Hydrogen Exchange Rates Research Articles

Insight into a Random Coil Conformation and an Isolated Helix: Structural and Dynamical Characterisation of the C-Helix Peptide from Hen Lysozyme

A 17 residue peptide corresponding to the C-helix of hen lysozyme (residues 86 to 102) has been investigated in detail to assess the factors that determine its conformation in both aqueous and trifluoroethanol (TFE) solutions. A thorough characterisation of the peptide by CD and NMR techniques under both conditions has been performed including the determination of complete NMR proton sequential assignments, and measurement of NOE effects, 3 J HNαcoupling constants, temperature coefficients and residue-specific hydrogen-exchange rates. In water, the peptide adopts a largely unstructured conformation and NMR data, particularly coupling constants and chemical shift deviations, have been shown to agree closely with predictions from a model for a random coil based on the φ,ψ distributions in a protein database. This indicates that under these conditions the intrinsic conformational preferences of the individual amino acid residues are the dominating factors that determine the population of conformers adopted. With increasing concentrations of TFE a cooperative transition to an extensively helical conformation occurs and the resultant changes in CαH chemical shifts have been shown to correlate with the changes in φ,ψ populations. Using NOE and coupling constant data for this state, an ensemble of structures has been calculated and provides a model for a helix in the absence of tertiary interactions. In this model fluctuations, which increase in amplitude towards the termini, occur about the average helical φ,ψ angles and are responsible for increasing the values of 3 J HNαcoupling constants above those anticipated for a static helix. The residue-specific rates of hydrogen exchange for the peptide in 50% TFE-d 3are consistent with such a model, the maximum protection from exchange being observed for residues in the centre of the helix.

H NMR probes for inter-segmental hydrogen bonds in myoglobins.

NMR signals arising from the HisB5 N delta H and HisEF5 N epsilon H protons in sperm whale skeletal and horse heart myoglobins have been located for the first time in the downfield shifted portion of the spectra. The shifts and hydrogen exchange rates indicate that these His imidazole ring NH protons are involved in the inter-segmental hydrogen bonds of the protein in solution, as demonstrated by a crystallographic study [Takano, T. (1977) J. Mol. Biol. 220, 381-399]. The assigned His imidazole ring NH proton resonances can serve as new sensitive structural probes in the study of the local conformation of myoglobin. The applicability of the NMR spectral parameters in the study of the tertiary structure of apomyoglobin, the denaturation of the protein, and the protein stability of sperm whale and horse myoglobins is presented in some detail.

Flexibility is a likely determinant of binding specificity in the case of ileal lipid binding protein

Background: The family of lipid binding proteins (LBPs) includes a large number of fatty acid binding proteins (FABPs) but only two proteins (ileal lipid binding protein, ILBP, and liver fatty acid binding protein) that can bind both fatty acids and bile acids. Bile acid transport is medically and pharmacologically important, but is poorly understood. To understand the binding properties of ILBP, we studied its solution structure with and without bound lipids and compared these with known structures of FABPs. Results The sequence-specific 1H resonance assignments for porcine ILBP have been determined by homonuclear two-dimensional (2D) NMR spectroscopy for the apo-protein as well as for ILBP complexes with fatty acid and bile acid ligands. From NOE spectra and hydrogen exchange data, similar secondary structure elements were identified for all three protein forms. ILBP is composed of ten antiparallel β strands arranged in two nearly orthogonal β sheets (a fold seen in other FABPs, and dubbed the ‘ β-clam shell’), covered on one side by two short, nearly parallel α helices. Binding of fatty acids or bile acids to ILBP alters mainly the side-chain proton resonances of amino acids within the protein cavity, indicating that both bile acids and fatty acids can bind in the interior of the protein between the two β sheets; binding of bile acids stabilizes the protein backbone by a small amount. Fast hydrogen exchange rates for the backbone amide protons of ILBP indicate that the hydrogen-bonding network of the β sheet in ILBP is weaker than the corresponding network in rat intestinal and bovine heart FABPs. Conclusion The tertiary structure of ILBP is similar to that of other LBPs, but appears to be unusually flexible, with a relatively weak hydrogen-bonding network. It is likely that this flexibility is important in allowing bile acids, which are larger and more rigid than fatty acids, to enter the central cavity of the protein.

