Influence of the ionic strength on the heat-induced aggregation of the globular protein beta-lactoglobulin at pH 7

The influence of the ionic strength on the structure of beta-lactoglobulin aggregates formed after heating at pH 7 has been studied using static and dynamic light scattering. The native protein depletion has been monitored using size exclusion chromatography. Above a critical association concentration (CAC) well-defined clusters are formed containing about 100 monomers. The CAC increases with decreasing ionic strength. The so-called primary aggregates associate to form self similar semi-flexible aggregates with a large scale structure that is only weakly dependent on the ionic strength. The local density of the aggregates increases with increasing ionic strength. At a critical gel concentration, Cg, the size of the aggregates diverges. Cg decreases from 100 g/l without added salt to 1 g/l at 0.4M NaCl. For C > Cg the system gels except at high ionic strength close to Cg where the gels collapse under gravity and a precipitate is formed.     

Further studies on the contribution of electrostatic and hydrophobic interactions to protein adsorption on dye-ligand adsorbents.

The adsorption equilibria of bovine serum albumin (BSA), gamma-globulin, and lysozyme to three kinds of Cibacron blue 3GA (CB)-modified agarose gels, 6% agarose gel-coated steel heads (6AS), Sepharose CL-6B, and a home-made 4% agarose gel (4AB), were studied. We show that ionic strength has irregular effects on BSA adsorption to the CB-modified affinity gels by affecting the interactions between the negatively charged protein and CB as well as CB and the support matrix. At low salt concentrations, the increase in ionic strength decreases the electrostatic repulsion between negatively charged BSA and the negatively charged gel surfaces, thus resulting in the increase of BSA adsorption. This tendency depends on the pore size of the solid matrix, CB coupling density, and the net negative charges of proteins (or aqueous - phase pH value). Sepharose gel has larger average pore size, so the electrostatic repulsion-effected protein exclusion from the small gel pores is observed only for the affinity adsorbent with high CB coupling density (15.4 micromol/mL) at very low ionic strength (NaCl concentration below 0.05 M in 10 mM Tris-HCl buffer, pH 7.5). However, because CB-6AS and CB-4AB have a smaller pore size, the electrostatic exclusion effect can be found at NaCl concentrations of up to 0.2 M. The electrostatic exclusion effect is even found for CB-6AS with a CB density as low as 2.38 micromol/mL. Moreover, the electrostatic exclusion effect decreases with decreasing aqueous-phase pH due to the decrease of the net negative charges of the protein. For gamma-globulin and lysozyme with higher isoelectric points than BSA, the electrostatic exclusion effect is not observed. At higher ionic strength, protein adsorption to the CB-modified adsorbents decreases with increasing ionic strength. It is concluded that the hydrophobic interaction between CB molecules and the support matrix increases with increasing ionic strength, leading to the decrease of ligand density accessible to proteins, and then the decrease of protein adsorption. Thus, due to the hybrid effect of electrostatic and hydrophobic interactions, in most cases studied there exists a salt concentration to maximize BSA adsorption.     

“Ordered” structure in solutions and gels of a globular protein as studied by small angle neutron scattering

Small-angle neutron scattering measurements were used for structural investigation of β-lactoglobulin solutions and heat-set gels in conditions of strong double layer repulsions. At pH 9 and low ionic strength, a correlation peak was observed on the scattering curves of the solutions whatever the protein concentration C used (in the range C = 0.02–0.10 g/mL). The wave vector value q_max corresponding to these maxima scaled as C^0.25. This exponent value is in agreement with those reported in the literature for other globular proteins in the same concentration range. Increasing the ionic strength decreased the peak which vanished without changing position at 0.1M NaCl. This polyelectrolyte-like behaviour suggests a local structure in the protein solution due to double layer repulsions. In the case of heat-set aggregates and gels (0.02–0.13 g/mL) formed at pH 9 and low ionic strength, a peak in the scattering curves was also observed indicating that even after gelation a correlation is still present; q_max varied as C^0.5. As in the case of the solutions, the correlation peak decreased with increasing ionic strength, and it vanished at 0.06M NaCl. The dilution of the aggregates in order to determine their intraparticle structure factor showed that the correlations were lost and that the aggregates displayed the same internal structure as the elementary subunit in the gels. At high ionic strength, fractal structures of the aggregates down to a length scale of about 40 Å were observed with d_f = 1.3–1.75 ± 0.05, increasing with protein concentration. Subsequent dilution didn't change the fractal dimension of these structures. © 1996 John Wiley & Sons, Inc.     

