Conformational stability of ribonuclease T1 determined by hydrogen-deuterium exchange.

The hydrogen-deuterium exchange kinetics of 37 backbone amide residues in RNase T1 have been monitored at 25, 40, 45, and 50 degrees C at pD 5.6 and at 40 and 45 degrees C at pD 6.6. The hydrogen exchange rate constants of the hydrogen-bonded residues varied over eight orders of magnitude at 25 degrees C with 13 residues showing exchange rates consistent with exchange occurring as a result of global unfolding. These residues are located in strands 2-4 of the central beta-pleated sheet. The residues located in the alpha-helix and the remaining strands of the beta-sheet exhibited exchange behaviors consistent with exchange occurring due to local structural fluctuations. For several residues at 25 degrees C, the global free energy change calculated from the hydrogen exchange data was over 2 kcal/mol greater than the free energy of unfolding determined from urea denaturation experiments. The number of residues showing this unexpected behavior was found to increase with temperature. This apparent inconsistency can be explained quantitatively if the cis-trans isomerization of the two cis prolines, Pro-39 and Pro-55, is taken into account. The cis-trans isomerization equilibrium calculated from kinetic data indicates the free energy of the unfolded state will be 2.6 kcal/mol higher at 25 degrees C when the two prolines are cis rather than trans (Mayr LM, Odefey CO, Schutkowski M, Schmid FX. 1996. Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped-flow double-mixing technique. Biochemistry 35: 5550-5561). The hydrogen exchange results are consistent with the most slowly exchanging hydrogens exchanging from a globally higher free energy unfolded state in which Pro-55 and Pro-39 are still predominantly in the cis conformation. When the conformational stabilities determined by hydrogen exchange are corrected for the proline isomerization equilibrium, the results are in excellent agreement with those from an analysis of urea denaturation curves.     

Tyrosine hydrogen bonds make a large contribution to protein stability

The aim of this study was to gain a better understanding of the contribution of hydrogen bonds by tyrosine -OH groups to protein stability. The amino acid sequences of RNases Sa and Sa3 are 69 % identical and each contains eight Tyr residues with seven at equivalent structural positions. We have measured the stability of the 16 tyrosine to phenylalanine mutants. For two equivalent mutants, the stability increases by 0.3 kcal/mol (RNase Sa Y30F) and 0.5 kcal/mol (RNase Sa3 Y33F) (1 kcal=4.184 kJ). For all of the other mutants, the stability decreases with the greatest decrease being 3.6 kcal/mol for RNase Sa Y52F. Seven of the 16 tyrosine residues form intramolecular hydrogen bonds and the average decrease in stability for these is 2.0(+/-1.0) kcal/mol. For the nine tyrosine residues that do not form intramolecular hydrogen bonds, the average decrease in stability is 0.4(+/-0.6) kcal/mol. Thus, most tyrosine -OH groups contribute favorably to protein stability even if they do not form intramolecular hydrogen bonds. Generally, the stability changes for equivalent positions in the two proteins are remarkably similar. Crystal structures were determined for two of the tyrosine to phenylalanine mutants of RNase Sa: Y80F (1.2 A), and Y86F (1.7 A). The structures are very similar to that of wild-type RNase Sa, and the hydrogen bonding partners of the tyrosine residues always form intermolecular hydrogen bonds to water in the mutants. These results provide further evidence that the hydrogen bonding and van der Waals interactions of polar groups in the tightly packed interior of folded proteins are more favorable than similar interactions with water in the unfolded protein, and that polar group burial makes a substantial contribution to protein stability.     

Folding subdomains of thioredoxin characterized by native-state hydrogen exchange

