NH Exchange Rates Research Articles

Residue-specific NH exchange rates studied by NMR diffusion experiments

We present a novel approach to the investigation of rapid (>2 s −1) NH exchange rates in proteins, based on residue-specific diffusion measurements. 1H, 15N-DOSY-HSQC spectra are recorded in order to observe resolved amide proton signals for most residues of the protein. Human ubiquitin was used to demonstrate the proposed method. Exchange rates are derived directly from the decay data of the diffusion experiment by applying a model deduced from the assumption of a two-site exchange with water and the “pure” diffusion coefficients of water and protein. The “pure” diffusion coefficient of the protein is determined in an experiment with selective excitation of the amide protons in order to suppress the influence of magnetization transfer from water to amide protons on the decay data. For rapidly exchanging residues a comparison of our results with the exchange rates obtained in a MEXICO experiment showed good agreement. Molecular dynamics (MD) and quantum mechanical calculations were performed to find molecular parameters correlating with the exchangeability of the NH protons. The RMS fluctuations of the amide protons, obtained from the MD simulations, together with the NH coupling constants provide a bilinear model which shows a good correlation with the experimental NH exchange rates.

Effects of Co-operative Ligand Binding on Protein Amide NH Hydrogen Exchange

Amide protection factors have been determined from NMR measurements of hydrogen/deuterium amide NH exchange rates measured on assigned signals from Lactobacillus casei apo-DHFR and its binary and ternary complexes with trimethoprim (TMP), folinic acid and coenzymes (NADPH/NADP(+)). The substantial sizes of the residue-specific DeltaH and TDeltaS values for the opening/closing events in NH exchange for most of the measurable residues in apo-DHFR indicate that sub-global or global rather than local exchange mechanisms are usually involved. The amide groups of residues in helices and sheets are those most protected in apo-DHFR and its complexes, and the protection factors are generally related to the tightness of ligand binding. The effects of ligand binding that lead to changes in amide protection are not localised to specific binding sites but are spread throughout the structure via a network of intramolecular interactions. Although the increase in protein stability in the DHFR.TMP.NADPH complex involves increased ordering in the protein structure (requiring TDeltaS energy) this is recovered, to a large extent, by the stronger binding (enthalpic DeltaH) interactions made possible by the reduced motion in the protein. The ligand-induced protection effects in the ternary complexes DHFR.TMP.NADPH (large positive binding co-operativity) and DHFR.folinic acid.NADPH (large negative binding co-operativity) mirror the co-operative effects seen in the ligand binding. For the DHFR.TMP.NADPH complex, the ligand-induced protection factors result in DeltaDeltaG(o) values for many residues being larger than the DeltaDeltaG(o) values in the corresponding binary complexes. In contrast, for DHFR.folinic acid.NADPH, the DeltaDeltaG(o) values are generally smaller than many of those in the corresponding binary complexes. The results indicate that changes in protein conformational flexibility on formation of the ligand complex play an important role in determining the co-operativity in the ligand binding.

Residue specific studies of NH exchange rates performed on ubiquitin

Residue specific studies of NH exchange rates performed on ubiquitin

Solution structure, mutagenesis, and NH exchange studies of the MutT enzyme–Mg 2+-8-oxo-dGMP complex