Protein interactions in solution characterized by light and neutron scattering: comparison of lysozyme and chymotrypsinogen.

The effects of pH and electrolyte concentration on protein-protein interactions in lysozyme and chymotrypsinogen solutions were investigated by static light scattering (SLS) and small-angle neutron scattering (SANS). Very good agreement between the values of the virial coefficients measured by SLS and SANS was obtained without use of adjustable parameters. At low electrolyte concentration, the virial coefficients depend strongly on pH and change from positive to negative as the pH increases. All coefficients at high salt concentration are slightly negative and depend weakly on pH. For lysozyme, the coefficients always decrease with increasing electrolyte concentration. However, for chymotrypsinogen there is a cross-over point around pH 5.2, above which the virial coefficients decrease with increasing ionic strength, indicating the presence of attractive electrostatic interactions. The data are in agreement with Derjaguin-Landau-Verwey-Overbeek (DLVO)-type modeling, accounting for the repulsive and attractive electrostatic, van der Waals, and excluded volume interactions of equivalent colloid spheres. This model, however, is unable to resolve the complex short-ranged orientational interactions. The results of protein precipitation and crystallization experiments are in qualitative correlation with the patterns of the virial coefficients and demonstrate that interaction mapping could help outline new crystallization regions.     

Conformational prerequisites for formation of amyloid fibrils from histones

We demonstrate that bovine core histones are natively unfolded proteins in solutions with low ionic strength due to their high net positive charge at pH 7.5. Using a variety of biophysical techniques we characterized their conformation as a function of pH and ionic strength, as well as correlating the conformation with aggregation and amyloid fibril formation. Tertiary structure was absent under all conditions except at pH 7.5 and high ionic strength. The addition of trifluoroethanol or high ionic strength induced significant alpha-helical secondary structure at pH 7.5. At low pH and high salt concentration, small-angle X-ray scattering and SEC HPLC indicate the histones are present as a hexadecamer of globular subunits. The secondary structure at low pH was independent of the ionic strength or presence of TFE, as judged by FTIR. The data indicate that histones are able to adopt five different relatively stable conformations; this conformational variability probably reflects, in part, their intrinsically disordered structure. Under most of the conditions studied the histones formed amyloid fibrils with typical morphology as seen by electron microscopy. In contrast to most aggregation/amyloidogenic systems, the kinetics of fibrillation showed an inverse dependence on histone concentration; we attribute this to partitioning to a faster pathway leading to non-fibrillar self-associated aggregates at higher protein concentrations. The rate of fibril formation was maximal at low pH, and decreased to zero by pH 10. The kinetics of fibrillation were very dependent on the ionic strength, increasing with increasing salt concentration, and showing marked dependence on the nature of the ions; interestingly Gdn.HCl increased the rate of fibrillation, although much less than NaCl. Different ions also differentially affected the rate of nucleation and the rate of fibril elongation.     

Effects of ionic strength on the folding and stability of SAMP1, a ubiquitin-like halophilic protein

Our knowledge of the folding behavior of proteins from extremophiles is limited at this time. These proteins may more closely resemble the primordial proteins selected in early evolution under extreme conditions. The small archaeal modifier protein 1 (SAMP1) studied in this report is an 87-residue protein with a β-grasp fold found in the halophile Haloferax volcanii from the Dead Sea. To gain insight into the effects of salt on the stability and folding mechanism of SAMP1, we conducted equilibrium and kinetic folding experiments as a function of sodium chloride concentration. The results revealed that increasing ionic strength accelerates refolding and slows down unfolding of SAMP1, giving rise to a pronounced salt-induced stabilization. With increasing NaCl concentration, the rate of folding observed via a combination of continuous-flow (0.1–2 ms time range) and stopped-flow measurements (>2 ms) exhibited a >100-fold increase between 0.1 and 1.5 M NaCl and leveled off at higher concentrations. Using the Linderström-Lang smeared charge formalism to model electrostatic interactions in ground and transition states encountered during folding, we showed that the observed salt dependence is dominated by Debye-Hückel screening of electrostatic repulsion among numerous negatively charged residues. Comparisons are also drawn with three well-studied mesophilic members of the β-grasp superfamily: protein G, protein L, and ubiquitin. Interestingly, the folding rate of SAMP1 in 3 M sodium chloride is comparable to that of protein G, ubiquitin, and protein L at lower ionic strength. The results indicate the important role of electrostatic interactions in protein folding and imply that proteins have evolved to minimize unfavorable charge-charge interactions under their specific native conditions.     