Native-state hydrogen exchange (HX) studies, used in conjunction with NMR spectroscopy, have been carried out on Escherichia coli thioredoxin (Trx) for characterizing two folding subdomains of the protein. The backbone amide protons of only the slowest-exchanging 24 amino acid residues, of a total of 108 amino acid residues, could be followed at pH 7. The free energy of the opening event that results in an amide hydrogen exchanging with solvent (DeltaG(op)) was determined at each of the 24 amide hydrogen sites. The values of DeltaG(op) for the amide hydrogens belonging to residues in the helices alpha(1), alpha(2), and alpha(4) are consistent with them exchanging with the solvent only when the fully unfolded state is sampled transiently under native conditions. The denaturant-dependences of the values of DeltaG(op) provide very little evidence that the protein samples partially unfolded forms, lower in energy than the unfolded state. The amide hydrogens belonging to the residues in the beta strands, which form the core of the protein, appear to have higher values of DeltaG(op) than amide hydrogens belonging to residues in the helices, suggesting that they might be more stable to exchange. This apparently higher stability to HX of the beta strands might be either because they exchange out their amide hydrogens in a high energy intermediate preceding the globally unfolded state, or, more likely, because they form residual structure in the globally unfolded state. In either case, the central beta strands-beta(3,) beta(2), and beta(4)-would appear to form a cooperatively folding subunit of the protein. The native-state HX methodology has made it possible to characterize the free energy landscape that Trx can sample under equilibrium native conditions.     

pK values of histidine residues in ribonuclease Sa: effect of salt and net charge

The primary goal of this study was to gain a better understanding of the effect of environment and ionic strength on the pK values of histidine residues in proteins. The salt-dependence of pK values for two histidine residues in ribonuclease Sa (RNase Sa) (pI=3.5) and a variant in which five acidic amino acids have been changed to lysine (5K) (pI=10.2) was measured and compared to pK values of model histidine-containing peptides. The pK of His53 is elevated by two pH units (pK=8.61) in RNase Sa and by nearly one pH unit (pK=7.39) in 5K at low salt relative to the pK of histidine in the model peptides (pK=6.6). The pK for His53 remains elevated in 1.5M NaCl (pK=7.89). The elevated pK for His53 is a result of screenable electrostatic interactions, particularly with Glu74, and a non-screenable hydrogen bond interaction with water. The pK of His85 in RNase Sa and 5K is slightly below the model pK at low salt and merges with this value at 1.5M NaCl. The pK of His85 reflects mainly effects of long-range Coulombic interactions that are screenable by salt. The tautomeric states of the neutral histidine residues are changed by charge reversal. The histidine pK values in RNase Sa are always higher than the pK values in the 5K variant. These results emphasize that the net charge of the protein influences the pK values of the histidine residues. Structure-based pK calculations capture the salt-dependence relatively well but are unable to predict absolute histidine pK values.     

Contribution of hydrogen bonds to protein stability

Our goal was to gain a better understanding of the contribution of the burial of polar groups and their hydrogen bonds to the conformational stability of proteins. We measured the change in stability, Δ(ΔG), for a series of hydrogen bonding mutants in four proteins: villin headpiece subdomain (VHP) containing 36 residues, a surface protein from Borrelia burgdorferi (VlsE) containing 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa (RNase Sa) and T1 (RNase T1). Crystal structures were determined for three of the hydrogen bonding mutants of RNase Sa: S24A, Y51F, and T95A. The structures are very similar to wild type RNase Sa and the hydrogen bonding partners form intermolecular hydrogen bonds to water in all three mutants. We compare our results with previous studies of similar mutants in other proteins and reach the following conclusions. (1) Hydrogen bonds contribute favorably to protein stability. (2) The contribution of hydrogen bonds to protein stability is strongly context dependent. (3) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (4) Polar group burial can make a favorable contribution to protein stability even if the polar groups are not hydrogen bonded. (5) The contribution of hydrogen bonds to protein stability is similar for VHP, a small protein, and VlsE, a large protein.     

Nuclear magnetic resonance studies of the internal dynamics in Apo, (Cd2+)1 and (Ca2+)2 calbindin D9k. The rates of amide proton exchange with solvent.

The backbone dynamics of the EF-hand Ca(2+)-binding protein, calbindin D9k, has been investigated in the apo, (Cd2+)1 and (Ca2+)2 states by measuring the rate constants for amide proton exchange with solvent. 15N-1H correlation spectroscopy was utilized to follow direct 1H-->2H exchange of the slowly exchanging amide protons and to follow indirect proton exchange via saturation transfer from water to the rapidly exchanging amide protons. Plots of experimental rate constants versus intrinsic rate constants have been analyzed to give qualitative insight into the opening modes of the protein that lead to exchange. These results have been interpreted within the context of a progressive unfolding model, wherein hydrophobic interactions and metal chelation serve to anchor portions of the protein, thereby damping fluctuations and retarding amide proton exchange. The addition of Ca2+ or Cd2+ was found to retard the exchange of many amide protons observed to be in hydrogen-bonding environments in the crystal structure of the (Ca2+)2 state, but not of those amide protons that were not involved in hydrogen bonds. The largest changes in rate constant occur for residues in the ion-binding loops, with substantial effects also found for the adjacent residues in helices I, II and III, but not helix IV. The results are consistent with a reorganization of the hydrogen-bonding networks in the metal ion-binding loops, accompanied by a change in the conformation of helix IV, as metal ions are chelated. Further analysis of the results obtained for the three states of metal occupancy provides insight into the nature of the changes in conformational fluctuations induced by ion binding.     

