Evidence for increased local flexibility in psychrophilic alcohol dehydrogenase relative to its thermophilic homologue

The psychrophilic alcohol dehydrogenase (psADH) cloned from Antarctic Moraxella sp. TAE123 exhibits distinctive catalytic parameters in relation to the homologous thermophilic alcohol dehydrogenase (htADH) from Bacillus stearothermophilus LLD-R. Amide hydrogen-deuterium (H/D) exchange studies using Fourier-transformed infrared (FTIR) spectroscopy and mass spectrometry (MS) were conducted to investigate whether the differences are caused by variation in either global or regional protein flexibility. The FTIR H/D exchange study suggested that psADH does not share similar global flexibility with htADH at their physiologically relevant temperatures as has been predicted by the "corresponding state" hypothesis. However, the MS H/D exchange study revealed a more complicated picture concerning the flexibility of the two homologous enzymes. Analysis of the deuteration and exchange rates of protein-derived peptides suggested that only some functionally important regions in psADH that are involved in substrate and cofactor binding exhibit greater flexibility compared to htADH at low temperature (10 degrees C). These observations strongly suggest that variable conformational flexibility between the two protein forms is a local phenomenon, and that global H/D exchange measurement by FTIR can be misleading and should be used with discretion. These results are supportive of the idea that functionally important local flexibility can be uncoupled from global thermal stability. The structural factors underlying the differences in local protein flexibility and catalysis between htADH and psADH are discussed in conjunction with results from crystallographic and fluorescence spectroscopy studies.     

Kinetics of unfolding and folding from amide hydrogen exchange in native ubiquitin

Amide hydrogen (NH) exchange is one of the few experimental techniques with the potential for determining the thermodynamics and kinetics of conformational motions at nearly every residue in native proteins. Quantitative interpretation of NH exchange in terms of molecular motions relies on a simple two-state kinetic model: at any given slowly exchanging NH, a closed or exchange-incompetent conformation is in equilibrium with an open or exchange-competent conformation. Previous studies have demonstrated the accuracy of this model in measuring conformational equilibria by comparing exchange data with the thermodynamics of protein unfolding. We report here a test of the accuracy of the model in determining the kinetics of conformational changes in native proteins. The kinetics of folding and unfolding for ubiquitin have been measured by conventional methods and compared with those derived from a comprehensive analysis of the pH dependence of exchange in native ubiquitin. Rate constants for folding and unfolding from these two very different types of experiments show good agreement. The simple model for NH exchange thus appears to be a robust framework for obtaining quantitative information about molecular motions in native proteins.     

Investigation of the structural stability of the human acidic fibroblast growth factor by hydrogen-deuterium exchange

The conformational stability of the human acidic fibroblast growth factor (hFGF-1) is investigated using amide proton exchange and temperature-dependent chemical shifts, monitored by two-dimensional NMR spectroscopy. The change in free energy of unfolding (DeltaG(u)) of hFGF-1 is estimated to be 5.00 +/- 0.09 kcal.mol(-)(1). Amide proton-exchange rates of 74 residues (in hFGF-1) have been unambiguously measured, and the exchange process occurs predominately according to the conditions of the EX2 limit. The exchange rates of the fast-exchanging amide protons exposed to the solvent have been measured using the clean SEA-HSQC technique. The amide proton protection factor and temperature coefficient estimates show reasonably good correlation. Residues in beta-strands II and VI appear to constitute the stability core of the protein. Among the 12 beta-strands constituting the beta-barrel architecture of hFGF-1, beta-strand XI, located in the heparin binding domain, exhibits the lowest average protection factor value. Amide protons involved in the putative folding nucleation site in hFGF-1, identified by quench-flow NMR studies, do not represent the slow-exchanging core. Residues in portions of hFGF-1 experiencing high conformational flexibility mostly correspond to those involved in receptor recognition and binding.     

Proton nuclear magnetic resonance measurement of the amide hydrogen exchange rates of group A streptococcal polysaccharide in H2O

The solvent exchange rates of the acetamido hydrogen of the 2-acetamido-2-deoxy-beta-D-glucopyranosyl unit of group A streptococcal polysaccharide dissolved in H2O have been measured and compared with the corresponding exchange rates in the solvated model compound 1-O-methyl-2-acetamido-2-deoxy-beta-D-glucopyranoside. Amide hydrogen exchange rates were measured at 25 degrees C over a wide pH range by a combination of two separate NMR techniques: the transfer of solvent saturation and the amide hydrogen saturation recovery NMR experiments. The data indicate that the acetamido hydrogen essentially exists in a solvated environment and that its contribution to the conformational stability of this polysaccharide through intramolecular hydrogen bonding is negligible.     