Effect of salt addition on the fractal structure of aggregates formed by heating dilute BSA solutions

The fractal dimension, Df, of aggregates in a dilute BSA system with added salt was evaluated by static light scattering (SLS). A fractal structure was observed for the system with NaCl addition. The values of Df increased with increasing heating time and ionic strength. The values of Df were larger than those (Df = 1.8 or 2.1) predicted by the conventional cluster-cluster aggregation model, probably due to a "restructuring" of aggregates during the aggregation process. On the other hand, a fractal structure was not apparent for the system with added CaCl2.     

Conformation of <Emphasis Type="Italic">Thermus thermophilus</Emphasis> ribosomal protein S1 in solution at different ionic strengths

Small-angle x-ray and neutron scattering were used to study the structure of the ribosomal protein S1 (61 kDa) from Thermus thermophilus in solution at low and moderate ionic strength (0 and 100 mM NaCl). The protein was found to be globular in both cases. Modeling of the S1 structure comprising six homologous domains on the basis of the NMR data for one domain showed that the best fit to scattering data was provided by compact domain packing. The calculated gyration radius was 28–29 Å, as typical of globular proteins about 60 kDa. The protein was prone to self-association, forming mainly dimers and trimers at moderate ionic strength and higher compact associates at low ionic strength. Neutron scattering assays in heavy water at 100 mM NaCl revealed markedly elongated associates. The translational diffusion coefficient calculated for S1 at 100 mM NaCl from dynamic light scattering was markedly lower than the one expected for its globular monomer (D _20,w = (2.7 ± 0.1)·10^?7 versus (5.8–6.0)·10^?7 cm² s^?1), confirming protein association under equilibrium conditions.     

Kinetics and structure during self-assembly of oppositely charged proteins in aqueous solution

Self-assembly in aqueous solution of two oppositely charged globular proteins, hen egg white lysozyme (LYS) and bovine calcium-depleted α-lactalbumin (apo α-LA), was investigated at pH 7.5. The aggregation rate of equimolar mixtures of the two proteins was determined using static and dynamic light scattering as a function of the ionic strength (15-70 mM) and protein concentration (0.28-2.8 g/L) at 25 and 45 °C. The morphology of formed supramolecular structures was observed by confocal laser scanning microscopy. When the two proteins are mixed, small aggregates were formed rapidly that subsequently grew by collision and fusion. The aggregation process led on larger length scales to irregularly shaped flocs at 25 °C, but to monodisperse homogeneous spheres at 45 °C. Both the initial rate of aggregation and the fraction of proteins that associated decreased strongly with decreasing protein concentration or increasing ionic strength but was independent of the temperature.     

Mg2+-paracrystal formation of tropomyosin as a condensation phenomenon. Effects of pH, salt, temperature, and troponin binding.

Tropomyosin (Tm) paracrystal formation induced by Mg2+ was studied by monitoring increases in light scattering. Paracrystals formed above a critical Tm concentration with lag phases in the time courses at pH 7.5 and 6.0, indicating that condensation polymerization processes are involved. The kinetic data at pH 7.5 reasonably fit a model in which nucleation and elongation are taken into account. The rate and extent of light scattering increased at low [Mg2+] and decreased at high [Mg2+] with a maximum at [Mg2+] = 15 mM, indicating different effects of Mg2+ in the two [Mg2+] ranges. The paracrystals were destabilized by increasing the salt concentration and decreasing the temperature. Mg2+ produces paracrystals at pH 6.0 and pH 7.5 by different kinetic mechanisms. Different Tm intermolecular interactions at the two pH values were indicated by studies of the excimer fluorescence of pyrene-labeled Tm and by effects of salt and temperature on the kinetics. At pH 6.0 Tm more readily formed paracrystals with decreased electrostatic effects. Effects of troponin on Mg2+-paracrystal formation of Tm at the two pH values correlated with the known differences in paracrystal structure when troponin is bound to Tm.     