Measurement of intrinsic exchange rates of amide protons in a 15N-labeled peptide

We have used a modified version of a previously proposed technique, MEXICO [Gemmecker et al. (1993) J. Am. Chem. Soc., 115, 11620], and improved data analysis procedures in order to measure rapid hydrogen exchange (HX) rates of amide protons in peptides labeled only with 15N. The requirement of 13C-/15N-labeled material has been circumvented by adjusting conditions so that NOE effects associated with amide protons can be neglected (i.e., ω0τc~1). The technique was applied to an unstructured 15N-labeled 12-residue peptide to measure intrinsic HX rates, which are the essential reference for examining protein and peptide structure and dynamics through deceleration of HX rates. The method provided accurate HX rates from 0.5 to 50 s-1 under the conditions used. The measured rates were in good agreement with those predicted using correction factors determined by Englander and co-workers [Bai et al. (1993) Proteins, 17, 75], with the largest deviations from the predicted rates found for residues close to the N-terminus. The exchange rates were found to exhibit significant sensitivity to the concentration of salt in the sample.

Mass spectrometric measurement of changes in protein hydrogen exchange rates that result from point mutations

Point mutations, as well as additions or deletions of entire domains, are frequently produced to study protein function; however, to infer function from mutant proteins, it is imperative that their structural integrity be verified. Although detailed structural studies can be performed by using NMR or crystallography, for practical reasons mutant proteins usually are characterized by using less rigorous techniques. Here it is shown that measurement of hydrogen exchange rates via electrospray ionization mass spectrometry is a sensitive and generally applicable method for detection of conformational or dynamic changes that result from point mutations. Hydrogen exchange experiments were performed on a bacterial phosphocarrier protein (HPr) and two variants produced by conversion of either serine-46 to aspartic acid (S46D) or serine-31 to alanine (S31A), where the differences in the ΔG of folding relative to the wild type were 1.5 and 0.5 kcal/mol, respectively. Whereas no significant differences were found for the intact mutant and wild-type proteins, changes in deuterium incorporation could be detected within specific regions produced by peptic proteolysis of the deuterium-labeled proteins. Thus, energetically small changes in conformation (or dynamics) that result from point mutations can be characterized by mass spectrometric measurements of hydrogen exchange rates. Furthermore, these changes can be localized to specific regions within the protein.

Hydrogen exchange and the unfolding pathway of ribonuclease A

Recently, when the kinetic unfolding process of ribonuclease A was monitored by hydrogen exchange (T. Kiefhaber and R.L. Baldwin, Proc. Natl. Acad. Sci. USA, 92 (1995) 2657–2661), all peptide hydrogen bonds were found to undergo rapid exchange in a single kinetic step under conditions where unfolding is slow and the intrinsic rate of hydrogen exchange is fast (pH 8.0, 10°C, 4.5 M guanidinium chloride). Comparison with the unfolding rate measured by circular dichroism indicates that hydrogen exchange is caused by the rate-limiting step of unfolding. No evidence was found for partly unfolded intermediates that are formed slowly enough to be observed by EX1 (unfolding-limited) hydrogen exchange. Some peptide NH protons were found to show, in addition to EX1 exchange, faster EX2 exchange that is base-catalyzed. The EX2 exchange is caused by species that equilibrate rapidly with the native protein at the start of the unfolding process. These species might include rapidly formed unfolding intermediates. We show here that any such unfolding intermediates must have large protection factors because the EX2 reactions of ribonuclease A under these unfolding conditions have protection factors ≥ 2500.