Ribonuclease Sa conformational stability studied by NMR-monitored hydrogen exchange

The conformational stability of ribonuclease Sa (RNase Sa) has been measured at the per-residue level by NMR-monitored hydrogen exchange at pH* 5.5 and 30 degrees C. In these conditions, the exchange mechanism was found to be EXII. The conformational stability calculated from the slowest exchanging amide groups was found to be 8.8 kcal/mol, in close agreement with values determined by spectroscopic methods. RNase Sa is curiously rich in acidic residues (pI = 3.5) with most basic residues being concentrated in the active-site cleft. The effects of dissolved salts on the stability of RNase Sa was studied by thermal denaturation experiments in NaCl and GdmCl and by comparing hydrogen exchange rates in 0.25 M NaCl to water. The protein was found to be stabilized by salt, with the magnitude of the stabilization being influenced by the solvent exposure and local charge environment at individual amide groups. Amide hydrogen exchange was also measured in 0.25, 0.50, 0.75, and 1.00 M GdmCl to characterize the unfolding events that permit exchange. In contrast to other microbial ribonucleases studied to date, the most protected, globally exchanging amides in RNase Sa lie not chiefly in the central beta strands but in the 3/10 helix and an exterior beta strand. These structural elements are near the Cys7-Cys96 disulfide bond.     

Non-covalent interactions between Fab and Fc regions in immunoglobulin G molecules. Hydrogen-deuterium exchange studies

To reveal non-covalent interactions between the Fab and Fc regions of IgG molecules the average conformational free-energy change (delta Go), associated with reversible micro-unfoldings, was measured by hydrogen-deuterium exchange for the Fab and Fc fragments and the complete molecule. Human monoclonal IgG1 and pooled IgG samples were used in these experiments. Hydrogen-deuterium exchange data were summarized and compared in the form of exchange relaxation spectra. The experimentally observed relaxation spectrum of intact IgG could not be deduced by weighted summation of spectra measured for Fab and Fc fragments. A comparison of the measured and calculated data revealed a 5-kJ/mol increase in the conformational free energy upon splitting the IgG molecule into two Fab and Fc pieces, i.e. an increase of conformational mobility occurred. This change can be explained either by related fluctuation patterns of the Fab and Fc pieces in the intact molecule or by a shielding effect on the contact surfaces. Both interpretations suppose non-covalent interactions between Fab and Fc that can be a means of information transduction between recognition and effector sites. The pH dependence of the hydrogen-deuterium exchange also indicates interactions between the Fab and Fc regions. A shift in the relaxation spectra of the Fab fragment was observed between pH 8.2 and 7.3 revealing destabilization of the structure at lower pH. This effect is absent in the intact molecule, reflecting interactions that stabilize the Fab structure. Comparison of the relaxation spectra of Fab and Fc shows a difference of about 10 kJ/mol in the microstability of these fragments: the Fab part possesses more conformational flexibility (i.e. its microstability is smaller) than the Fc part.     

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

This study compares the local conformational dynamics and physical stability of an IgG1 mAb (mAb-A) with its corresponding YTE (M255Y/S257T/T259E) mutant (mAb-E), which was engineered for extended half-life in vivo. Structural dynamics was measured using hydrogen/deuterium (H/D) exchange mass spectrometry while protein stability was measured with differential scanning calorimetry (DSC) and size exclusion chromatography (SEC). The YTE mutation induced differences in H/D exchange kinetics at both pH 6.0 and 7.4. Segments covering the YTE mutation sites and the FcRn binding epitopes showed either subtle or no observable differences in local flexibility. Surprisingly, several adjacent segments in the C_H2 and distant segments in the V_H, C_H1, and V_L domains had significantly increased flexibility in the YTE mutant. Most notable among the observed differences is increased flexibility of the 244–254 segment of the C_H2 domain, where increased flexibility has been shown previously to correlate with decreased conformational stability and increased aggregation propensity in other IgG1 mAbs (e.g., presence of destabilizing additives as well as upon de-glycosylation or methionine oxidation). DSC analysis showed decreases in both thermal onset (T_onset) and unfolding (T_m1) temperatures of 7°C and 6.7°C, respectively, for the C_H2 domain of the YTE mutant. In addition, mAb-E aggregated faster than mAb-A under accelerated stability conditions as measured by SEC analysis. Hence, the relatively lower physical stability of the YTE mutant correlates with increased local flexibility of the 244–254 segment, providing a site-directed mutant example that this segment of the C_H2 domain is an aggregation hot spot in IgG1 mAbs.     