Osmotic observations on chemically cross-linked DNA gels in physiological salt solutions

Neutralized DNA gels exhibit a reversible volume transition when CaCl2 is added to the surrounding aqueous NaCl solution. In this paper, a systematic study of the osmotic and mechanical properties of Na-DNA gels is presented to determine, qualitatively and quantitatively, the effect of Ca-Na exchange on the volume transition. It is found that in the absence of CaCl2 the DNA gels exhibit osmotic behavior similar to that of DNA solutions with reduced DNA concentration. At low CaCl2 concentration, the gel volume gradually decreases as the CaCl2 concentration increases. Below the volume transition, the concentration dependence of the osmotic pressure can be satisfactorily described by a Flory-Huggins-type equation. The Ca2+ ions primarily affect the third-order interaction term, which strongly increases upon the introduction of Ca2+ ions. The second-order interaction term only slightly depends on the CaCl2 concentration. It is demonstrated that DNA gels cross-linked in solutions containing CaCl2 exhibit reduced osmotic mixing pressure. The concentration dependence of the shear modulus of DNA gels can be described by a single power law. The scaling exponent is practically independent of the NaCl concentration and increases with increasing CaCl2 content.     

Electrostatic Interactions Contribute to the Folded-back Conformation of Wild Type Human Factor H

Factor H (FH), a major serum regulator of C3b in the complement alternative pathway, is composed of 20 short complement regulator (SCR) domains. Earlier solution structures for FH showed that this has a folded-back domain arrangement and exists as oligomers. To clarify the molecular basis for this, analytical ultracentrifugation and X-ray scattering studies of native FH were performed as a function of NaCl concentration and pH. The sedimentation coefficient for the FH monomer decreased from 5.7 S to 5.3 S with increase in NaCl concentration, showing that weak electrostatic inter-domain interactions affect its folded-back structure. FH became more elongated at pH 9.4, showing the involvement of histidine residue(s) in its folded-back structure. Similar studies of partially deglycosylated FH suggested that oligosaccharides were not significant in determining the FH domain structure. The formation of FH oligomers decreased with increased NaCl concentration, indicating that electrostatic interactions also affect this. X-ray scattering showed that the maximum length of FH increased from 32 nm in low salt to 38 nm in high salt. Constrained X-ray scattering modelling was used to generate significantly improved FH molecular structures at medium resolution. In 50 mM NaCl, the modelled structures showed that inter-SCR domain contacts are likely, while these contacts are fewer in 250 mM NaCl. The results of this study show that the conformation of FH is affected by its local environment, and this may be important for its interactions with C3b and when bound to polyanionic cell surfaces.     

Effect of secondary structure on the potential of mean force for poly-L-lysine in the alpha-helix and beta-sheet conformations

Because poly-L-lysine (PLL) can exist in the alpha-helix or beta-sheet conformation depending on solution preparation and solution conditions, PLL is a suitable candidate to probe the dependence of protein interactions on secondary structure. The osmotic second virial coefficient and weight-average molecular weight are reported from low-angle laser-light scattering measurements for PLL as a function of NaCl concentration, pH, and alpha-helix or beta-sheet content. Interactions between PLL molecules become more attractive as salt concentration increases due to screening of PLL charge by salt ions and at low salt concentration become more attractive as pH increases due to decreased net charge on PLL. The experimental results show that interactions are stronger for the beta-sheet conformation than for the alpha-helix conformation. A spherically-symmetric model for the potential of mean force is used to account for specific interactions not described by DLVO theory and to show how differences in secondary structure affect PLL interactions.     

Spatial optimization of electrostatic interactions between the ionized groups in globular proteins

A model approach is suggested to estimate the degree of spatial optimization of the electrostatic interactions in protein molecules. The method is tested on a set of 44 globular proteins, representative of the available crystallographic data. The theoretical model is based on macroscopic computation of the contribution of charge–charge interactions to the electrostatic term of the free energy for the native proteins and for a big number of virtual structures with randomly distributed on protein surface charge consetellations (generated by a Monte-Carlo technique). The statistical probability of occurrence of random structures with electrostatic energies lower than the energy of the native protein is suggested as a criterion for spatial optimization of the electrostatic interactions. The results support the hypothesis that the folding process optimizes the stabilizing effect of electrostatic interactions, but to very different degree for different proteins. A parallel analysis of ion pairs shows that the optimization of the electrostatic term in globular proteins has increasingly gone in the direction of rejecting the repulsive short contacts between charges of equal sign than of creating of more salt bridges (in comparison with the statistically expected number of shortrange ion pairs in the simulated random structures). It is observed that the decrease in the spatial optimization of the electrostatic interactions is usually compensated for by an appearance of disulfide bridges in the covalent structure of the examined proteins. © 1994 Wiley-Liss, Inc.     