Investigation of the structural stability of cardiotoxin analogue III from the Taiwan cobra by hydrogen-deuterium exchange kinetics.

The conformational stability of a small ( approximately 7 kDa), all beta-sheet protein, cardiotoxin analogue III (CTX III), from the venom of the Taiwan cobra has been investigated by hydrogen-deuterium (H/D) exchange using two-dimensional NMR spectroscopy. The H/D exchange kinetics of backbone amide protons in CTX III has been monitored at pD 3.6 and 6.6 (at 25 degrees C), for over 5000 h. Examination of H/D exchange kinetics in the protein showed that a number of slowly exchanging residues are in the hydrophobic core of the protein. The average protection factor of the amide protons of residues belonging to the triple-stranded beta-sheet domain is about 20 times greater than that of those in the double-stranded beta-sheet segment. The residues in the C-terminal tail of the molecule, though structureless, have been found to exhibit significant protection against H/D exchange. Comparison of the quenched-flow H/D exchange data on CTX III with those obtained in the present study reveals that the most slowly exchanging portion constitutes the folding core of the protein.     

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI=3.5) and a basic variant (pI=10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulomb's Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.     

Structural mobility in human manganese superoxide dismutase

Human manganese superoxide dismutase (MnSOD) is a homotetramer of 22 kDa subunits, a dimer of dimers containing dimeric and tetrameric interfaces. We have investigated conformational mobility at these interfaces by measuring amide hydrogen/deuterium (H/D) exchange kinetics and 19F NMR spectra, both being excellent methods for analyzing local environments. Human MnSOD was prepared in which all nine tyrosine residues in each subunit are replaced with 3-fluorotyrosine. The 19F NMR spectrum of this enzyme showed five sharp resonances that have been assigned by site-specific mutagenesis by replacing each 3-fluorotyrosine with phenylalanine; four 19F resonances not observed are near the paramagnetic manganese and extensively broadened. The temperature dependence of the line widths and chemical shifts of the 19F resonances were used to estimate conformational mobility. 3-Fluorotyrosine 169 at the dimeric interface showed little conformational mobility and 3-fluorotyrosine 45 at the tetrameric interface showed much greater mobility by these measures. In complementary studies, H/D exchange mass spectrometry was used to measure backbone dynamics in human MnSOD. Using this approach, amide hydrogen exchange kinetics were measured for regions comprising 78% of the MnSOD backbone. Peptides containing Tyr45 at the tetrameric interface displayed rapid exchange of hydrogen with deuterium while peptides containing Tyr169 in the dimeric interface only displayed moderate exchange. Taken together, these studies show that residues at the dimeric interface, such as Tyr169, have significantly less conformational freedom or mobility than do residues at the tetrameric interface, such as Tyr45. This is discussed in terms of the role in catalysis of residues at the dimeric interface.     

Hydrogen/deuterium exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSFbeta)

Studies with the homodimeric recombinant human macrophage colony-stimulating factor beta (rhM-CSFbeta), show for the first time that a large number (9) of disulfide linkages can be reduced after amide hydrogen/deuterium (H/D) exchange, and the protein digested and analyzed successfully for the isotopic composition by electrospray mass spectrometry. Analysis of amide H/D after exchange-in shows that in solution the conserved four-helix bundle of (rhM-CSFbeta) has fast and moderately fast exchangeable sections of amide hydrogens in the alphaA helix, and mostly slow exchanging sections of amide hydrogens in the alphaB, alphaC, and alphaD helices. Most of the amide hydrogens in the loop between the beta1 and beta4 sheets exhibited fast or moderately fast exchange, whereas in the amino acid 63-67 loop, located at the interface of the two subunits, the exchange was slow. Solvent accessibility as measured by H/D exchange showed a better correlation with the average depth of amide residues calculated from reported X-ray crystallographic data for rhM-CSFalpha than with the average B-factor. The rates of H/D exchange in rhM-CSFbeta appear to correlate well with the exposed surface calculated for each amino acid residue in the crystal structure except for the alphaD helix. Fast hydrogen isotope exchange throughout the segment amino acids 150-221 present in rhM-CSFbeta, but not rhM-CSFalpha, provides evidence that the carboxy-terminal region is unstructured. It is, therefore, proposed that the anomalous behavior of the alphaD helix is due to interaction of the carboxy-terminal tail with this helical segment.     