The role of interchain disulphide bridges in the conformational stability of human immunoglobulin G1 subclass. Hydrogen-deuterium exchange studies.

The hydrogen-deuterium exchange data of human immunoglobulin G1 (IgG1) are interpreted by assuming fast fluctuations of the protein conformation, through which the peptide groups become exposed to the solvent. The probability of solvent exposure of peptide hydrogens reflects a rather loose conformation for native IgG in comparison with other globular proteins. The probability of solvent exposure is greater than 10(-3) for half of the peptide groups, which shows that the conformational transitions by which these groups are exposed to the solvent are accompanied by changes in standard free energy less than 17 kJ/mol (4 kcal/mol). In the range of pH 6.2-8.45, at 25 degrees C no gross conformational changes are reflected in the hydrogen-deuterium exchange behaviour of the native, the reduced-nonalkylated-reassociated and the reduced-S-alkylated-reassociated IgG1. No difference could be detected in the conformational stability of the native and reoxidised reassociated IgG1 proteins. The lack of inter-subunit disulphide bridges in S-alkylated-reassociated molecules results in an increased conformational motility. This destabilization of protein conformation affects about 90% of the peptide groups covered by the measurements, and corresponds to changes in standard free energy of 8 kJ/mol on the average.     

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

This study compares the local conformational dynamics and physical stability of an IgG1 mAb (mAb-A) with its corresponding YTE (M255Y/S257T/T259E) mutant (mAb-E), which was engineered for extended half-life in vivo. Structural dynamics was measured using hydrogen/deuterium (H/D) exchange mass spectrometry while protein stability was measured with differential scanning calorimetry (DSC) and size exclusion chromatography (SEC). The YTE mutation induced differences in H/D exchange kinetics at both pH 6.0 and 7.4. Segments covering the YTE mutation sites and the FcRn binding epitopes showed either subtle or no observable differences in local flexibility. Surprisingly, several adjacent segments in the C_H2 and distant segments in the V_H, C_H1, and V_L domains had significantly increased flexibility in the YTE mutant. Most notable among the observed differences is increased flexibility of the 244–254 segment of the C_H2 domain, where increased flexibility has been shown previously to correlate with decreased conformational stability and increased aggregation propensity in other IgG1 mAbs (e.g., presence of destabilizing additives as well as upon de-glycosylation or methionine oxidation). DSC analysis showed decreases in both thermal onset (T_onset) and unfolding (T_m1) temperatures of 7°C and 6.7°C, respectively, for the C_H2 domain of the YTE mutant. In addition, mAb-E aggregated faster than mAb-A under accelerated stability conditions as measured by SEC analysis. Hence, the relatively lower physical stability of the YTE mutant correlates with increased local flexibility of the 244–254 segment, providing a site-directed mutant example that this segment of the C_H2 domain is an aggregation hot spot in IgG1 mAbs.     

Probing the nature of the blue-shifted intermediate of photoactive yellow protein in solution by NMR: hydrogen-deuterium exchange data and pH studies

The nature of the pB intermediate of photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been probed by NMR. pH-dependent changes in the NMR spectrum of the dark state of PYP are shown to closely mimic exchange broadening effects observed previously in the NMR spectrum of the pB intermediate in solution. Amide H-D exchange data show that while pB retains a solid protected core, two regions become significantly less protected than the dark state. The amide exchange data help to rationalize why the conformational exchange process affects the N-terminal 28-residue segment of the protein, which is not close to the site of chromophore rearrangement. At very low pH (pH 1.7), the dark state NMR spectrum displays approximately 30 very sharp signals, which are characteristic of a portion of the molecule becoming unfolded. Similarities between the dark state spectra at pH approximately 3.2 and the spectra of pB suggest a model for pB in solution where the protein exists in an equilibrium between a well-ordered state and a state in which a region is unfolded. Such a two-state model accounts for the exchange phenomena observed in the NMR spectra of pB, and the hydrophobic exposure and lability inferred from thermodynamic data. It is likely that in the crystalline environment the ordered form of pB is strongly favored.     

