Anion modulation of the 1H/2H exchange rates in backbone amide protons monitored by NMR spectroscopy

The ability of three anionic cosolutes (sulfate, thiocyanate, and chloride) in modulating the (1)H/(2)H exchange rates for backbone amide protons has been investigated using nuclear magnetic resonance (NMR) for two different proteins: the IGg-binding domain of protein L (ProtL) and the glucose-galactose-binding protein (GGBP). Our results show that moderate anion concentrations (0.2 M-1 M) regulate the exchange rate following the Hofmeister series: Addition of thiocyanate increases the exchange rates for both proteins, while sulfate and chloride (to a less extent) slow down the exchange reaction. In the presence of the salt, no alteration of the protein structure and minimal variations in the number of measurable peaks are observed. Experiments with model compounds revealed that the unfolded state is modulated in an equivalent way by these cosolutes. For ProtL, the estimated values for the local free energy change upon salt addition (m (3,DeltaG )) are consistent with the previously reported free energy contribution from the cosolute's preferential interaction/exclusion term indicating that nonspecific weak interactions between the anion and the amide groups constitute the dominant mechanism for the exchange-rate modulation. The same trend is also found for GGBP in the presence of thiocyanate, underlining the generality of the exchange-rate modulation mechanism, complementary to more investigated effects like the electrostatic interactions or specific anion binding to protein sites.     

Protein Stabilization and the Hofmeister Effect: The Role of Hydrophobic Solvation

Using the IGg binding domain of protein L from Streptoccocal magnus (ProtL) as a case study, we investigated how the anions of the Hofmeister series affect protein stability. To that end, a suite of lysine-to-glutamine modifications were obtained and structurally and thermodynamically characterized. The changes in stability introduced with the mutation are related to the solvent-accessible area of the side chain, specifically to the solvation of the nonpolar moiety of the residue. The thermostability for the set of ProtL mutants was determined in the presence of varying concentrations (0-1 M) of six sodium salts from the Hofmeister series: sulfate, phosphate, fluoride, nitrate, perchlorate, and thiocyanate. For kosmotropic anions (sulfate, phosphate, and fluoride), the stability changes induced by the cosolute (encoded in ) are proportional to the surface changes introduced with the mutation. In contrast, the m₃ values measured for chaotropic anions are much more independent of such surface modifications. Our results are consistent with a model in which the increase in the solution surface tension induced by the anion stabilizes the folded conformation of the protein. This contribution complements the nonspecific and weak interactions between the ions and the protein backbone that shift the equilibrium toward the unfolded state.     

Crowding alone cannot account for cosolute effect on amyloid aggregation

Amyloid fiber formation is a specific form of protein aggregation, often resulting from the misfolding of native proteins. Aimed at modeling the crowded environment of the cell, recent experiments showed a reduction in fibrillation halftimes for amyloid-forming peptides in the presence of cosolutes that are preferentially excluded from proteins and peptides. The effect of excluded cosolutes has previously been attributed to the large volume excluded by such inert cellular solutes, sometimes termed "macromolecular crowding". Here, we studied a model peptide that can fold to a stable monomeric β-hairpin conformation, but under certain solution conditions aggregates in the form of amyloid fibrils. Using Circular Dichroism spectroscopy (CD), we found that, in the presence of polyols and polyethylene glycols acting as excluded cosolutes, the monomeric β-hairpin conformation was stabilized with respect to the unfolded state. Stabilization free energy was linear with cosolute concentration, and grew with molecular volume, as would also be predicted by crowding models. After initiating the aggregation process with a pH jump, fibrillation in the presence and absence of cosolutes was followed by ThT fluorescence, transmission electron microscopy, and CD spectroscopy. Polyols (glycerol and sorbitol) increased the lag time for fibril formation and elevated the amount of aggregated peptide at equilibrium, in a cosolute size and concentration dependent manner. However, fibrillation rates remained almost unaffected by a wide range of molecular weights of soluble polyethylene glycols. Our results highlight the importance of other forces beyond the excluded volume interactions responsible for crowding that may contribute to the cosolute effects acting on amyloid formation.     