The MutT pyrophosphohydrolase from E. coli (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP), including 8-oxo-dGTP, by substitution at Pβ, to yield NMP and pyrophosphate. The product, 8-oxo-dGMP is an unusually tight binding, slowly exchanging inhibitor with a K D=52 nM, (Δ G°=−9.8 kcal/mol) which is 6.1 kcal/mol tighter than the binding of dGMP (Δ G°=−3.7 kcal/mol). The higher affinity for 8-oxo-dGMP results from a more favorable Δ H binding (−32 kcal/mol) despite an unfavorable − TΔ S° binding (+22 kcal/mol). The solution structure of the MutT–Mg 2+-8-oxo-dGMP complex shows a narrowed, hydrophobic nucleotide-binding cleft with Asn-119 and Arg-78 among the few polar residues. The N119A, N119D, R78K and R78A single mutations, and the R78K+N119A double mutant all showed largely intact active sites, on the basis of small changes in the kinetic parameters of dGTP hydrolysis and in 1H– 15N HSQC spectra. However, the N119A mutation profoundly weakened the active site binding of 8-oxo-dGMP by 4.3 kcal/mol (1650-fold). The N119D mutation also weakened 8-oxo-dGMP binding but only by 2.1 kcal/mol (37-fold), suggesting that Asn-119 functioned both as a hydrogen bond donor to C8O, and a hydrogen bond acceptor from N7H of 8-oxo-dGMP, while aspartate at position −119 functioned as an acceptor of a single hydrogen bond. Much smaller weakening effects (0.3–0.4 kcal/mol) on the binding of dGMP and dAMP were found, indicating specific hydrogen bonding of Asn-119 to 8-oxo-dGMP. While formation of the wild type MutT–Mg 2+-8-oxo-dGMP complex slowed the backbone NH exchange rates of 45 residues distributed throughout the protein, the same complex of the N119A mutant slowed the exchange rates of only 11 residues at or near the active site, indicating an increase in conformational flexibility of the N119A mutant. The R78K and R78A mutations weakened the binding of 8-oxo-dGMP by 1.7 and 1.1 kcal/mol, respectively, indicating a lesser role of Arg-78 than of Asn-119 in the selective binding of 8-oxo-dGMP, likely donating a single hydrogen bond to its C6O. The R78K+N119A double mutant weakened the binding of 8-oxo-dGMP ( K I slope=3.1 mM) by 6.5±0.2 kcal/mol which overlaps, within error with the sum of the effects of the two single mutants (6.0±0.3 kcal/mol). Such additive effects of the two single mutants in the double mutant are most simply explained by the independent functioning of Asn-119 and Arg-78 in the binding of 8-oxo-dGMP. Independent functioning of these two residues in nucleotide binding is consistent with their locations in the MutT–Mg 2+-8-oxo-dGMP complex, on opposite sides of the active site cleft, with a distance of 8.4±0.5 Å between their side chain nitrogens.

Mutational, NMR, and NH exchange studies of the tight and selective binding of 8-oxo-dGMP by the MutT pyrophosphohydrolase.

The solution structure of the ternary MutT enzyme-Mg(2+)-8-oxo-dGMP complex showed the proximity of Asn119 and Arg78 and the modified purine ring of 8-oxo-dGMP, suggesting specific roles for these residues in the tight and selective binding of this nucleotide product [Massiah, M. A., Saraswat, V., Azurmendi, H. F., and Mildvan, A. S. (2003) Biochemistry 42, 10140-10154]. These roles are here tested by mutagenesis. The N119A, N119D, R78K, and R78A single mutations and the R78K/N119A double mutant showed very small effects on k(cat) (<or=2-fold) and K(m) (<or=4-fold) in the hydrolysis of dGTP, indicating largely intact active sites. (1)H-(15)N HSQC spectra showed largely intact protein structures for all of these mutants. However, the N119A mutation profoundly and selectively weakened the active site binding of 8-oxo-dGMP, increasing the K(I)(slope) of this product inhibitor 1650-fold, while increasing the K(I)(slope) of dGMP and dAMP less than 2-fold. The N119D mutation also selectively weakened 8-oxo-dGMP binding but only by 37-fold, suggesting that Asn119 both donated a hydrogen bond to the C8=O group and accepted a hydrogen bond from the N7H group of 8-oxo-dGMP, while Asp119 functioned as only an acceptor. Direct binding of 8-oxo-dGMP to N119A, monitored by continuous changes in the (15)N and/or NH chemical shifts of 12 residues, revealed fast exchange, and a K(D) of 237 +/- 130 microM for 8-oxo-dGMP, comparable to its K(I)(slope) of 81 +/- 22 microM. While formation of the wild-type MutT-Mg(2+)-8-oxo-dGMP complex slowed the backbone NH exchange rates of 45 residues distributed throughout the protein, the same complex of the N119A mutant slowed the exchange rates of only 11 residues at or near the active site, indicating an increase in the conformational flexibility of the N119A mutant. The R78K and R78A mutations selectively increased the K(I)(slope) of 8-oxo-dGMP by factors of 17 and 6.6, respectively, indicating a smaller role for Arg78 than for Asn119 in the binding of 8-oxo-dGMP, likely donating a hydrogen bond to its C6=O group. The much greater contribution of Asn119 (4.0 kcal/mol) than of Arg78 (1.0 kcal/mol) to the selectivity of binding of 8-oxo-dGMP versus dGMP indicates a 2 order of magnitude smaller contribution of a structure with the reversed orientation of the 8-oxo-dG ring. The R78K/N119A double mutant weakened the binding of 8-oxo-dGMP by a factor (63,000 +/- 22,000) which overlaps within error with the product of the effects of the two single mutants (28,000 +/- 15,000). Such additive effects of the two single mutants in the double mutant are most simply explained by the independent functioning of Asn119 and Arg78 in the binding of 8-oxo-dGMP. Independent functioning of these two residues in nucleotide binding is consistent with their locations in the MutT-Mg(2+)-8-oxo-dGMP complex, on opposite sides of the active site cleft, with a minimal distance of 8.4 +/- 0.5 A between their side chain nitrogens.

