Influence of the Hofmeister anions on protein stability as studied by thermal denaturation and chemical shift perturbation

The influence of external cosolutes on the thermal stability of the B1 domain of protein L (ProtL) has been studied by circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. The thermal denaturation midpoint is effectively modulated by the addition of a suite of anions and follows the Hofmeister series. The maximum increase in thermostability (corresponding to 14 degrees C) was observed in the presence of 1 M sodium sulfate. After conversion of the experimental data into the change in the virial coefficient, a mechanistic model was used to estimate the relative contributions from excluded volume and preferential anion solvation for each anion. As expected, the excluded volume term stabilizes the native conformation of ProtL for all the cosolutes, but opposite effects on protein stability arise from the anion's solvation depending on their tendency to interact with or to become excluded from the protein surface. This behavior is in agreement with the results of independent NMR experiments: the anions that strongly interact with the protein surface produce significant perturbations in the amide protein chemical shift (delta d23(HN)). A correlation obtained between delta d23(HN) and the temperature coefficients for the different amide protons provides qualitative information about the structural determinants for the interaction between the protein surface and the cosolute.     

Effects of denaturants on amide proton exchange rates: a test for structure in protein fragments and folding intermediates

A method for detecting structure in marginally stable forms of a protein is described. The principle is to measure amide proton exchange rates in the absence and presence of varying concentrations of a denaturant. Unfolding of structure by the denaturant is reflected by an acceleration of amide proton exchange rates, after correction for the effects of the denaturant on the intrinsic rate of exchange. This exchange-rate test for structure makes no assumptions about the rate of exchange in the unfolded state. The effects of 0-8 M urea and 0-6 M guanidinium chloride (GdmCl) on acid- and base-catalyzed exchange from model compounds have been calibrated. GdmCl does not appear to be well-suited for use in the exchange-rate test; model compound studies show that the effects of GdmCl on intrinsic exchange rates are complicated. In contrast, the effects of urea are a more uniform function of denaturant concentration. Urea increases acid-catalyzed, and decreases base-catalyzed, rates in model compounds. The exchange-rate test is used here to study structure formation in the S-protein (residues 21-124 of ribonuclease A). In conditions where an equilibrium folding intermediate of S-protein (I3) is known to be populated (pH 1.7, 0 degree C), the exchange-rate test for structure is positive. At higher temperatures (greater than 32 degrees C) I3 is unfolded, but circular dichroism data suggest that residual structure remains [Labhardt, A. M. (1982) J. Mol. Biol. 157, 357-371].(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of viruses using novel protein A wash buffers

Low pH viral inactivation is typically performed in the eluate pool following the protein A capture step during the manufacturing of monoclonal antibodies and Fc‐fusion proteins. However, exposure to low pH has the potential to alter protein quality. To avoid these difficulties, novel wash buffers capable of inactivating viruses while antibodies or Fc‐fusion proteins were bound to protein A or mixed mode resins were developed. By equilibrating the column in high salt buffer (2 M ammonium sulfate or 3 M sodium chloride) after loading, the hydrophobic interactions between antibodies and protein A ligands were increased enough to prevent elution at pH 3. The ammonium sulfate was also found to cause binding of an antibody to a mixed mode cation exchange and a mixed mode anion exchange resin at pH values that caused elution in conventional cation and anion exchange resins (pH 3.5 for Capto Adhere and pH 8.0 for Capto MMC), indicating that retention was due to enhanced hydrophobic interactions. The potential of the 2 M ammonium sulfate pH 3 buffer, a 1 M arginine buffer, and a buffer containing the detergent LDAO to inactivate XMuLV virus when used as protein A wash buffers with a 1 hour contact time were studied. The high salt and detergent containing wash buffers provided about five logs of removal, determined using PCR, and complete combined removal and inactivation (> 6 logs), determined by measuring infectivity. The novel protein A washes could provide more rapid, automated viral inactivation steps with lower pool conductivities. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:406–413, 2015     

Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes

Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutions of ammonium sulfate, sodium chloride, potassium isothiocyanate, and potassium chloride. The two-body interactions between ovalbumin molecules in concentrated ammonium-sulfate solutions can be described by the DLVO potentials plus a potential that accounts for the decrease in free volume available to the protein due to the presence of the salt ions. The interaction between ovalbumin and ammonium sulfate is unfavorable, reflecting the kosmotropic nature of sulfate anions. Lysozyme-lysozyme interactions cannot be described by the above potentials because anion binding to lysozyme alters these interactions. Lysozyme-isothiocyanate complexes are strongly attractive due to electrostatic interactions resulting from bridging by the isothiocyanate ion. Lysozyme-lysozyme interactions in sulfate solutions are more repulsive than expected, possibly resulting from a larger excluded volume of a lysozyme-sulfate bound complex or perhaps, hydration forces between the lysozyme-sulfate complexes.     

The use of spin desalting columns in DMSO‐quenched H/D‐exchange NMR experiments

Dimethylsulfoxide (DMSO)‐quenched hydrogen/deuterium (H/D)‐exchange is a powerful method to characterize the H/D‐exchange behaviors of proteins and protein assemblies, and it is potentially useful for investigating non‐protected fast‐exchanging amide protons in the unfolded state. However, the method has not been used for studies on fully unfolded proteins in a concentrated denaturant or protein solutions at high salt concentrations. In all of the current DMSO‐quenched H/D‐exchange studies of proteins so far reported, lyophilization was used to remove D₂O from the protein solution, and the lyophilized protein was dissolved in the DMSO solution to quench the H/D exchange reactions and to measure the amide proton signals by two‐dimensional nuclear magnetic resonance (2D NMR) spectra. The denaturants or salts remaining after lyophilization thus prevent the measurement of good NMR spectra. In this article, we report that the use of spin desalting columns is a very effective alternative to lyophilization for the medium exchange from the D₂O buffer to the DMSO solution. We show that the medium exchange by a spin desalting column takes only about 10 min in contrast to an overnight length of time required for lyophilization, and that the use of spin desalting columns has made it possible to monitor the H/D‐exchange behavior of a fully unfolded protein in a concentrated denaturant. We report the results of unfolded ubiquitin in 6.0M guanidinium chloride.     

Erythrocyte bisulfite transport

The wide range of transport rates for anions of differing chemical structure by the human erythrocyte anion transport protein (Band 3 protein) suggests that this protein is highly selective for anions that chemically resemble its natural substrate bicarbonate. To test this hypothesis, the influx of bisulfite (HSO3-), a bicarbonate analog, was compared to influxes of chloride, sulfate, and bicarbonate, as measured by the technique of colloid osmotic lysis in isotonic ammonium salt solution. The lysis time induced in chloride solution (much greater than 10 min) was markedly accelerated to 0.6 min by the addition of small amounts (5 mM) of bicarbonate, an effect characteristic of colloid osmotic lysis induced by the anion transport pathway. Lysis in bicarbonate solution was extremely rapid (0.09 min), and was markedly inhibited by acetazolamide (2.9 min). Lysis in bisulfite solution occurred spontaneously (2.2 min) but was markedly accelerated to a time similar to that of chloride (0.56 min) by addition of 5 mM bicarbonate. In contrast, sulfate induced lysis was extremely slow (less than 10% lysis at 40 min in the presence of bicarbonate). Preincubation of erythrocytes with SITS, an inhibitor of anion exchange, prevented lysis by chloride, but had no effect on lysis by bicarbonate, indicating that lysis by bicarbonate was predominantly through diffusion and not anion transport. SITS treatment of erythrocytes eliminated the catalytic effect of bicarbonate during lysis by bisulfite, indicating that anion transport of bisulfite and diffusion of the conjugate acid in the form of SO2 both contribute to the total membrane flux. When the contribution of diffusion is taken into account, the rate of bisulfite influx through the anion exchange pathway is at least 100-fold faster than that for sulfate.     