Global Changes in Amide Hydrogen Exchange Rates for a Protein Antigen in Complex with Three Different Antibodies

The binding of anti-lysozyme monoclonal antibodies, D44.1 or D1.3, to their antigen reduces the rate of exchange for many amide hydrogens in lysozyme. The D44.1 antibody contacts a similar region of lysozyme to the HyHEL-5 antibody, while the D1.3 antibody binds to the side of lysozyme which is opposite to the HyHEL-5 and D44.1 epitopes. We compare the effects of binding these antibodies on amide hydrogen exchange rates in lysozyme. These comparisons suggest that there are regions of lysozyme that fluctuate in a coordinated manner such that the effects of binding can be propagated to regions that are distant from the epitope. The activation enthalpies for hydrogen exchange for 36 of the 126 amide hydrogens in lysozyme and for 25 of 126 lysozyme amide hydrogens in the lysozyme-D1.3 complex are also reported. These data suggest that the reduction in amide hydrogen exchange rates upon antibody binding reflect changes in the dynamics of the antigen. These changes contribute to a reduction in the specific heat capacity upon binding.

A general two-process model describes the hydrogen exchange behavior of RNase A in unfolding conditions.

When NMR hydrogen exchange was used previously to monitor the kinetics of RNase A unfolding, some peptide NH protons were found to show EX2 exchange (detected by base catalysis) in addition to the expected EX1 exchange, whose rate is limited by the kinetic unfolding process. In earlier work, two groups showed independently that a restricted two-process model successfully fits published hydrogen exchange rates of native RNase A in the range 0-0.7 M guanidinium chloride. We find that this model predicts properties that are very different from the observed properties of the EX2 exchange reactions of RNase A in conditions where guanidine-induced unfolding takes place. The model predicts that EX2 exchange should be too fast to measure by the technique used, whereas it is readily measurable. Possible explanations for the contradiction are considered here, and we show that removing the restriction from the earlier two-process model is sufficient to resolve the contradiction; instead of specifying that exchange caused by global unfolding occurs by the EX2 mechanism, we allow it to occur by the general mechanism, which includes both the EX1 and EX2 cases. It is logical to remove this restriction because global unfolding of RNase A is known to give rise to EX1 exchange in these unfolding conditions. Resolving the contradiction makes it possible to determine whether populated unfolding intermediates contribute to the EX2 exchange, and this question is considered elsewhere. The results and simulations indicate that moderate or high denaturant concentrations readily give rise to EX1 exchange in native proteins. Earlier studies showed that hydrogen exchange in native proteins typically occurs by the EX2 mechanism but that high temperatures or pH values above 7 may give rise to EX1 exchange. High denaturant concentrations should be added to the list of variables likely to cause EX1 exchange.

Determination of the Activation Volume of the Uncatalyzed Hydrogen Exchange Reaction between N-Methylacetamide and Water

The amide−water hydrogen exchange rates of the individual backbone amide protons are very sensitive to the local environment of the protons and can be used to probe fluctuations in the local conformations of proteins. Hydrogen exchange between N-methylacetamide (NMA) and water is important in that it serves as a model for the amide−water hydrogen exchange in peptides and proteins. The rate constants for amide−water hydrogen exchange between 16 mol % NMA and water at 66 °C were measured at pressures ranging from 1 bar to 5 kbar at three different pH values using the NMR magnetization transfer technique, and the apparent activation volumes of hydrogen exchange were obtained at each pH. The activation volume of the acid-catalyzed hydrogen exchange reaction was calculated to be +1.7 cm3 mol-1, and the activation volume of the base-catalyzed hydrogen exchange reaction was calculated to be +11.0 cm3 mol-1. The hydrogen exchange rate constants were also measured as a function of concentration and pH at ambient p...

Amide hydrogen exchange determined by mass spectrometry: application to rabbit muscle aldolase.