Combining conformational flexibility and continuum electrostatics for calculating pK(a)s in proteins

Protein stability and function relies on residues being in their appropriate ionization states at physiological pH. In situ residue pK(a)s also provides a sensitive measure of the local protein environment. Multiconformation continuum electrostatics (MCCE) combines continuum electrostatics and molecular mechanics force fields in Monte Carlo sampling to simultaneously calculate side chain ionization and conformation. The response of protein to charges is incorporated both in the protein dielectric constant (epsilon(prot)) of four and by explicit conformational changes. The pK(a) of 166 residues in 12 proteins was determined. The root mean square error is 0.83 pH units, and >90% have errors of <1 pH units whereas only 3% have errors >2 pH units. Similar results are found with crystal and solution structures, showing that the method's explicit conformational sampling reduces sensitivity to the initial structure. The outcome also changes little with protein dielectric constant (epsilon(prot) 4-20). Multiconformation continuum electrostatics titrations show coupling of conformational flexibility and changes in ionization state. Examples are provided where ionizable side chain position (protein G), Asn orientation (lysozyme), His tautomer distribution (RNase A), and phosphate ion binding (RNase A and H) change with pH. Disallowing these motions changes the calculated pK(a).     

Intrinsic local destabilization of the C‐terminus predisposes integrin α1 I domain to a conformational switch induced by collagen binding

Integrin–collagen interactions play a critical role in a myriad of cellular functions that include immune response, and cell development and differentiation, yet their mechanism of binding is poorly understood. There is increasing evidence that conformational flexibility assumes a central role in the molecular mechanisms of protein–protein interactions and here we employ NMR hydrogen–deuterium exchange (HDX) experiments to explore the impact of slower timescale dynamic events. To gain insight into the mechanisms underlying collagen‐induced conformational switches, we have undertaken a comparative study between the wild type integrin α1 I and a gain‐of‐function E317A mutant. NMR HDX results suggest a relationship between regions exhibiting a reduced local stability in the unbound I domain and those that undergo significant conformational changes upon binding. Specifically, the αC and α7 helices within the C‐terminus are at the center of such major perturbations and present reduced local stabilities in the unbound state relative to other structural elements. Complementary isothermal titration calorimetry experiments have been performed to derive complete thermodynamic binding profiles for association of the collagen‐like triple‐helical peptide with wild type α1 I and E317A mutant. The differential energetics observed for E317A are consistent with the HDX experiments and support a model in which intrinsically destabilized regions predispose conformational rearrangement in the integrin I domain. This study highlights the importance of exploring different timescales to delineate allosteric and binding events.     

Thermal unfolding studies of a phytocyanin

The thermal stability of umecyanin, a stellacyanin from horseradish roots, has been investigated by differential scanning calorimetry, optical absorption and fluorescence spectroscopy at neutral and alkaline pH. Above pH 9 the Cu(II) protein experiences a blue shift of the main visible absorption band at approximately 600 nm and changes colour from blue to violet. The thermal transition of the protein is irreversible and occurs between 61.4 and 68.8 degrees C at pH 7.5 and between 50.7 and 57.4 degrees C at pH 9.8. The calorimetric data indicates that at both pH values the thermally induced transition of the protein between the native and denaturated states can be described in terms of the classical Lumry-Eyring unfolding model Native<-->Unfolded-->Final. The analysis of the reversible step in the unfolding pathway demonstrates a significant reduction in conformational stability (DeltaG) of the alkaline form of the protein. Such a reduction is consistent with an enhanced flexibility of UMC at high pH and has mainly entropic character.     

Role of native-state structure in rubredoxin native-state hydrogen exchange

Amide exchange rates were measured for Pyrococcus furiosus (Pf) rubredoxin substituted with either Zn(II), Ga(III), or Ge(IV). Base-catalyzed exchange rate constants increase up to 3000-fold per unit charge for the highly protected amides surrounding the active site metal, yielding apparent residue-specific conformational energy decreases of more than 8 kcal/mol in a comparison of the Zn(II)- and Ge(IV)-substituted proteins. However, the exchange kinetics for many of the other amides of the protein are insensitive to these metal substitutions. These differential rates are inversely correlated with the distance between the amide nitrogen and the metal in the X-ray structure, out to a distance of at least 12 A, consistent with an electrostatic potential-dependent shifting of the amide nitrogen pK. This strongly correlated distance dependence is consistent with a nativelike structure for the exchange-competent conformations. The electric field potential within the interior of the rubredoxin structure gives rise to a change of as much as a million-fold in the rate for the exchange-competent state of the individual amide hydrogens. Nevertheless, the strength of these electrostatic interactions in Pf rubredoxin appears to be comparable to those previously reported within other proteins. As a result, contrary to the conventional analysis of hydrogen exchange data, for exchange processes that occur via nonglobal transitions, the residual conformational structure will often modulate the observed rates. Although this necessarily complicates the estimation of the conformational equilibria of these exchange-competent states, this dependence on residual structure can provide insight into the conformation of these transient states.     