Solution structures of micelle-bound amyloid β-(1-40) and β-(1-42) peptides of Alzheimer’s disease

The amyloid β-peptide is the major protein constituent of neuritic plaques in Alzheimer’s disease. The β-peptide varies slightly in length and exists in two predominant forms: (1) the shorter, 40 residue β-(1-40), found mainly in cerebrovascular amyloid; and (2) the longer, 42 residue β-(1-42), which is the major component in amyloid plaque core deposits. We report here that the sodium dodecyl sulphate (SDS) micelle, a membrane-mimicking system for biophysical studies, prevents aggregation of the β-(1-40) and the β-(1-42) into the neurotoxic amyloid-like, β-pleated sheet structure, and instead encourages folding into predominantly α-helical structures at pH 7.2. Analysis of the nuclear Overhauser enhancement (NOE) and the αH NMR chemical shift data revealed no significant structural differences between the β-(1-40) and the β-(1-42). The NMR-derived, three-dimensional structure of the β-(1-42) consists of an extended chain (Asp1-Gly9), two α-helices (Tyr10-Val24 and Lys28-Ala42), and a looped region (Gly25-Ser26-Asn27). The most stable α-helical regions reside at Gln15-Val24 and Lys28-Val36. The majority of the amide (NH) temperature coefficients were less than 5, indicative of predominately strong NH backbone bonding. The lack of a persistent region with consistently low NH coefficients, together with the rapid NH exchange rates in deuterated water and spin-labeled studies, suggests that the β-peptide is located at the lipid-water interface of the micelle and does not become inbedded within the hydrophobic interior. This result has implications for the circulation of membrane-bound β-peptide in biological fluids, and may also facilitate the design of amyloid inhibitors to prevent an α-helix→β-sheet conversion in Alzheimer’s disease.

Determination of the secondary structure and global topology of the 44 kda ectodomain of gp41 of the simian immunodeficiency virus by multidimensional nuclear magnetic resonance spectroscopy

The gp41 protein of the human (HIV) and simian (SIV) immunodeficiency viruses is part of the envelope glycoprotein complex gp41/gp120 which plays an essential role in viral infection. We present a multidimensional NMR study on the trimeric 44 kDa soluble ectodomain of SIV gp41 (e-gp41), comprising residues 27 to 149. Despite the large molecular weight and very limited spectral dispersion, complete backbone 1H, 13C, 13CO and 15N assignments have been made using a combination of triple resonance experiments on uniformly 13C/ 15N and 2H/ 13C/ 15N-labeled samples. The secondary structure of SIV e-gp41, derived on the basis of 13C chemical shifts, NH exchange rates, medium range nuclear Overhauser enhancements (NOE), and 3 J HNα coupling constants, consists of a 49 residue helix at the N terminus (residues 29 to 77) and a 40 residue helix at the C terminus (residues 108 to 147), connected by a 30 residue loop which does not display any of the characteristics of regular secondary structure. The cross-peak intensities of the loop region in scalar correlation experiments suggests that it is more mobile than the core helical regions. The presence, however, of numerous long range NOEs, both intra and inter-subunit, within the loop indicates that it adopts a well-defined structure in which the loops from the three subunits interact with each other. Based on a number of long range intra and inter-subunit NOEs, a topological model is presented for the symmetric SIV e-gp41 trimer in which the N-terminal helices are packed within the protein interior in a parallel trimeric coiled-coil arrangement, while the C-terminal helices are located on the protein exterior, oriented antiparallel to the N-terminal helices.

Structural transitions of a GG-platinated DNA duplex induced by pH, temperature and box A of high-mobility-group protein 1.