Electrostatic interaction between anions bound to site I and the retinal Schiff base of halorhodopsin

The influence of different anions on the deprotonation of the retinal Schiff base of halorhodopsin in the dark was investigated. We find that a large number of anions cause a significant increase of the pKa of the Schiff base, an effect attributed to binding to "site I" on the protein. The concentration dependencies of the spectroscopic shifts associated with the changes of the pKa yielded dissociation constants (and thus binding energies) for the anions, which were related to the Stokes radii. The data fit the predictions of electrostatic interaction between the anions and the positive charge associated with site I, if the latter is located within a few angstroms from the surface of the protein. The specificity of site I toward various anions is quantitatively explained by the differences in the change of Born energy upon transfer of the anions from water to the binding site. The changes in the deprotonation energy of the Schiff base upon the binding of anions, delta delta Gdeprot, could be calculated from the delta pKa at infinite anion concentration. Unexpectedly, the delta delta Gdeprot values were remarkably close to the energies of binding to site I. Thus, site I and the Schiff base are strongly electrostatically coupled, either because of close proximity or because of the possibility of allosteric energy transfer between them.     

Studying the effect of crowding and dehydration on DNA G-quadruplexes

Intracellular environment is crowded with biomolecules that occupy a significant fraction (up to 40%) of the cellular volume, with a total concentration in the range 300-400mg/ml. Recently, the effect of crowding/dehydrating agents on the DNA G-quadruplexes has become a subject of an increasing interest. Crowding and/or dehydrating agents have been used to simulate how G-quadruplexes behave under cell-mimicking conditions characterized by a large excluded volume and a lower water activity. Indeed, the presence of both steric crowding and a lower water activity can affect G-quadruplex stability, their folding/unfolding kinetics, as well as their binding processes with proteins or small ligands. Many of these effects can be explored experimentally by measuring the dependence of the conformational stability, isomerisation kinetics and equilibria on the concentration of cosolutes which do not interact with the molecules (G-quadruplexes) under investigation. Spectroscopic methodologies, like circular dichroism, UV and fluorescence, have been widely employed to study G-quadruplexes in dilute solution. Here we focus on some aspects that need to be taken into account when employing such techniques in the presence of large amount of a cosolute. Additionally, we discuss possible problems/artifacts that arise in setting experiments in presence of these commonly employed cosolutes and in interpreting the results.     

Second virial coefficients as a measure of protein--osmolyte interactions

The cytoplasm contains high concentrations of cosolutes. These cosolutes include macromolecules and small organic molecules called osmolytes. However, most biophysical studies of proteins are conducted in dilute solutions. Two broad classes of models have been used to describe the interaction between osmolytes and proteins. One class focuses on excluded volume effects, while the other focuses on binding between the protein and the osmolyte. To better understand protein--smolyte interactions, we have conducted sedimentation equilibrium analytical ultracentrifugation experiments using ferricytochrome c as a model protein. From these experiments, we determined the second virial coefficients for a series of osmolytes. We have interpreted the second virial coefficient as a measure of both excluded volume and protein--osmolyte binding. We conclude that simple models are not sufficient to understand the interactions between osmolytes and proteins.     

Influence of stabilizers cosolutes on catalase conformation

The effects of sucrose, mannitol and betaine on the thermodynamic stability and the conformational state of the catalase enzyme were analyzed in order to understand the molecular mechanism whereby the solutes stabilized the enzyme. Catalase was selected as the model enzyme because it is used in several biotechnological processes. In the presence of each cosolute, our data have shown that there was a significant increase in the thermal stability of catalase. A minor stabilization in the enzyme secondary structure were induced by these cosolutes, as circular dichroism in the far UV region has demonstrated. Furthermore, our results support the idea that the overall native structure of catalase becomes more rigid, at least in certain surface areas, in the presence of the assayed stabilizers. This last finding can be reasonably explained by the exclusion mechanism of cosolutes from the protein surface which increases the structured water around this area.     