The protein fragmentation/mass spectrometry method described by Zhang and Smith [(1993) Protein Sci. 2, 522-531] has been extended to measure amide hydrogen exchange rates in rabbit muscle aldolase, a homotetramer with M(r) = 157,000. Following a period of deuterium exchange, the partially deuterated protein was proteolytically fragmented into peptides whose deuterium contents were determined by directly coupled HPLC fast atom bombardment mass spectrometry. Hydrogen exchange rates were determined for amide hydrogens located in short segments derived from 85% of the aldolase backbone. Isotopic exchange rate constants spanning the range from 100 to 0.001 h-1 were determined for the exchange-in times used in this study (2.5 min to 44 h). The exchange rates for amide hydrogens located within short segments differed by as much as 10(4), demonstrating that local structural features dramatically affect the isotopic exchange rates in large proteins. A high level of correlation between the slowing of hydrogen exchange and intramolecular hydrogen bonding in aldolase was found. An exception to this correlation occurs at the subunit interface, where the amide hydrogens in one peptide segment with few amide hydrogen bonds have slower exchange rates than expected, suggesting that the amide hydrogens in this region are effectively shielded from the deuterated solvent. Isotope patterns observed for most peptides were binomial, indicating that hydrogen exchange proceeds through the EX2 mechanism (uncorrelated exchange). However, bimodal isotope patterns were found for peptides derived from three short segments of aldolase (including residues 58-64, 279-283, and 326-337), suggesting structural differences in these regions. A high level of correlation was found between crystallographic B-factors and amide hydrogen exchange rates, suggesting an isotopic exchange mechanism involving localized low-amplitude, high-frequency motions that do not require collective motion of many residues. From a methodology viewpoint, these results demonstrate that the combination of protein fragmentation with mass spectrometry is a useful method for determining the rates at which amide hydrogens located over major portions of large proteins undergo isotopic exchange.

Protonation-state dependence of hydrogen bond strengths and exchange rates in a serine protease catalytic triad: bovine chymotrypsinogen A.

Hydrogen-1 nuclear magnetic resonance spectroscopy was used to measure D/H fractionation factors and the temperature dependence of the rate of hydrogen exchange at two sites in the catalytic triad of chymotrypsinogen (hydrogen bond between aspartate-102 and histidine-57 and hydrogen bond between histidine-57 and serine-195) as a function of the protonation states of the constituent residues. Connectivities in one-dimensional spectra used to assign NMR data were collected at three pH values: pH 9, at which His-57 is neutral and Asp-102 is negatively charged; pH 3.5, at which His-57 is positively charged and Asp-102 is negatively charged; and pH 1, at which His-57 is positively charged and Asp-102 is neutral. The signal from H epsilon 2 of histidine-57 was assigned by reference to 1H-1H NOE connectivities at pH 3.5 to the previously assigned signals from the H epsilon 1 and H delta 2 of the same residue. The D/H fractionation factor, phi, for the hydrogen bond between Asp-102 and His-57 changed from phi = 2 at pH 9 to phi = 0.4 at pH 3.5. From studies of model systems, it may be concluded that a change of phi of this magnitude corresponds to a large increase in hydrogen bond strength. A signal from the hydrogen bond between Ser-195 and His-57 was detected only at the lower pH values studied. The D/H fractionation factor for this hydrogen bond was phi = 0.7 at pH 3.5, indicative of a moderately strong interaction. Data obtained at pH 1 indicate that the hydrogen bond between Asp-102 and His-57 is weakened but that the hydrogen bond between His-57 and Ser-195 persists. The results are consistent with the hypothesis that changes in hydrogen bonding strength serve to lower barriers along the reaction coordinate in the catalytic mechanism. Large pH-dependent changes were found in the activation enthalpy (delta H ++) for exchange with protons from the solvent at the hydrogen bond between aspartate-102 and histidine-57: delta H ++ was approximately 10-12 kcal.mol-1 higher at pH 3.5 than at pH 1 or 9.

Intramolecular Electrostatic Interactions Accelerate Hydrogen Exchange in Diketopiperazine Relative to 2-Piperidone

1H NMR saturation transfer and T1 experiments were used to monitor the amide hydrogen exchange rates of 2,5-piperazinedione (diketopiperazine, DKP) and 2-piperidone (δ-valerolactam) in water. Speci...