[1H, 15N] and 1H NMR, and CD spectroscopy are used to show that the duplex d(A-T-A-C-A-T-Pt 7G-Pt7G-T-A-C-A-T-A).d(T-A-T-G-T-A-C-C-A-T-G-T-A-T), where Pt7G is platinated guanine, containing the cis-[Pt(NH3)2]2+ adduct, undergoes reversible temperature-induced (T0.5 310 K) and pH-induced (pKa approximately 4.8) transitions between kinked-duplex and distorted forms, with the latter forms predominating at high temperature and low pH. A related pH-induced structural change was observed for the unplatinated duplex (pKa 4.69, Hill coefficient n = 1.4) but was less cooperative than for the platinated duplex (n = 2). The pH-induced transition is attributed to protonation of cytosine residues and has wider implications, since many reported NMR studies of DNA are carried out near pH 5 to minimize NH-exchange rates. The [Pt(en)]2+ (where en is 1,2-ethanediamine) GG chelate of the same duplex is shown to exist in kinked and distorted forms, and the [1H,15N]-NMR shifts for the kinked form are indicative of the presence of highly stereospecific interactions with the Pt-NH protons. On binding of the duplex platinated with [Pt(NH3)2]2+ to high-mobility-group protein 1 (HMG1) box A, similar changes in shifts of the Pt-NH3 resonances to those induced by raising the temperature or lowering the pH were observed. The specific changes in 1H-NMR chemical shifts of HMG1 box A are consistent with binding of the platinated duplex (intermediate exchange rate on the 1H-NMR time-scale) to the concave face of the protein via helices I and II and the intervening loop.

Sequential 1H and 15N nuclear magnetic resonance assignments and secondary structure of the lipoyl domain of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii. Evidence for high structural similarity with the lipoyl domain of the pyruvate dehydrogenase complex.

A 79-amino-acid polypeptide, corresponding to the lipoyl domain of the succinyltransferase component of the 2-oxoglutarate dehydrogenase multienzyme complex from Azotobacter vinelandii, has been sub-cloned and produced in Escherichia coli. Complete sequential 1H and 15N resonance assignments for the lipoyl domain have been obtained by using homo- and hetero-nuclear NMR spectroscopy. Two antiparallel beta-sheets of four strands each were identified from characteristic NOE connectivities and 3JHN alpha values. The lipoyl-lysine residue is found in a type-I turn connecting two beta-strands. The secondary structure of the lipoyl domain very much resembles the secondary solution structure of the N-terminal lipoyl domain of the A. vinelandii pyruvate dehydrogenase complex, despite the sequence identity of 25%. A detailed comparison of the NMR-derived parameters of both lipoyl domains, i.e. chemical shifts, NH-exchange rates, NOEs, and 3JHN alpha values suggests a high structural similarity in solution between the two lipoyl domains. Preliminary tertiary-structure calculations confirm that these lipoyl domains have very similar overall folds. The observed specificity of the 2-oxo acid dehydrogenase components of both complexes for these lipoyl domains is discussed in this respect.

A heteronuclear correlation experiment for simultaneous determination of 15N longitudinal decay and chemical exchange rates of systems in slow equilibrium

A heteronuclear correlation experiment is described which permits simultaneous characterization of both 15N longitudinal decay rates and slow conformational exchange rates. Data pertaining to the exchange between folded and unfolded forms of an SH3 domain is used to illustrate the technique. Because the unfolded form of the molecule, on average, shows significantly higher NH exchange rates than the folded form, an approach which minimizes the degree of water saturation is employed, enabling the extraction of accurate rate constants.

Exchange kinetics of individual amide protons in 15N-labeled helical peptides measured by isotope-edited NMR.

Amide proton exchange measured by one-dimensional 15N-edited proton NMR has been used to probe helical structure in an alanine-based peptide. This study is the first report of individual peptide NH exchange rates determined in a simple, repeating sequence peptide whose helical structure can be predicted by helix-coil theory. Measured protection factors directly demonstrate that the ends of the helix are frayed. The protection factors are compared to the Lifson-Roig theory, modified to include N-capping, using known values for helix propensities and N-cap propensities. Base-catalyzed exchange rates are shown to measure the extent of hydrogen bonding of the peptide NHs, and the results are fitted by a simple model in which hydrogen bonding of the peptide NH group provides protection and no exchange occurs from the hydrogen-bonded state. Protection from acid-catalyzed exchange correlates with hydrogen bonding by both the NH and CO groups of a peptide unit: the data are fitted by a model in which exchange occurs only when both hydrogen bonds formed by a peptide unit are broken. This result indicates that acid-catalyzed exchange occurs by the O-protonation mechanism, in agreement with earlier work [Perrin & Arrhenius (1982) J. Am. Chem. Soc. 104, 6693-6696; Perrin et al. (1984) J. Am. Chem. Soc. 106, 2749-2753; Tüchsen & Woodward (1985) J. Mol. Biol. 185, 421-430].