Electrostatic effects on the stability of discoidal high-density lipoproteins

High-density lipoproteins (HDL) remove cholesterol from peripheral tissues and thereby help to prevent atherosclerosis. Nascent HDL are discoidal complexes composed of a phospholipid bilayer surrounded by protein alpha-helices that are thought to form extensive stabilizing interhelical salt bridges. Earlier we showed that HDL stability, which is necessary for HDL functions, is modulated by kinetic barriers. Here we test the role of electrostatic interactions in the kinetic stability by analyzing the effects of salt, pH, and point mutations on model discoidal HDL reconstituted from human apolipoprotein C-1 (apoC-1) and dimyristoyl phosphatidylcholine (DMPC). Circular dichroism, Trp fluorescence, and light scattering data show that molar concentrations of NaCl or Na(2)SO(4) increase the apparent melting temperature of apoC-1:DMPC complexes by up to 20 degrees C and decelerate protein unfolding. Arrhenius analysis shows that 1 M NaCl stabilizes the disks by deltaDeltaG* approximately equal 3.5 kcal/mol at 37 degrees C and increases the activation energy of their denaturation and fusion by deltaE(a) approximately equal deltaDeltaH* approximately equal 13 kcal/mol, indicating that the salt-induced stabilization is enthalpy-driven. Denaturation studies in various solvent conditions (pH 5.7-8.2, 0-40% sucrose, 0-2 M trimethylamine N-oxide) suggest that the salt-induced disk stabilization results from ionic screening of unfavorable short-range Coulombic interactions. Thus, the dominant electrostatic interactions in apoC-1:DMPC disks are destabilizing. Comparison of the salt effects on the protein:lipid complexes of various composition reveals an inverse correlation between the lipoprotein stability and the salt-induced stabilization and suggests that short-range electrostatic interactions significantly contribute to lipoprotein stability: the better-optimized these interactions are, the more stable the complex is.     

Experimental assignment of the structure of the transition state for the association of barnase and barstar

Association of a protein complex follows a two step reaction mechanism, with the first step being the formation of an encounter complex which evolves into the final complex. Here we present new experimental data for the association of the bacterial ribonuclease barnase and its polypeptide inhibitor barstar which shed light on the thermodynamics and structure of the transition state and preceding encounter complex of association at diminishing electrostatic attraction. We show that the activation entropy at the transition state is close to zero, with the activation enthalpy being equal to the free energy of binding. This observation was independent of the magnitude of the mutual electrostatic attraction, which were altered by mutagenesis or by addition of salt. The low activation entropy implies that the transition state is mostly solvated at all ionic strengths. The structure of the transition state was probed by measuring pairwise interaction energies using double-mutant-cycles. While at low ionic strength all proximal charge-pairs form contacts, at high salt only a subset of these interactions are maintained. More specifically, charge-charge interactions between partially buried residues are lost, while exposed charged residues maintain their ability to form specific interactions even at the highest salt concentration. Uncharged residues do not interact at any ionic strength. The results presented here suggest that the barnase-barstar binding sites are correctly aligned during the transition state even at diminishing electrostatic attraction, although specific short range interactions of uncharged residues are not yet formed. Furthermore, most of the interface desolvation (which contributes to the entropy of the system) has not yet occurred. This picture seems to be valid at low and high salt. However, at high salt, interactions of the activated complex are limited to a more restricted set of residues which are easier approached during diffusion, prior to final docking. This suggest that the steering region at high salt is more limited, albeit maintaining its specificity.     

Light scattering study of heat-denatured globular protein aggregates

The structure of aggregates formed by the globular protein beta-lactoglobulin (beta-lg) after heat induced denaturation was studied using light scattering and size exclusion chromatography. The influence of varying the pH above the iso-electric point (pH 5.2) was investigated in the absence of added salt. Stable aggregates could be formed and characterized between pH 5.8 and pH 9. The large-scale structure of the aggregates was self-similar and remarkably insensitive to the pH. Below a critical association concentration, which decreased with decreasing pH from 10 to 1g/L, denatured monomers and small oligomers were formed. At higher concentrations larger so-called preaggregates formed containing roughly 100 monomers. With increasing concentration the size of the aggregates varied little until it rose sharply close to the gelation concentration that decreased with decreasing pH (50 g/L相似文献   