The pH dependence of hydrogen exchange in proteins

The static accessibility modified discrete charge model for electrostatic interactions in proteins is extended to the prediction of the pH dependence of hydrogen exchange reactions. The exchange rate profiles of buried amide protons are shown to follow the calculated pH dependence of the electrostatic component of protein stability. Rate profiles are calculated for individual buried amide protons in ribonuclease S and bovine pancreatic trypsin inhibitor. The electrostatic free energy of stabilization of the protein and the energy required to bring the catalytic ion to an exchange site are expressed as an apparent, pH-dependent contribution to the activation energy. Changes in the electrostatic stabilization of the proteins affect the calculated exchange rate for buried amide protons by more than 1000, while local field effects raise or lower the predicted exchange rates by less than 100. The pH dependence of exchangeable protons at the protein surface, such as the C-2 imidazole protons, is shown to follow the estimated energy required to introduce the catalytic ion at the exchange site. These calculations are discussed in terms of current models for proton exchange which incorporate the dynamic nature of the structure to explain exchange data from the interior of a protein.     

Protein free energy landscapes remodeled by ligand binding

Glucose/galactose binding protein (GGBP) functions in two different larger systems of proteins used by enteric bacteria for molecular recognition and signaling. Here we report on the thermodynamics of conformational equilibrium distributions of GGBP. Three fluorescence components appear at zero glucose concentration and systematically transition to three components at high glucose concentration. Fluorescence anisotropy correlations, fluorescent lifetimes, thermodynamics, computational structure minimization, and literature work were used to assign the three components as open, closed, and twisted conformations of the protein. The existence of three states at all glucose concentrations indicates that the protein continuously fluctuates about its conformational state space via thermally driven state transitions; glucose biases the populations by reorganizing the free energy profile. These results and their implications are discussed in terms of the two types of specific and nonspecific interactions GGBP has with cytoplasmic membrane proteins.     

Amide proton hydrogen exchange rates for sperm whale myoglobin obtained from 15N-1H NMR spectra

The hydrogen exchange behavior of exchangeable protons in proteins can provide important information for understanding the principles of protein structure and function. The positions and exchange rates of the slowly-exchanging amide protons in sperm whale myoglobin have been mapped using 15N-1H NMR spectroscopy. The slowest-exchanging amide protons are those that are hydrogen bonded in the longest helices, including members of the B, E, and H helices. Significant protection factors were observed also in the A, C, and G helices, and for a few residues in the D and F helices. Knowledge of the identity of slowly-exchanging amide protons forms the basis for the extensive quench-flow kinetic folding experiments that have been performed for myoglobin, and gives insights into the tertiary interactions and dynamics in the protein.     

Fluctuations and the Hofmeister effect

The Hofmeister effect consists in changes of protein solubility triggered by neutral electrolyte cosolutes. Based on the assumption that salts cause stochastic fluctuations of the free energy barrier profiles, a kinetic theory of this phenomenon is proposed. An exponentially correlated noise, of amplitude proportional to the salt concentration, is added to each energy level, and the time-dependence of the mean protein concentration is calculated. It is found that the theory yields the well-known Setschenow equation if the noise correlation time is low in comparison to the aggregation time constant. Experimental data on salting-in agents are well fitted, whereas, in the case of salting-out cosolutes, two independent dichotomic fluctuations are needed to fit the data. This may result from the fact that, in both cases, the low-concentration regime is dominated by salting-in electrostatic contributions, whereas, at high salt concentrations, electron donor/acceptor interactions become important; these have opposite effects. The theory offers a novel way to metricate Hofmeister effects and also leads to thermodynamic quantities, which account for the influence of salts. The formalism may also be applied to describe kinetic phenomena in the presence of cosolutes.     

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.     

