings are contrary to findings with other antimicrobial peptides, which have demonstrated decreased antibacterial efficacy due to HSA-binding, (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), however, these are completely different peptides with different sizes compared to EntEJ97s, which likely influence the results.\u003c/p\u003e\u003cp\u003eTaken together, our results indicate that there is an interaction with low affinity between EntEJ97s and HSA, most likely involving both of the most important binding sites; Sudlow site I and II. However, this interaction does not seem to interfere with the antibacterial effect of EntEJ97s. The potent effect of EntEJ97s in the presence of HSA is promising for developing EntEJ97s into an intravenous treatment against infections, such as those caused by VRE.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThe work was supported by a grant from the Research Council of Norway (FRIMEDBIO grant #275190).\u003c/p\u003e\n\u003cp\u003eConflict of Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003eData Availability Statement\u003c/p\u003e\n\u003cp\u003eAll data are incorporated into the article and its online supplementary material.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eIR designed and performed the experiments, analysed the data, and wrote the manuscript. 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Carlsen, Exploring the feasibility of bacteriocins EntK1 and EntEJ97s in treatment of systemic vancomycin resistant enterococci infections in mice. \u003cem\u003eJ Appl Microbiol\u003c/em\u003e \u003cstrong\u003e135\u003c/strong\u003e (2024).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"0d162466-a797-4e01-83b7-672d7b5dbefc","identifier":"10.13039/501100005416","name":"Norges Forskningsråd","awardNumber":"275190","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Norwegian University of Life Sciences","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"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":"bacteriocins, antimicrobial peptides, albumin binding, nano differential scanning calorimetry, molecular modelling, nano isothermal titration calorimetry","lastPublishedDoi":"10.21203/rs.3.rs-8041595/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8041595/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEnterocin EJ97s is a bacteriocin with potent activity against vancomycin-resistant \u003cem\u003eEnterococcus faecalis\u003c/em\u003e. Since EntEJ97 is active in the presence of blood and has been shown to circulate at therapeutic concentrations without causing damage in a murine model, it is considered a promising candidate for treatment of \u003cem\u003eE. faecalis\u003c/em\u003e infections. The aim of this study was to investigate the interaction of EntEJ97s with human serum albumin (HSA) as a step in the pre-clinical assessment of this bacteriocin. The binding association between EntEJ97s and HSA was investigated with fluorescence, nano differential scanning calorimetry (nano DSC) and nano isothermal titration calorimetry (nano ITC). The results show low to moderate binding affinity between EntEJ97s and HSA, with a stoichiometry of less than one EntEJ97s per HSA molecule. Nano ITC competition experiments with competitive ligands indicated that the peptide binds to both the Sudlow I site and the Sudlow II site, the two main drug binding sites on HSA. Molecular modelling efforts corroborated the findings of a binding in the vicinity of these sites. The antibacterial effect of the peptide was preserved in the presence of physiological and experimental concentrations of HSA, as indicated by minimum inhibitory concentration experiments, and spot-on-lawn assays. These findings accentuate that the properties EntEJ97s has in association with HSA are favourable for intravenous treatment.\u003c/p\u003e","manuscriptTitle":"The bacteriocin enterocin EJ97s retains its antibacterial effect upon interaction with human serum albumin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-07 08:57:24","doi":"10.21203/rs.3.rs-8041595/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":"b462ddc0-47fb-4963-9b4f-57a4fc3921a2","owner":[],"postedDate":"November 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57509903,"name":"Applied \u0026 Industrial Microbiology"}],"tags":[],"updatedAt":"2025-11-07T08:57:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-07 08:57:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8041595","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8041595","identity":"rs-8041595","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}