Effects of anions on the activation thermodynamics and fluorescence emission spectrum of alkaline phosphatase: evidence for enzyme hydration changes during catalysis

The effect of anions on the thermodynamic activation functions for a model enzyme, calf intestinal alkaline phosphatase (EC 3.1.3.1), have been studied in order to examine the role of protein hydration changes in establishing the energetics of enzyme catalysis. The influences of these anions on the activation volume (delta V) and activation free energy (delta G) reflected clear Hofmeister (lyotropic) series effects, in the order F- greater than Cl- greater than Br- greater than I- (order of increasing salting-out potential). A pronounced covariation was observed between the influences of these anions on Vmax, which is proportional to delta G, and on the negative activation volume of the reaction. Fluoride was able to counteract the influences of Br- and I- on both Vmax and delta V when combinations of these anions were employed. The effects of Br- and I- on Vmax and delta V were more pronounced at lower temperatures. The control delta V was increasingly negative at reduced temperatures. The effects of the neutral salts and propanol on delta V and delta G, as well as the effects of salting-in anions on the activation enthalpy and the negative activation entropy of the reaction, are consistent with a model which proposes that peptide groups or polar side chains on the native enzyme exergonically increase their exposure to solvent during the catalytic activation event. These conclusions are in accord with the known free energy, enthalpy, entropy, and volume changes which occur when model peptide groups are transferred between water and concentrated salt solutions. Consistent with the kinetic results, the fluorescence emission wavelength maximum of alkaline phosphatase increased in the presence of anions in the order F- greater than Cl- greater than Br- greater than I-. The salting-out ion (F-) and the salting-in ions (Br- and I-) shifted lambda max in different directions, and these lambda max shifts could be counterbalanced by using equimolar combinations of salting-in and salting-out anions. Control experiments with a model compound, N-acetyltryptophanamide, showed that the spectra shifts caused by the salts did not result solely from differential quenching by the anions of the solvent-exposed tryptophan(s) on the enzyme. Hofmeister additivity phenomena indicated that the solvent is at the basis of these salt-induced enzyme structural changes. It is concluded that changes in protein solvation during enzymic reactions contribute significantly to the thermodynamic activation parameters in both the native and the salt-perturbed enzyme.     

The effects of anions on the solution structure of Na,K-ATPase

Anions interact with protein to induce structural changes at ligand binding sites. The effects of anion complexation include structural stabilization and promote cation-protein interaction. This study was designed to examine the interaction of aspirin and ascorbate anions with the Na+, K+-dependent adenosine triphosphatase (Na,K-ATPase) in H2O and D2O solutions at physiological pH, using anion concentrations of 0.1 microM to 1 mM with final protein concentration of 0.5 to 1 mg/ml. Absorption spectra and Fourier transform infrared (FTIR) difference spectroscopy with its self-deconvolution, second derivative resolution enhancement and curve-fitting procedures were applied to characterize the anion binding mode, binding constant, and the protein secondary structure in the anion-ATPase complexes. Spectroscopic evidence showed that the anion interaction is mainly through the polypeptide C=O and C-N groups with minor perturbation of the lipid moiety. Evidence for this came from major spectral changes (intensity variations) of the protein amide I and amide II vibrations at 1651 and 1550 cm(-1). respectively. The anion-ATPase binding constants were K=6.45 x 10(3) M(-1) for aspirin and K=1.04 x 10(4) M(-1) for ascorbate complexes. The anion interaction resulted in major protein secondary structural changes from that of the alpha-helix 19.8%; beta-pleated sheet 25.6%; turn 9.1%; beta-antiparallel 7.5% and random 38% in the free Na,K-ATPase to that of the alpha-helix 24-26%; beta-pleated 17-18%; turn 8%; beta-antiparallel 5-3% and random 45.0% in the anion-ATPase complexes.     

Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: a calorimetric and spectroscopic study

The effect of methylurea, N,N'-dimethylurea, ethylurea, and butylurea as well as guanidine hydrochloride (GuHCl), urea and pH on the thermal stability, structural properties, and preferential solvation changes accompanying the thermal unfolding of ribonuclease A (RNase A) has been investigated by differential scanning calorimetry (DSC), UV, and circular dichroism (CD) spectroscopy. The results show that the thermal stability of RNase A decreases with increasing concentration of denaturants and the size of the hydrophobic group substituted on the urea molecule. From CD measurements in the near- and far-UV range, it has been observed that the tertiary structure of RNase A melts at about 3 degrees C lower temperature than its secondary structure, which means that the hierarchy in structural building blocks exists for RNase A even at conditions at which according to DSC and UV measurements the RNase A unfolding can be interpreted in terms of a two-state approximation. The far-UV CD spectra also show that the final denatured states of RNase A at high temperatures in the presence of different denaturants including 4.5 M GuHCl are similar to each other but different from the one obtained in 4.5 M GuHCl at 25 degrees C. The concentration dependence of the preferential solvation change delta r23, expressed as the number of cosolvent molecules entering or leaving the solvation shell of the protein upon denaturation and calculated from DSC data, shows the same relative denaturation efficiency of alkylureas as other methods.     

Analysis of thermodynamic non-ideality in terms of protein solvation.

The effects of thermodynamic non-ideality on the forms of sedimentation equilibrium distributions for several isoelectric proteins have been analysed on the statistical-mechanical basis of excluded volume to obtain an estimate of the extent of protein solvation. Values of the effective solvation parameter delta are reported for ellipsoidal as well as spherical models of the proteins, taken to be rigid, impenetrable macromolecular structures. The dependence of the effective solvated radius upon protein molecular mass exhibits reasonable agreement with the relationship calculated for a model in which the unsolvated protein molecule is surrounded by a 0.52-nm solvation shell. Although the observation that this shell thickness corresponds to a double layer of water molecules may be of questionable relevance to mechanistic interpretation of protein hydration, it augurs well for the assignment of magnitudes to the second virial coefficients of putative complexes in the quantitative characterization of protein-protein interactions under conditions where effects of thermodynamic non-ideality cannot justifiably be neglected.     

Identification and modulation of a voltage-dependent anion channel in the plasma membrane of guard cells by high-affinity ligands.

Guard cell anion channels (GCAC1) catalyze the release of anions across the plasma membrane during regulated volume decrease and also seem to be involved in the targeting of the plant growth hormones auxins. We have analyzed the modulation and inhibition of these voltage-dependent anion channels by different anion channel blockers. Ethacrynic acid, a structural correlate of an auxin, caused a shift in activation potential and simultaneously a transient increase in the peak current amplitude, whereas other blockers shifted and blocked the voltage-dependent activity of the channel. Comparison of dose-response curves for shift and block imposed by the inhibitor, indicate two different sites within the channel which interact with the ligand. The capability to inhibit GCAC1 increases in a dose-dependent manner in the sequence: probenecid less than A-9-C less than ethacrynic acid less than niflumic acid less than IAA-94 less than NPPB. All inhibitors reversibly blocked the anion channel from the extracellular side. Channel block on the level of single anion channels is characterized by a reduction of long open transitions into flickering bursts, indicating an interaction with the open mouth of the channel. IAA-23, a structural analog of IAA-94, was used to enrich ligand-binding polypeptides from the plasma membrane of guard cells by IAA-23 affinity chromatography. From this protein fraction a 60 kDa polypeptide crossreacted specifically with polyclonal antibodies raised against anion channels isolated from kidney membranes. In contrast to guard cells, mesophyll plasma membranes were deficient in voltage-dependent anion channels and lacked crossreactivity with the antibody.     

Comparison of atomic solvation parametric sets: applicability and limitations in protein folding and binding.

Atomic solvation parameters (ASP) are widely used to estimate the solvation contribution to the thermodynamic stability of proteins as well as the free energy of association for protein-ligand complexes. They are also included in several molecular mechanics computer programs. In this work, a total of eight atomic solvation parametric sets has been employed to calculate the solvation contribution to the free energy of folding delta Gs for 17 proteins. A linear correlation between delta Gs and the number of residues in each protein was found for each ASP set. The calculations also revealed a great variety in the absolute value and in the sign of delta Gs values such that certain ASP sets predicted the unfolded state to be more stable than the folded, whereas others yield precisely the opposite. Further, the solvation contribution to the free energy of association of helix pairs and to the disassociation of loops (connection between secondary structural elements in proteins) from the protein tertiary structures were computed for each of the eight ASP sets and discrepancies were evident among them.     