An inserted Gly residue fine tunes dynamics between mesophilic and thermophilic ribonucleases H

Dynamic processes are inherent properties of proteins and are crucial for a wide range of biological functions. To address how changes in protein sequence and structure affect dynamic processes, a quantitative comparison of microsecond-to-microsecond time scale conformational changes, measured by solution NMR spectroscopy, within homologous mesophilic and thermophilic ribonuclease H (RNase H) enzymes is presented. Kinetic transitions between the observed major state (high population) and alternate (low population) conformational state(s) of the substrate-binding handle region in RNase H from the mesophile Escherichia coli (ecRNH) and thermophile Thermus thermophilus (ttRNH) occur with similar kinetic exchange rate constants, but the difference in stability between exchanging conformers is smaller in ttRNH compared to ecRNH. The altered thermodynamic equilibrium between kinetically exchanging conformers in the thermophile is recapitulated in ecRNH by the insertion of a Gly residue within a putative hinge between alpha-helices B and C. This Gly insertion is conserved among thermophilic RNases H, and allows the formation of additional intrahelical hydrogen bonds. A Gly residue inserted between alpha-helices B and C appears to relieve unfavorable interactions in the transition state and alternate conformer(s) and represents an important adaptation to adjust conformational changes within RNase H for activity at high temperatures.     

Reduced temperature dependence of collective conformational opening in a hyperthermophile rubredoxin.

Spatially localized differences in the conformational dynamics of the rubredoxins from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophile Clostridium pasteurianum (Cp) are monitored via amide exchange measurements. As shown previously for the hyperthermophile protein, nearly all backbone amides of the Cp rubredoxin exhibit EX(2) hydrogen exchange kinetics with conformational opening rates of >1 s(-)(1). Significantly slower amide exchange is observed for Pf rubredoxin in the region surrounding the metal site and the proximal end of the three-stranded beta-sheet, while for the rest of the structure, the exchange rates at 23 degrees C are similar for both proteins. For the multiple-turn region comprising residues 14-32 in both rubredoxins, the uniformity of both the exchange rate constants and the values of the activation energy at the slowly exchanging sites is consistent with a model of solvent exposure via a subglobal cooperative conformational opening. In contrast to the common expectation of increased rigidity in the hyperthermophile proteins, below room temperature Pf rubredoxin exhibits a larger apparent flexibility in this multiple-turn region. The smaller enthalpy for the conformational opening process of this region in Pf rubredoxin reflects the much weaker temperature dependence of the underlying conformational equilibrium in the hyperthermophile protein.     

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.     

Increasing protein conformational stability by optimizing beta-turn sequence

Protein conformational stability is an important concern in many fields. Here, we describe a strategy for significantly increasing conformational stability by optimizing beta-turn sequence. Proline and glycine residues are statistically preferred at several beta-turn positions, presumably because their unique side-chains contribute favorably to conformational stability in certain beta-turn positions. However, beta-turn sequences often deviate from preferred proline or preferred glycine. Therefore, our strategy involves replacing non-proline and non-glycine beta-turn residues with preferred proline or preferred glycine residues. Here, we develop guidelines for selecting appropriate mutations, and present results for five mutations (S31P, S42G, S48P, T76P, and Q77G) that significantly increase the conformational stability of RNase Sa. The increases in stability ranged from 0.7 kcal/mol to 1.3 kcal/mol. The strategy was successful in overlapping or isolated beta-turns, at buried (up to 50%) or completely exposed sites, and at relatively flexible or inflexible sites. Considering the significant number of beta-turn residues in every globular protein and the frequent deviation of beta-turn sequences from preferred proline and preferred glycine residues, this simple, efficient strategy will be useful for increasing the conformational stability of proteins.     