GuHCl and NaCl-dependent hydrogen exchange in MerP reveals a well-defined core with an unusual exchange pattern

We have analysed hydrogen exchange at amide groups to characterise the energy landscape of the 72 amino acid residue protein MerP. From the guanidine hydrochloride (GuHCl) dependence of exchange in the pre-transitional region we have determined free energy values of exchange (DeltaG(HX)) and corresponding m-values for individual amide protons. Detailed analysis of the exchange patterns indicates that for one set of amide protons there is a weak dependence on denaturant, indicating that the exchange is dominated by local fluctuations. For another set of amide protons a linear, but much stronger, denaturant dependence is observed. Notably, the plots of free energy of exchange versus [GuHCl] for 16 amide protons show pronounced upward curvature, and a close inspection of the structure shows that these residues form a well-defined core in the protein. The hydrogen exchange that was measured at various concentrations of NaCl shows an apparent selective stabilisation of this core. Detailed analysis of this exchange pattern indicates that it may originate from selective destabilisation of the unfolded state by guanidinium ions and/or selective stabilisation of the core in the native state by chloride ions.     

Tracking lysozyme unfolding during salt-induced precipitation with hydrogen exchange and mass spectrometry

We utilized electrospray ionization mass spectrometry (ESI-MS) and hydrogen-deuterium exchange (HX) to detect unfolding of hen egg white lysozyme during salt-induced precipitation. Deuterated lysozyme was dissolved in protonated buffer at pH 2.16 and precipitated with ammonium sulfate, sodium chloride, and potassium thiocyanate. ESI-MS was used to detect mass differences in lysozyme due to the loss of deuterons for solvent protons, providing insight on the conformational history of the protein during the labeling experiment. Precipitation with ammonium sulfate and sodium chloride did not unfold lysozyme, consistent with the known stabilizing effects of kosmotropic salts. Potassium thiocyanate, an aggressive chaotrope, was an effective precipitant at 0.2 M, but also disrupted lysozyme structure and caused the formation of precipitate fractions that did not readily redissolve into aqueous solution without the use of a chemical denaturant. Precipitation with 1.0 M thiocyanate resulted in faster rates of unfolding and larger amounts of the insoluble precipitate. The unfolding kinetics were biphasic, exhibiting a slow phase after a few hours that presumably reflected a smaller propensity for lysozyme to unfold in the precipitated state. Bimodal mass distributions in the ESI-MS spectra for the thiocyanate precipitates indicate two states for lysozyme in this system, a native and a molten globule-like partially unfolded state. ESI-MS analysis of the insoluble precipitates indicated that they consisted primarily of protein molecules that had unfolded. Investigation of the HX behavior of lysozyme in a KSCN solution at low protein concentrations confirmed the destabilizing effect of the salt on the protein structure, even when there was almost no solid phase present. The HX/ESI-MS results provide insight into the mechanism combining precipitation and denaturation for such a system, both in terms of obtaining quantitative kinetic and stability information and the identification of the conformers present.     

Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: implications for redox potential regulation and flavodoxin folding.

As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly wound protein Che Y.     

Rapid Analysis of Protein Structure and Dynamics by Hydrogen/Deuterium Exchange Mass Spectrometry

An automated approach for the rapid analysis of protein structure has been developed and used to study acid-induced conformational changes in human growth hormone. The labeling approach involves hydrogen/deuterium exchange (H/D-Ex) of protein backbone amide hydrogens with rapid and sensitive detection by mass spectrometry (MS). Briefly, the protein is incubated for defined intervals in a deuterated environment. After rapid quenching of the exchange reaction, the partially deuterated protein is enzymatically digested and the resulting peptide fragments are analyzed by liquid chromatography mass spectrometry (LC-MS). The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. Additional analyses allow mapping of the free energy of folding on localized segments along the protein sequence affording unique dynamic and structural information. While amide H/D-Ex coupled with MS is recognized as a powerful technique for studying protein structure and protein–ligand interactions, it has remained a labor-intensive task. The improvements in the amide H/D-Ex methodology described here include solid phase proteolysis, automated liquid handling and sample preparation, and integrated data reduction software that together improve sequence coverage and resolution, while achieving a sample throughput nearly 10-fold higher than the commonly used manual methods.     