An NMR study of anion binding to yeast phosphoglycerate kinase

Anion binding to yeast phosphoglycerate kinase has been investigated using 1H-NMR spectroscopy. The use of anionic paramagnetic probes. [Cr(CN)6]3- and [Fe(CN)6]3-, has enabled the location of the primary anion binding site in the 'basic-patch' region of the amino-terminal domain. The anions interact most closely with Arg-65 and Arg-168. The binding of these and a variety of other anions to this site is directly competitive with the binding of the substrate, 3-phosphoglycerate. Binding of 3-phosphoglycerate and 1.3-bisphosphoglycerate is, however, stronger than expected on the basis of anionic charge and causes conformational changes in the protein not seen with any of the other simple spherical anions investigated. This must be part, at least, of the substrate specificity. Evidence for a secondary anion binding site involving the side chains of surface lysine residues is also presented. It is suggested that the primary anion site is responsible for the observed activation by anions at low concentrations while the secondary site leads to inhibition at higher anion concentrations. The kinetics fit these deductions and a scheme for kinase activity is presented.     

Thermal stability of proteins in the presence of poly(ethylene glycols)

Thermal unfolding of ribonuclease, lysozyme, chymotrypsinogen, and beta-lactoglobulin was studied in the absence or presence of poly(ethylene glycols). The unfolding curves were fitted to a two-state model by a nonlinear least-squares program to obtain values of delta H, delta S, and the melting temperature Tm. A decrease in thermal transition temperature was observed in the presence of poly(ethylene glycol) for all of the protein systems studied. The magnitude of such a decrease depends on the particular protein and the molecular size of poly(ethylene glycol) employed. A linear relation can be established between the magnitude of the decrease in transition temperature and the average hydrophobicity of these proteins; namely, the largest observable decrease is associated with the protein of the highest hydrophobicity. Further analysis of the thermal unfolding data reveals that poly(ethylene glycols) significantly effect the relation between delta H degrees of unfolding and temperature for all the proteins studied. For beta-lactoglobulin, a plot of delta H versus Tm indicates a change in slope from a negative to a positive value, thus implying a change in delta Cp in thermal unfolding caused by the presence of poly(ethylene glycols). Results from solvent-protein interaction studies indicate that at high temperature poly(ethylene glycol) 1000 preferentially interacts with the denatured state of protein but is excluded from the native state at low temperature. These observations are consistent with the fact that poly(ethylene glycols) are hydrophobic in nature and will interact favorably with the hydrophobic side chains exposed upon unfolding; thus, it leads to a lowering of thermal transition temperature.     

Assembly-dependent conformational changes in a viral capsid protein. Calorimetric comparison of successive conformational states of the gp23 surface lattice of bacteriophage T4

Inter- and intra-subunit bonding within the surface lattice of the capsid of bacteriophage T4 has been investigated by differential scanning calorimetry of polyheads, in conjunction with electron microscopy, limited proteolysis and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The bonding changes corresponding to successive stages of assembly of the major capsid protein gp23, including its maturation cleavage, were similarly characterized. The uncleaved/unexpanded surface lattice exhibits two endothermic transitions. The minor event, at 46 degrees C, does not visibly affect the surface lattice morphology and probably represents denaturation of the N-terminal domain of gp23. The major endotherm, at 65 degrees C, represents denaturation of the gp23 polymers. Soluble gp23 from dissociated polyheads is extremely unstable and exhibits no endotherm. Cleavage of gp23 to gp23* and the ensuing expansion transformation effects a major stabilization of the surface lattice of polyheads, with single endotherms whose melting temperatures (t*m) range from 73 to 81 degrees C, depending upon the mutant used and the fraction of gp23 that is cleaved to gp23* prior to expansion. Binding of the accessory proteins soc and hoc further modulates the thermograms of cleaved/expanded polyheads, and their effects are additive. hoc binding confers a new minor endotherm at 68 degrees C corresponding to at least partial denaturation of hoc. Denatured hoc nevertheless remains associated with the surface lattice, although in an altered, protease-sensitive state which correlates with delocalization of hoc subunits visualized in filtered images. While hoc binding has little effect on the thermal stability of the gp23* matrix, soc binding further stabilizes the surface lattice (delta Hd approximately +50%; delta t*m = +5.5 degrees C). It is remarkable that in all states of the surface lattice, the inter- and intra-subunit bonding configurations of gp23 appear to be co-ordinated to be of similar thermal stability. Thermodynamically, the expansion transformation is characterized by delta H much less than 0; delta Cp approximately 0, suggesting enhancement of van der Waals' and/or H-bonding interactions, together with an increased exposure to solvent of hydrophobic residues of gp23* in the expanded state. These findings illuminate hypotheses of capsid assembly based on conformational properties of gp23: inter alia, they indicate a role for the N-terminal portion of gp23 in regulating polymerization, and force a reappraisal of models of capsid swelling based on the swivelling of conserved domains.     