Structure and dynamics of cytochrome c in nonaqueous solvents by 2D NH-exchange NMR spectroscopy

Recent biocatalysis studies have shown that many enzymes exhibit catalytic activity and enhanced thermal stability in essentially anhydrous nonpolar solvents. At present we do not understand the important role that water plays in protein structure stabilization and in enzyme mechanisms in nonaqueous environments. In this study, the NH-exchange rates of oxidized horse cytochrome c suspended in tetrahydrofuran (THF) containing 1% D 2 O (vol/vol) at 37 o C, pH 8.9, have been determined indirectly by 2D NH-exchange NMR spectroscopy. In such a solvent system, we have allowed just enough water molecules to form approximately a monolayer surrounding the protein

Conformational isomerism of endothelin in acidic aqueous media: a quantitative NOESY analysis

The conformational features of endothelin-1 (ET-1) in mixed water/ethylene glycol media have been studied by two-dimensional 1H NMR experiments throughout the pH range 3.2-7.2. At pH less than 5 all backbone NH signals can be observed, and NOESY experiments provided a large set of dipolar cross-peaks. Cross-peak intensities from each experiment (different mixing times and H2O versus D2O) were converted to distance constraints using a novel algorithm (program DISCON) for removing spin diffusion effects and thus obtain cross-rates rather than cross-peak intensities. A set of 168 nonstereospecific distance bounds (average experimental precision, +/- 0.38 A) was used in dynamics simulated annealing refinements. Two consensus structural features were found--a reverse turn at Ser5----Asp8 and an alpha-helical stretch from Lys9 to Cys15; however, after constraint-free minimization, structures generated using XPLOR-1.5, CONGEN, and DISCOVER all violated at least 32% of the bounds by more than 0.2 A, which we ascribe to conformational isomerism. When the constraints were modified to reflect subsequent experimental data and to eliminate constraints that could not be obeyed by any single conformer structure, the relaxed structures still violated at least 15% of this more limited and looser set of constraints. Therefore, a modified procedure for constrained dynamics refinement (using XPLOR-2.1), which allows for conformational isomerism outside of the central helical core region, was developed. This "conformer search procedure" produced structures which fell into five tightly defined conformational clusters. The two most populated clusters correspond to a rotation of the 8,9-amide unit. The conformer which we propose as the major contributor at pH 3.2-5.8 was defined to a backbone rmsd of 0.51 A over residues 1----15. An alternative description of the motional averaging in segments of the endothelin structure as extensive randomization rather than rapid interconversion between a small number of discreet conformers was ruled out by an analysis of NH shift-temperature gradients and exchange rates. This analysis suggests that small delta delta/delta T values need not correlate with H-bonding for conformational mixtures. In ET-1 the greatest motional averaging occurs from Ser2 through Ser5 (not in the C-terminus) and may be so extensive as to approximate a flexible random coil population as high as 30%. The C-terminus shows less rapid and less extensive conformational averaging, but no definitive structures for individual conformers could be derived in the absence of stereospecific constraints. The pharmacological implications of the consensus structural features are discussed.

Two- and three-dimensional 1H NMR studies of a wheat phospholipid transfer protein: sequential resonance assignments and secondary structure.