Conformation of the ribosomal protein S1 of Thermus thermophilus in solution under different ionic conditions

The structure of protein SI of Thermus thermophilus (M = 61 kDa) in solution at low and moderate ionic strengths (0 M and 100 mM NaCl, respectively) has been studied by small-angle X-ray and neutron scattering. It was found that protein S1 has a globular conformation under both ionic conditions. The modelling of different packing of six homologous domains of S1 on the basis of the NMR-resolved structure of one domain showed that the best fit of calculated scattering patterns from such complexes to experimental ones is observed at a compact package of the domains. The calculated value of the radius of gyration of the models is 28-29 angtroms, which is characteristic for globular proteins with a molecular mass of about 60 kDa. It was found that protein S1 has a tendency to form associates, and the type of the associate depends on ionic strength. These associates have, in general, two or three monomers at a moderate ionic strength, while at a low ionic strength the number of monomers exceeds three and they are packed in a compact manner. Strongly elongated associates were observed in neutron experiments at a moderate ionic strength in heavy water. The association of protein molecules was also confirmed by the data of dynamic light scattering. From these data, the translational diffusion coefficient of protein S1 at a moderate ionic strength was calculated to be (D20,w = (2.7 +/- 0.1) x 10(-7)cm2/s). This value is essentially smaller than the expected value (D20,w = (5.8 - 6.0) x 10(-7)cm2/s) for the S1 monomer in the globular conformation, indicating the association of protein molecules under equilibrium conditions.     

Critical Examination of the Colloidal Particle Model of Globular Proteins

Recent studies of globular protein solutions have uniformly adopted a colloidal view of proteins as particles, a perspective that neglects the polymeric primary structure of these biological macromolecules, their intrinsic flexibility, and their ability to sample a large configurational space. While the colloidal perspective often serves as a useful idealization in many cases, the macromolecular identity of proteins must reveal itself under thermodynamic conditions in which the native state is no longer stable, such as denaturing solvents and high protein concentrations where macromolecules tend to have screened excluded volume, charge, and hydrodynamic interactions. Under extreme pH conditions, charge repulsion interactions within the protein chain can overcome the attractive hydrogen-bonding interactions, holding it in its native globular state. Conformational changes can therefore be expected to have great significance on the shear viscosity and other rheological properties of protein solutions. These changes are not envisioned in conventional colloidal protein models and we have initiated an investigation of the scattering and rheological properties of model proteins. We initiate this effort by considering bovine serum albumin because it is a globular protein whose solution properties have also been extensively investigated as a function of pH, temperature, ionic strength, and concentration. As we anticipated, near-ultraviolet circular dichroism measurements and intrinsic viscosity measurements clearly indicate that the bovine serum albumin tertiary structure changes as protein concentration and pH are varied. Our findings point to limited validity of the colloidal protein model and to the need for further consideration and quantification of the effects of conformational changes on protein solution viscosity, protein association, and the phase behavior. Small-angle Neutron Scattering measurements have allowed us to assess how these conformational changes influence protein size, shape, and interprotein interaction strength.     

Electrostatic interactions modulate the conformation of collagen I

The pH- and electrolyte-dependent charging of collagen I fibrils was analyzed by streaming potential/streaming current experiments using the Microslit Electrokinetic Setup. Differential scanning calorimetry and circular dichroism spectroscopy were applied in similar electrolyte solutions to characterize the influence of electrostatic interactions on the conformational stability of the protein. The acid base behavior of collagen I was found to be strongly influenced by the ionic strength in KCl as well as in CaCl(2) solutions. An increase of the ionic strength with KCl from 10(-4) M to 10(-2) M shifts the isoelectric point (IEP) of the protein from pH 7.5 to 5.3. However, a similar increase of the ionic strength in CaCl(2) solutions shifts the IEP from 7.5 to above pH 9. Enhanced thermal stability with increasing ionic strength was observed by differential scanning calorimetry in both electrolyte systems. In line with this, circular dichroism spectroscopy results show an increase of the helicity with increasing ionic strength. Better screening of charged residues and the formation of salt bridges are assumed to cause the stabilization of collagen I with increasing ionic strength in both electrolyte systems. Preferential adsorption of hydroxide ions onto intrinsically uncharged sites in KCl solutions and calcium binding to negatively charged carboxylic acid moieties in CaCl(2) solutions are concluded to shift the IEP and influence the conformational stability of the protein.