Conformational stability and integrity of alpha-amylase from mung beans: evidence of kinetic intermediate in GdmCl-induced unfolding

alpha-Amylase from mung beans (Vigna radiata) being one of the few plant alpha-amylases purified so far was studied with respect to its conformational stability by CD and fluorescence spectroscopy. The enzyme was shown to bind 3-4 Ca(2+) ions, which all are important for its activity. In contrast to other alpha-amylases no inhibition was observed at high Ca(2+) concentrations (100 mM). Depletion of calcium decreased the transition temperature from 87 to 48 degrees C. Kinetic stopped-flow fluorescence measurements allowed detecting two unfolding phases at >6 M GdmCl, whereas only one phase was observed at <5 M GdmCl. These results suggest that the first (reversible) step of unfolding is slower than the second (irreversible) step at low GdmCl concentrations, whereas the rates of these two steps are opposite at high GdmCl concentrations.     

Native state hydrogen exchange study of suppressor and pathogenic variants of transthyretin

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for numerous familial amyloid diseases, the exact phenotype being dependent on the sequence deposited. Many familial disease variants display decreased stability in vitro, and early onset pathology in vivo. Only subtle structural differences were observed upon crystallographic comparison of the disease-associated variants to the T119M interallelic trans-suppressor. Herein three human TTR single amino acid variant homotetramers including two familial amyloidotic polyneuropathy (FAP) causing variants (V30M and L55P), and a suppressor variant T119M (known to protect V30M carriers from disease by trans-suppression) were investigated in a residue-specific fashion by monitoring (2)H-(1)H exchange employing NMR spectroscopy. The measured protection factors for slowly exchanging amide hydrogen atoms reveal destabilization of the protein core in the FAP variants, the core consisting of strands A, B, E and G and the loop between strands A and B. The same core exhibits much slower exchange in the suppressor variant. Accelerated exchange rates were observed for residues at the subunit interfaces in L55P, but not in the T119M or V30M TTR. The correlation between destabilization of the TTR core strands and the tendency for amyloid formation supports the view that these strands are involved in amyloidogenicity, consistent with previous (2)H-(1)H exchange analysis of the WT-TTR amyloidogenic intermediate.     

Carbon-13 NMR method for the detection of correlated hydrogen exchange at adjacent backbone peptide amides and its application to hydrogen exchange in five antiparallel beta strands within the hydrophobic core of Streptomyces subtilisin inhibitor (SSI)

A novel method for monitoring proton-deuteron (H/D) exchange at backbone amides is based on the observation of H/D isotope effects on the (13)C NMR signals from peptide carbonyls. The line shape of the carbonyl (13)C(i) signal is influenced by differential H/D occupancy at the two adjacent amides: the H(N)(i)(+1) (beta site) and the H(N)(i) (gamma site). At a carbon frequency of 75.4 MHz, the H --> D isotope shifts on the (13)C signal are about 5-7 Hz for exchange at the beta site and 2 Hz or less for exchange at the gamma site. Because the effects at the two sites are additive, the time dependence of the line shape of a particular carbonyl resonance can report not only the exchange rates at the individual sites but also the level of dual exchange. Therefore, the data can be analyzed to determine the rate (k(c)) and degree of correlated exchange (X(betagamma)) at the two sites. We have applied this approach to the investigation of the pH dependence of hydrogen exchange at several adjacent residues in Streptomyces subtilisin inhibitor (SSI). Two selectively labeled SSI proteins were produced: one with selective (13)C' labeling at all valyl residues and one with selective (13)C' labeling at all leucyl residues. This permitted the direct observation by one-dimensional (13)C NMR of selected carbonyl signals from residues with slowly exchanging amides at the i and i + 1 positions. The residues investigated were located in an alpha helix and in a five-stranded antiparallel beta sheet. Samples of the two labeled proteins were prepared at various pH values, and (13)C NMR spectra were collected at 50 degrees C prior to and at various times after transferring the sample from H(2)O to (2)H(2)O. Most of the slowly exchanging amides studied were intramolecular hydrogen-bond donors. In agreement with prior studies, the results indicated that the exchange rates of the amide hydrogens in proteins are governed not only by hydrogen bonding but also by other factors. For example, the amide hydrogen of Thr34 exchanges rapidly even though it is an intramolecular hydrogen-bond donor. Over nearly the whole pH range studied, the apparent rates of uncorrelated exchange (k(beta) and k(gamma)) were proportional to [OH(-)] and the apparent rates of correlated exchange at two adjacent sites (k(c)) were roughly proportional to [OH(-)](2). This enabled us to extract the pH-independent exchange rates (k(beta) degrees , k(gamma) degrees , and k(c) degrees ). In all cases in which correlated exchange could be measured, the observed sigmoidal pH dependence of X(betagamma) could be replicated roughly from the derived pH-independent rates.     