Collagen-Based Structures Containing the Peptoid Residue N-Isobutylglycine (Nleu). 6. Conformational Analysis of Gly-Pro-Nleu Sequences by 1H NMR, CD, and Molecular Modeling

Molecular modeling, 1H NMR, and CD were employed to study the structure and stability of collagen-like triple helices composed of Gly-Pro-Nleu repeats. The compounds studied include the acetyl analogs Ac-(Gly-Pro-Nleu)n-NH2 (where n = 1, 6, 9) and the KTA conjugates KTA-[Gly-(Gly-Pro-Nleu)n-NH2]3 (where n = 1, 3, 6, 9 and KTA denotes the Kemp triacid). The presence of collagen-like assembled structures was supported by a consistent set of experimental observations, including the appearance of a distinct set of resonances, low hydrogen exchange rates for Gly NH, KTA signal splitting, cooperative melting transition, and analysis of NOESY cross peaks. In this regard, the concept of ensemble interchain NOEs was introduced and used to establish the close packing of Gly, Pro, and Nleu residues in triple helices composed of Gly-Pro-Nleu repeats. In addition, the ensemble interchain NOEs gave insight into the puckering of the Pro ring and the conformations accessible to the Nleu side chain. The effect of the KTA template on triple helicity was studied and shown to consist in a net gain in the free energy of triple-helix formation, as also seen for Gly-Pro-Hyp sequences. This free energy gain led to the induction of an assembled collagen-like structure in the KTA conjugate containing six Gly-Pro-Nleu repeats per chain and to an increase in thermal stability of the compound containing nine Gly-Pro-Nleu repeats per chain.

Measurement of intrinsic exchange rates of amide protons in a 15N-labeled peptide

We have used a modified version of a previously proposed technique, MEXICO [Gemmecker et al. (1993) J. Am. Chem. Soc., 115, 11620], and improved data analysis procedures in order to measure rapid hydrogen exchange (HX) rates of amide protons in peptides labeled only with 15N. The requirement of 13C-/15N-labeled material has been circumvented by adjusting conditions so that NOE effects associated with amide protons can be neglected (i.e., omega 0 tau c approximately 1). The technique was applied to an unstructured 15N-labeled 12-residue peptide to measure intrinsic HX rates, which are the essential reference for examining protein and peptide structure and dynamics through deceleration of HX rates. The method provided accurate HX rates from 0.5 to 50 s-1 under the conditions used. The measured rates were in good agreement with those predicted using correction factors determined by Englander and co-workers [Bai et al. (1993) Proteins, 17, 75], with the largest deviations from the predicted rates found for residues close to the N-terminus. The exchange rates were found to exhibit significant sensitivity to the concentration of salt in the sample.

Effects of a naturally occurring compatible osmolyte on the internal dynamics of ribonuclease A.

Osmolytes are small organic solutes accumulated intracellularly by many organisms as they adapt to environmental stresses. Compatible osmolytes, a functional class of osmolytes, increase protein stability while having little or no effect on protein function. To investigate the interrelationships between protein stability, function, and internal dynamics, a hydrogen exchange (HX) quench method was established and used to study the effects of sucrose (a typical compatible osmolyte) on the structural fluctuations of ribonuclease A. It was found that the HX rates of the amide protons with intermediate rates are not affected by 1 M sucrose, but the slow-exchanging amide protons exchange even slower in 1 M sucrose. The protection factors of the slow-exchanging protons fall into a comparatively narrow range while those of the intermediate-exchanging protons vary widely. In agreement with the two-process model [Woodward, C.K., & Hilton, B. D. (1980) Biophys. J. 32, 561-575], we conclude that for those slow-exchanging amide protons, the exchange occurs mainly from the compact unfolded state ensemble of the protein. The internal dynamics leading to slow exchange involve exposure of large protein surface areas, similar to that which occurs upon the unfolding of protein. Because sucrose opposes such an increase in protein surface area exposure, both the slow HX rates and the protein stability are affected by sucrose. For those amide protons with fast and intermediate HX rates, the exchange occurs mainly from the native state ensemble of the protein. The internal dynamics involved in the exchange are localized without much surface area change, and functionally important structural fluctuations are likely to occur within this dynamic range.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure Effects of Double D-Amino Acid Replacements: A Nuclear Magnetic Resonance and Circular Dichroism Study Using Amphipathic Model Helixes