A globular protein with slower amide proton exchange from an alpha helix than from antiparallel beta sheets

In proteinase inhibitor IIA from bull seminal plasma, which is a small globular protein with 57 amino acid residues, measurements of individual amide proton exchange rates by two-dimensional correlated 1H NMR spectroscopy (COSY) showed that the exchange was slowest for some hydrogen bonded amide groups in an alpha-helix. This contrasts with all other proteins which were so far studied in detail, where the slowest exchange rates were observed for hydrogen bonded amide protons in antiparallel beta-sheets.     

Equilibrium hydrogen exchange reveals extensive hydrogen bonded secondary structure in the on-pathway intermediate of Im7

The four-helical immunity protein Im7 folds through an on-pathway intermediate that has a specific, but partially misfolded, hydrophobic core. In order to gain further insight into the structure of this species, we have identified the backbone hydrogen bonds formed in the ensemble by measuring the amide exchange rates (under EX2 conditions) of the wild-type protein and a variant, I72V. In this mutant the intermediate is significantly destabilised relative to the unfolded state (deltadeltaG(ui) = 4.4 kJ/mol) but the native state is only slightly destabilised (deltadeltaG(nu) = 1.8 kJ/mol) at 10 degrees C in 2H2O, pH* 7.0 containing 0.4 M Na2SO4, consistent with the view that this residue forms significant non-native stabilising interactions in the intermediate state. Comparison of the hydrogen exchange rates of the two proteins, therefore, enables the state from which hydrogen exchange occurs to be identified. The data show that amides in helices I, II and IV in both proteins exchange slowly with a free energy similar to that associated with global unfolding, suggesting that these helices form highly protected hydrogen-bonded helical structure in the intermediate. By contrast, amides in helix III exchange rapidly in both proteins. Importantly, the rate of exchange of amides in helix III are slowed substantially in the Im7* variant, I72V, compared with the wild-type protein, whilst other amides exchange more rapidly in the mutant protein, in accord with the kinetics of folding/unfolding measured using chevron analysis. These data demonstrate, therefore, that local fluctuations do not dominate the exchange mechanism and confirm that helix III does not form stable secondary structure in the intermediate. By combining these results with previously obtained Phi-values, we show that the on-pathway folding intermediate of Im7 contains extensive, stable hydrogen-bonded structure in helices I, II and IV, and that this structure is stabilised by both native and non-native interactions involving amino acid side-chains in these helices.     

Stopped-flow NMR measurement of hydrogen exchange rates in reduced horse cytochrome c under strongly destabilizing conditions

A procedure to measure exchange rates of fast exchanging protein amide hydrogens by time-resolved NMR spectroscopy following in situ initiation of the reaction by diluting a native protein solution into an exchanging deuterated buffer is described. The method has been used to measure exchange rates of a small set of amide hydrogens of reduced cytochrome c, maintained in a strictly anaerobic atmosphere, in the presence of an otherwise inaccessible range of guanidinium deuterochloride concentrations. The results for the measured protons indicate that hydrogen exchange in the unfolding transition region of cytochrome c reach the EX2 limit, but emphasize the difficulty in interpretation of the exchange mechanism in protein hydrogen exchange studies. Comparison of free energies of structure opening for the measured hydrogens with the global unfolding free energy monitored by far-UV CD measurements has indicated the presence of at least one partially unfolded equilibrium species of reduced cytochrome c. The results provide the first report of measurement of free energy of opening of structure to exchange in the 0–2-kcal/mol range. Proteins 32:241–247, 1998. © 1998 Wiley-Liss, Inc.