The effect of solvated ions on the thermal denaturation of human serum albumin in water-dimethylsulfoxide solutions

The effects of solvated ions on the thermal denaturation of human serum albumin (HAS) in water-dimethylsulfoxide (DMSO) solutions were studied by the method of electron absorption spectroscopy. It was shown that depending on the DMSO concentration, electrolytes (LiCl, LiNO₃, LiClO₄, NaCl, and NaNO₃) contained in these solutions were characterized by different anion and cation solvation degrees: unlike cations, anions were only negligibly solvated, which affected HAS thermal denaturation. Electrostatic interactions between anions and positively charged amino acid residues supporting protein denaturation subsided in the line Cl⁻ > NO₃⁻ > ClO₄⁻.     

Hofmeister effects on activity and stability of alkaline phosphatase

We have studied the effects on alkaline phosphatase of adding high concentrations (normally 1.0 M) of simple salts. It is necessary to allow for significant effects of salts on the extinction coefficient of the reaction product, and on the apparent pH of the buffer. Both activity and stability of the enzyme correlate well with the Hofmeister series in terms of the salt's kosmotropic/chaotropic properties, which are assessed by the Jones–Dole viscosity B coefficients (B₊ for cations and B₋ for anions). The catalytic activity or V_max/K_m of the enzyme showed a bell-shaped relationship with the (B₋ − B₊) values of the salts present, being optimal with salts (such as NaCl, KCl, and KNO₃) where the anion and cation have similar kosmotropic/chaotropic properties. This effect is believed to be enzyme-specific and relates to the impact of both cations and anions on the enzyme's surface pH, active site, and catalytic mechanism. Anions play a more predominant role than cations in affecting enzyme stability. The rate of irreversible thermal inactivation is strongly reduced by addition of kosmotropic anions like SO₄²⁻ (half-life increased from 8 to 580 min at 60 °C). This effect is general and the mechanism probably involves the ability of the ions to affect the water solvation layer around the enzyme molecule and to interact with both the surface and internal structure of the enzyme.     

Excluded volume in solvation: sensitivity of scaled-particle theory to solvent size and density

Changes in solvent environment greatly affect macromolecular structure and stability. To investigate the role of excluded volume in solvation, scaled-particle theory is often used to calculate delta G(tr)(ev), the excluded-volume portion of the solute transfer free energy, delta G(tr). The inputs to SPT are the solvent radii and molarities. Real molecules are not spheres. Hence, molecular radii are not uniquely defined and vary for any given species. Since delta G(tr)(ev) is extremely sensitive to solvent radii, uncertainty in these radii causes a large uncertainty in delta G(tr)(ev)-several kcal/mol for amino acid solutes transferring from water to aqueous mixtures. This uncertainty is larger than the experimental delta G(tr) values. Also, delta G(tr)(ev) can be either positive or negative. Adding neutral crowding molecules may not necessarily reduce solubility. Lastly, delta G(tr)(ev) is very sensitive to solvent density, rho. A few percent error in rho may even cause qualitative deviations in delta G(tr)(ev). For example, if rho is calculated by assuming the hard-sphere pressure to be constant, then delta G(tr)(ev) values and uncertainties are now only tenths of a kcal/mol and are positive. Because delta G(tr)(ev) values calculated by scaled-particle theory are strongly sensitive to solvent radii and densities, determining the excluded-volume contribution to transfer free energies using SPT may be problematic.