D-Amino acid replacements and the determination of resulting structural changes are a useful tool to recognize amphipathic helices in biologically active peptides such as neuropeptide Y and corticotropin-releasing factor. In this paper the secondary structures of one amphipathic alpha-helical peptide and its double D-amino acid analog have been determined by means of 1H NMR and CD spectroscopies under equivalent conditions. The chemical shifts (NH and C alpha H) and the analysis of nuclear Overhauser effects show a split of the continuous helix for the all-L peptide into two helices at the position of double D-amino acid replacement. Hydrogen exchange rates correlate with water accessibilities in the hydrophobic/hydrophilic face and confirm the amphipathic helical structure in the all-L peptide as well as in its double D-amino acid analog. A significantly accelerated hydrogen isotope exchange rate is observed for the D-Ala9 backbone proton, implying an increased flexibility at that position. These results show that the incorporation of an adjacent pair of D-amino acids only causes a local change in structure and flexibility, which makes the double D replacement interesting as a tool for specific helix-disturbing modifications to search for helical conformations in biologically active peptides.

Determination of the rotational dynamics and pH dependence of the hydrogen exchange rates of the arginine guanidino group using NMR spectroscopy

The dynamic behaviour of the guanidino group of arginine has been investigated quantitatively with the intention of providing a set of basis values for the interpretation of data acquired for arginine residues in proteins. At room temperature, a single broad resonance line is observed for the four η-NH(2) protons. Upon cooling the sample (≈10°C at 500 MHz), two η-NH(2) proton resonances are resolved which were shown by HMQC spectroscopy to be the result of slowed rotation about the N(ε)-C(ζ) partial double bond. The flip rate (k(NC)) about the N(ε)-C(ζ) bond was measured as a function of temperature using line-shape analysis of both (1)H and (15)N NMR spectra; at 25°C, k(NC) is between 900 and 1000 s(-1). The exchange broadening, due to N(ε)-C(ζ) bond flips, typically results in weak or missing signals for the η-NH(2) protons of arginine residues in HMQC or INEPT experiments recorded at room temperature, unless the motion is restricted in some way. In a related series of experiments, the pH dependence of the hydrogen exchange rates of the ε-NH and η-NH(2) protons of arginine was measured using saturation transfer (1)H NMR spectroscopy and compared with the equivalent NH(2) protons of the guanidinium ion. As expected, OH ion catalysis dominates over most of the pH range and proceeds at a rate close to the diffusion limit for both types of proton (k(OH)=2×10(9)-1×10(10) M(-1)s(-1), depending on conditions). At low pH values, however, catalysis by H(3)O(+) becomes important and leads to characteristic rate minima in the exchange versus pH profiles. Acid catalysis is significantly more effective for the η-NH(2) protons than for the ε-NH proton; at low ionic strength (50 mM KCl) the rate minima occurred at pH 3.6 and 2.3, respectively. Under these conditions, acid-catalysed rate constants (k(H)) of 706 M(-1)s(-1) (η-NH(2)) and 3 M(-1)s(-1) (ε-NH) were obtained at 25°C. At high ionic strength (1 M KCl) the rate of OH(-) ion catalysis is decreased slightly, whereas the H(3)O(+)-catalysed rate is unchanged. The k(OH) value of the free guanidinium ion is identical to that of the η-NH(2) protons but acid catalysis occurs less easily, leading to a rate minimum at pH 3.3.