Detecting structural changes in viral capsids by hydrogen exchange and mass spectrometry

Amide hydrogen exchange and mass spectrometry have been used to study the pH-induced structural changes in the capsid of brome mosaic virus (BMV). Capsid protein was labeled in a structurally sensitive way by incubating intact viral particles in D(2)O at pH 5.4 and 7.3. Deuterium levels in the intact coat protein and its proteolytic fragments were determined by mass spectrometry. The largest deuterium increases induced by structural alteration occurred in the regions around the quasi-threefold axes, which are located at the center of the asymmetric unit. The increased levels of deuterium indicate loosening of structure in these regions. This observation confirms the previously proposed swelling model for BMV and cowpea chlorotic mottle virus (CCMV) and is consistent with the structure of swollen CCMV recently determined by cryo-electron microscopy and image reconstruction. Structural changes in the extended N- and C-terminal arms were also detected and compared with the results obtained with other swollen plant viruses. This study demonstrates that protein fragmentation/amide hydrogen exchange is a useful tool for probing structural changes in viral capsids.     

The conformational stability and flexibility of insulin with an additional intramolecular cross-link

The conformational stability and flexibility of insulin containing a cross-link between the alpha-amino group of the A-chain to the epsilon-amino group of Lys29 of the B-chain was examined. The cross-link varied in length from 2 to 12 carbon atoms. The conformational stability was determined by guanidine hydrochloride-induced equilibrium denaturation and flexibility was assessed by H2O/D2O amide exchange. The cross-link has substantial effects on both conformational stability and flexibility which depend on its length. In general, the addition of a cross-link enhances conformational stability and decreases flexibility. The optimal length for enhanced stability and decreased flexibility was the 6-carbon link. For the 6-carbon link the Gibbs free energy of unfolding was 8.0 kcal/mol compared to 4.5 kcal/mol for insulin, and the amide exchange rate decreased by at least 3-fold. A very short cross-link (i.e. the 2-carbon link) caused conformational strain that was detectable by a lack of stabilization in the Gibbs free energy of unfolding and enhancement in the amide exchange rate compared to insulin. The effect of the cross-link length on insulin hydrodynamic properties is discussed relative to previously obtained receptor binding results.     

The influence of protonation states on the dynamics of the NhaA antiporter from Escherichia coli

The crystal structure of NhaA Na(+)/H(+) antiporter of Escherichia coli has provided a basis to explore the mechanism of Na(+) and H(+) exchange and its regulation by pH. However, the dynamics and nature of the pH-induced changes in the proteins remained unknown. Using molecular mechanics methods, we studied the dynamic behavior of the hydrogen-bonded network in NhaA on shifting the pH from 4 to 8. The helical regions preserved the general architecture of NhaA throughout the pH change. In contrast, large conformational drifts occurred at pH 8 in the loop regions, and an increased flexibility of helix IVp was observed on the pH shift. A remarkable pH-induced conformational reorganization was found: at acidic pH helix X is slightly curved, whereas at alkaline pH, it is kinked around residue Lys(300). The barrier that exists between the cytoplasmic and periplasmic funnels at low pH is removed, and the two funnels are bridged by hydrogen bonds between water molecules and residues located in the TMSs IV/XI assembly and helix X at alkaline pH. In the variant Gly(338)Ser that lost pH control, a hydrogen-bonded chain between Ser(338) and Lys(300) was found to block the pH-induced conformational reorganization of helix X.     

pH dependence of individual tryptophan N-1 hydrogen exchange rates in lysozyme and its chemically modified derivatives

Nuclear magnetic resonance analyses have been made of the individual hydrogen-deuterium exchange rates of tryptophan indole N-1 hydrogens in native lysozyme and its chemically modified derivatives including lysozyme with an ester cross-linkage between Glu-35 and Trp-108, lysozyme with an internal amide cross-linking between the epsilon-amino group of Lys-13 and the alpha-carboxyl group of Leu-129, and lysozyme with the beta-aspartyl sequence at Asp-101. The pH dependence curves of the exchange rates for Trp-63 and Trp-108 are different from those expected for tryptophan. The pH dependence curve for Trp-108 exchange exhibits the effects from molecular aggregation at pH above 5 and from a transition between the two conformational fluctuations at around pH 4. The exchange rates for tryptophan residues in native lysozyme and modified derivatives are not correlated with the thermodynamic or kinetic parameters in protein denaturation, suggesting that the fluctuations responsible for the exchange are not global ones. The exchange rates for tryptophan residues remote from the modification site are perturbed. Such tryptophan residues are found to be involved in a small but distinct conformational change due to the modification. Therefore, the perturbations of the N-1 hydrogen exchange rates are related to the minor change in local conformation or in conformational strain induced by the chemical modification.