Two- and three-dimensional 1H NMR experiments have been used to sequentially assign nearly all proton resonances of the 90 residues of wheat phospholipid transfer protein. Only a few side-chain protons were not identified because of degeneracy or overlapping. The identification of spin systems and the sequential assignment were made at the same time by combining the data of the two- and three-dimensional experiments. The classical two-dimensional COSY, HOHAHA, and NOESY experiments benefit from both good resolution and high sensitivity, allowing the detection of long-range dipolar connectivities. The three-dimensional HOHAHA-NOESY experiment offers the advantage of a faster and unambiguous assignment. As a matter of fact, homonuclear three-dimensional NMR spectroscopy proved to be a very efficient method for resonance assignments of protein 1H NMR spectra which cannot be unraveled by 2D methods. An assignment strategy which overcomes most of the ambiguities has been proposed, in which each individual assignment toward the C-terminal end is supported by another in the opposite direction originating from a completely different part of the spectrum. Location of secondary structures of the phospholipid transfer protein was determined by using the method of analysis introduced here and was confirmed by 3J alpha NH coupling and NH exchange rates. Except for the C-terminal part, the polypeptide chain appears to be organized mainly as helical fragments connected by disulfide bridges. Further modeling will display the overall folding of the protein and should provide a better understanding of its interactions with lipids.

Calcium binding to calbindin D9k strongly affects backbone dynamics: measurements of exchange rates of individual amide protons using proton NMR

One- and two-dimensional 1H NMR have been used to study the backbone dynamics in Ca2(+)-free (apo) and Ca2(+)-loaded (Ca2) calbindin D9k at pH 7.5 and 25 degrees C. Hydrogen exchange rates of all 71 backbone amide protons (NH's) have been measured for the Ca2 form by both a direct exchange-out experiment and another experiment that measures the transfer of saturation from water protons to amide protons. A large number of NH's are found to be highly protected against exchange with solvent protons. The results for the Ca2 form are related to solvent accessibility and hydrogen bonding obtained in molecular dynamics simulations of calcium-loaded calbindin. The correlation with these parameters is strong within the N-terminal half of calbindin, which is found to be more stable than the C-terminal half. The amide proton exchange in the apo form is much faster than in the Ca2 form and was studied in a series of experiments in which the exchange was quenched after different times by Ca2+ addition. This experiment is applicable to all amide hydrogens that exchange slowly in the Ca2 form. For these NH's the effects of Ca2+ removal span from a 10(2)-fold decrease to a 10(5)-fold increase of the exchange rate, and the average is a 220-fold increase. The effects on individual NH exchange rates show that the four alpha-helices are almost intact after calcium removal and that the changes in dynamics involve not only the Ca2(+)-binding region. Hydrogen bonds involving backbone NH's in the Ca2+ loops appear to be broken or weakened when calbindin releases Ca2+, whereas the beta-sheet between the Ca2+ loops is found to be present in both the Ca2 and apo forms. Large Ca2(+)-induced effects on NH exchange rates were measured for a few residues at alpha-helix ends far from the two Ca2(+)-binding sites. This may be the result of a change in interhelix angles (or the rate of interhelix angle fluctuations) on calcium binding.

Proton magnetic resonance study of the influence of chemical modification, mutation, quaternary state, and ligation state on dynamic stability of the heme pocket in hemoglobin as reflected in the exchange of the proximal histidyl ring labile proton

Proton nuclear magnetic resonance spectroscopy has been utilized to investigate the rates of exchange with deuterium of the proximal histidyl ring protons in a series of chemically modified and mutated forms of Hb A. Differences in rates of exchange are related to differences in the stability of the deformed or partially unfolded intermediates from which exchange with bulk solvent takes place. Each modified/mutated Hb exhibited kinetic subunit heterogeneity in the reduced ferrous state, with the alpha subunit exhibiting faster exchange than the beta subunit. Modification or mutation resulted in significant increases in the His F8 ring NH exchange rates primarily for the affected subunit and only if the modification/mutation occurs at the allosterically important alpha 1 beta 2 subunit interface. Moreover, this enhancement in exchange rate is observed primarily in that quaternary state of the modified/mutated Hb in which the modified/substituted residue makes the intersubunit contact. This confirms the importance of allosteric constraints in determining the dynamic properties of the heme pocket. Using modified or mutated Hbs that can switch between the alternate quaternary states within a given ligation state or ligate within a given quaternary state, we show that the major portion of the enhanced exchange rate in R-state oxy Hb relative to T-state deoxy Hb originates from the quaternary switch rather than from ligation. However, solely ligation effects are not negligible. The exchange rates of the His F8 ring labile protons increase dramatically upon oxidizing the iron to the ferric state, and both the subunit kinetic heterogeneity and the allosteric sensitivity to the quaternary state are essentially abolished.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphate catalyzed exchange rates of NH-N protons of DNA.