Three-dimensional solution structure and stability of thioredoxin m from spinach.

Proton NMR spectral resonances of thioredoxin m from spinach have been assigned, and its solution structure has been determined on the basis of 1156 nuclear Overhauser effect- (NOE-) derived distance constraints by using restrained molecular dynamics calculations. The average pairwise root-mean-square deviation (RMSD) for the 25 best NMR structures for the backbone was 1.0 +/- 0.1, when the structurally well-defined residues were considered. The N- and C-terminal segments (1-13 and 118-119) and residues 41-49, comprising the active site, are highly disordered. At the time of concluding this work, a crystal structure of this protein was reported, in which thioredoxin m was found to crystallize as noncovalent dimers. Although the solution and crystal structures are very similar, no evidence was found about the existence of dimers in solution, thus confirming that dimerization is not needed for the regulatory activity of thioredoxin m. The spinach thioredoxin m does not unfold by heat in the range 25-85 degrees C, as revealed by thermal circular dichroic (CD) measurements. However, its unfolding free energy (9.1 +/- 0.8 kcal mol(-1), at pH 5.3 and 25 degrees C) could be determined by extrapolating the free energy values obtained at different concentrations of guanidinium chloride (GdmCl). The folding-unfolding process is two-state as indicated by the coincidence of the CD denaturation curves obtained at far and near UV. The H/D exchange behavior of backbone amide protons was analyzed. The slowest-exchanging protons, requiring a global-unfolding mechanism in order to exchange, are those from beta2, beta3, and beta4, the central strands of the beta-sheet, which constitute the main element of the core of the protein. The free energies obtained from exchange measurements of protons belonging to the alpha-helices are lower than those derived from GdmCl denaturation studies, indicating that those protons exchange by local-unfolding mechanisms.     

Cores and pH-dependent dynamics of ferredoxin-NADP+ reductase revealed by hydrogen/deuterium exchange

NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP(+) reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP(+))-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD(r) values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD(r) values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP(+)-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP(+)-binding domain at pD(r) 8.0, the daytime pH in chloroplasts, than at pD(r) 6.0 is likely to be involved in the increased binding of NADP(+) for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.     

Multi-state unfolding of the alpha subunit of tryptophan synthase, a TIM barrel protein: insights into the secondary structure of the stable equilibrium intermediates by hydrogen exchange mass spectrometry

The urea-induced unfolding of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, an eight-stranded (beta/alpha)(8) TIM barrel protein, has been shown to involve two stable equilibrium intermediates, I1 and I2, well populated at approximately 3 M and 5 M urea, respectively. The characterization of the I1 intermediate by circular dichroism (CD) spectroscopy has shown that I1 retains a significant fraction of the native ellipticity; the far-UV CD signal for the I2 species closely resembles that of the fully unfolded form. To obtain detailed insight into the disruption of secondary structure in the urea-induced unfolding process, a hydrogen exchange-mass spectrometry study was performed on alphaTS. The full-length protein was destabilized in increasing concentration of urea, the amide hydrogen atoms were pulse-labeled with deuterium, the labeled samples were quenched in acid and the products were analyzed by electrospray ionization mass spectrometry. Consistent with the CD results, the I1 intermediate protects up to approximately 129 amide hydrogen atoms against exchange while the I2 intermediate offers no protection. Electrospray ionization mass spectrometry analysis of the peptic fragments derived from alphaTS labeled at 3 M urea indicates that most of the region between residues 12-130, which constitutes the first four beta strands and three alpha helices, (beta/alpha)(1-3)beta(4), is structured. The (beta/alpha)(1-3)beta(4) module appears to represent the minimum sub-core of stability of the I1 intermediate. A 4+2+2 folding model is proposed as a likely alternative to the earlier 6+2 folding mechanism for alphaTS.