Hydrogen exchange studies of the Arc repressor: Evidence for a monomeric folding intermediate

The hydrogen exchange rates of the backbone amide and labile side-chain protons of the dimeric Arc repressor have been measured. For the slowly exchanging amides in the alpha-helical regions, these rates show a concentration dependence. To account for this dependence, the role of the monomer-dimer equilibrium was considered. Extrapolating the observed exchange rates to zero dimer concentration provides estimates of these rates in the monomer and shows that they are significantly retarded compared to those of an unfolded polypeptide. This suggests that the monomer is in a structured "molten globule" like state. In particular, the two helices of Arc retain a high degree of their secondary structure and it is proposed that the two amphiphilic helices are packed together with their hydrophobic faces. Evidence for a partially folded structure in the Arc monomer was reported earlier in two other studies [J. L. Silva, C. F. Silveira, A. Correia, Jr., and L. Pontes (1992) Journal of Molecular Biology, Vol. 223, pp. 545-555; X. Peng, J. Jonas, and J. L. Silva (1993) Proceedings of the National Academy of Science USA, Vol. 90, pp. 1776-1780]. By combining the results of these studies and ours, a folding pathway of the dimeric Arc repressor involving four different stages is proposed. Due to the low concentration of Arc repressor in the cell, the protein is present either as a free monomer or it is bound to DNA presumably as a tetramer. Therefore the folding pathway can be regarded as an integral part of the overall DNA binding process.

Hydrogen Exchange and Protein Hydration: The Deuteron Spin Relaxation Dispersions of Bovine Pancreatic Trypsin Inhibitor and Ubiquitin

Water deuteron ( 2H) spin relaxation was used to study hydrogen exchange hydration, and protein dynamics in aqueous soltuions of the globular proteins bovine pancreatic trypsin inhibitor (BPTI) and ubiquitin. The frequency dispersion of the longitudinal 2H relaxation rate was measured in the pD range 2 to 11 at 27°C. In contrast to the previously reported water 17O relaxation dispersion from the same samples, the 2H dispersion depends strongly on pD. This pD dependence is due to labile protein deuterons in acidic side-chains and surface peptide groups, which exchange rapidly with water deuterons. The pD dependence of the 2H relaxation in BPTI solutions could be quantitatively accounted for in terms of known p Kvalues and hydrogen exchange rate constants. For ubiquitin, labile protein deuterons contribute importantly to the 2H relaxation dispersion even in the neutral pD range. The 2H relaxation data also provided information about the orientational order and internal motion of OD and ND bonds in side-chains and surface peptides. A comparison of the water contribution to the 2H dispersion with the 17O dispersion indicates that one of the four internal water molecules of BPTI, presumably the deeply buried water molecule W122, exchanges more slowly (10 −6to 10 −4second) than the other three (10 −8to 10 −6second).

Mutations of surface residues in Anabaena vegetative and heterocyst ferredoxin that affect thermodynamic stability as determined by guanidine hydrochloride denaturation.

The stability properties of oxidized wild-type (wt) and site-directed mutants in surface residues of vegetative (Vfd) and heterocyst (Hfd) ferredoxins from Anabaena 7120 have been characterized by guanidine hydrochloride (Gdn-HCl) denaturation. For Vfd it was found that mutants E95K, E94Q, F65Y, F65W, and T48A are quite similar to wt in stability. E94K is somewhat less stable, whereas E94D, F65A, F65I, R42A, and R42H are substantially less stable than wt. R42H is a substitution found in all Hfds, and NMR comparison of the Anabaena 7120 Vfd and Hfd showed the latter to be much less stable on the basis of hydrogen exchange rates (Chae YK, Abildgaard F, Mooberry ES, Markley JL, 1994, Biochemistry 33:3287-3295); we also find this to be true with respect to Gdn-HCl denaturation. Strikingly, the Hfd mutant H42R is more stable than the wt Hfd by precisely the amount of stability lost in Vfd upon mutating R42 to H (2.0 kcal/mol). On the basis of comparison of the X-ray crystal structures of wt Anabaena Vfd and Hfd, the decreased stabilities of F65A and F65I can be ascribed to increased solvent exposure of interior hydrophobic groups. In the case of Vfd mutants E94K and E94D, the decreased stabilities may result from disruption of a hydrogen bond between the E94 and S47 side chains. The instability of the R42 mutants is also most probably due to decreased hydrogen bonding capabilities.(ABSTRACT TRUNCATED AT 250 WORDS)