We have studied the catalysis of the exchange of the hydrogen-bonded NH-N protons of the short DNA helix (d-CCAAGCTTGG)2 by phosphate addition. The NH exchange rates were monitored by the line widths of the corresponding NH resonances in the 1H nmr spectra. The exchange catalyst phosphate is most effective on the exchange rate of the terminal CG1 base pairs. However, all internal base pairs are also moderately affected by phosphate which suggests an exchange mechanism governed by a fast equilibrium between opened and closed states of the duplex. Within the limits of error the same effectiveness of phosphate on the exchange rate of all internal NH-N protons has been observed. With the exception of the terminal base pairs, no sequence and/or position specificity of the exchange rates of the NH-N protons of the base pairs has been found.

High-resolution NMR studies of fibrinogen-like peptides in solution: resonance assignments and conformational analysis of residues 1-23 of the A alpha chain of human fibrinogen.

The proton resonances of the following synthetic linear human fibrinogen-like peptides were completely assigned with two-dimensional NMR techniques in solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp(7)-Phe-Leu-Ala-Glu-Gly(12)-Gly(13)-Gly(14)- Val(15)-Arg(16)-Gly-Pro-Arg-Val-Val-Glu-Arg (F10), Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly(13)-Gly(14)-Val-Arg (F11), and Gly-Pro-Arg-Val-Val-Glu-Arg (F12). No predominant structure was found in the chain segment from Ala(1) to Gly(6) for F10 in both H2O and dimethyl sulfoxide. The previous suggestion that there is a hairpin loop involving residues Gly(12) to Val(15) in the A alpha chain of human fibrinogen is supported by the slow backbone NH exchange rates of Gly(14) and Val(15), by an unusually small NH chemical shift of Val(15), and by strong sequential NOE's involving this region in F10. This local chain fold within residues Asp(7) to Val(20) may place the distant Phe residue near the Arg(16)-Gly(17) peptide bond which is cleaved by thrombin.

Amide proton exchange rates of a bound pepsin inhibitor determined by isotope-edited proton NMR experiments

From a series of isotope-edited proton NMR spectra, amide proton exchange rates were measured at 20 degrees C, 30 degrees C, and 40 degrees C for a tightly bound 15N-labeled tripeptide inhibitor of porcine pepsin (IC50 = 1.7 X 10(-) M). Markedly different NH exchange rates were observed for the three amide protons of the bound inhibitor. The P1 NH exchanged much more slowly than the P2 NH and P3 NH. These results are discussed in terms of the relative solvent accessibility in the active site and the role of the NH protons of the inhibitor for hydrogen bonding to the enzyme. In this study a useful approach is demonstrated for obtaining NH exchange rates on ligands bound to biomacromolecules, the knowledge of which could be of potential utility in the design of therapeutically useful nonpeptide enzyme inhibitors from peptide leads.

Nuclear magnetic resonance study of the isotope exchange of the proximal histidyl ring labile protons in hemoglobin A: The exchange rates and mechanisms of individual subunits in deoxy and oxy-hemoglobin

Proton nuclear magnetic resonance spectroscopy has been used to investigate the rates and mechanism of exchange with deuterium of the proximal histidyl imidazole labile ring proton in deoxy and oxy-hemoglobin A. The resolved signals for the two subunits indicate dynamic heterogeneity, with the exchange rate always faster in the a than the β subunits, suggesting a lower dynamic stability for the α subunit. The activation energy for the exchange in both subunits (~25 kcal; 1 cal = 4.184 J) indicates that exchange proceeds via an intermediate far from denaturation or global unfolding. The pH profiles for both hemoglobin states reflect the EX 2 mechanism for both subunits. While the base catalysis expected for an iron-bound imidazole is observed in all cases, there are important differences in both rates and mechanisms between the subunits. In deoxy-hemoglobin, both base-catalyzed and water-assisted exchange contribute to the α subunit, but only the former to the β subunit. For oxy-hemoglobin, the base-catalysis is retained for both subunits, but the slope is considerably less for the α relative to the β subunit. Thus the two subunits in the two states of hemoglobin differ both in mechanisms and in the inherent dynamic stability reflected in any one mechanism. The relationships of the proximal histidyl ring NH exchange rates to previously characterized subsets of allosterically responsive protons in hemoglobin A is briefly discussed.