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1.
With proton nuclear magnetic resonance spectroscopy at 22 degrees C and pD 4.5, individual exchange rates in the range from 2 X 10(-5) to 1 X 10(-1) min-1 were observed for 23 amide protons in recombinant desulfatohirudin. The remaining 38 backbone amide protons exchange more rapidly than 1 X 10(-1) min-1. All 23 slowly exchanging protons are located in the polypeptide segment from residue 4 to residue 42, which forms a well-defined globular domain. Three different breathing modes of this molecular region are manifested in the exchange data, which appear to be correlated with the location of the three disulfide bonds. Chemical shift changes larger than 0.15 ppm between pH 2.5 and pH 5.0 arising from through-space interactions with carboxyl groups were observed for seven backbone amide protons. Two of these shifts can be explained by hydrogen bonds in the core of the protein, Gly 25 NH-Glu 43 O epsilon and Ser 32 NH-Asp 33 O delta, and two others by intraresidual NH-O epsilon interactions in Glu 61 and Glu 62. The remaining three pH shifts for Glu 35, Cys 39, and Ile 59 imply the existence of transient interactions between the molecular core and the flexible C-terminal segment 49-65, which have so far not been characterized by nuclear Overhauser effects or other conformational constraints. 相似文献
2.
The non-detectability of NH...N hydrogen bonds in nucleic acids due to exchange broadened imino/amino protons has recently been addressed via the use of non-exchangeable protons for detecting internucleotide 2hJ(NN) couplings. In these applications, the appropriate non-exchangeable proton is separated by two bonds from the NH...N bond. In this paper, we extend the scope of this approach to protons which are separated by four bonds from the NH...N moiety. Specifically, we consider the case of the commonly occurring sheared G x A mismatch alignment, in which we use the adenine H2 proton to report on the (A)N6H6(1.2)...N3(G) hydrogen bond, in the presence of undetectable, exchange broadened N6H6(1.2) protons. Two sequences, the 'straight-through' (H6)N6N3H2 and 'out-and-back' H2N6N3 experiments, are presented for observing these correlations in H2O and D2O solution, respectively. The sequences are demonstrated on two uniformly 15N,13C labelled DNA samples: d(G1G2G3T4T5C6A7G8G9)2, containing a G3 x (C6-A7) triad involving a sheared G3 x A7 mismatch, and d(G1G2G3C4A5G6G7T8)4, containing an A5 x (G3 x G6 x G3 x G6) x A5 hexad involving a sheared G3 x A5 mismatch. 相似文献
3.
NMR spin grouping and correlation exchange analysis. Application to low hydration NaDNA paracrystals. 总被引:3,自引:3,他引:0
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The NMR spin-grouping technique is applied to low hydration oriented fibers of NaDNA to study the role of exchange in determining the apparent (observed) spin relaxation of the system. The analysis proceeds in three steps: first, the apparent proton relaxation is measured at high fields, with both selective and nonselective inversion pulse sequences, and in the rotating frame. The spin-grouping technique is used in all spin-lattice relaxation measurements to provide the optimum apparent relaxation characterization of the sample. Next, all apparent results are analyzed for exchange. In this analysis the results from the high field and rotating frame experiments (which probe the exchange at two different time scales) are correlated to determine the inherent (or true) spin relaxation parameters of each of the proton groups in the system. The results of selective inversion T1 measurements are also incorporated into the exchange analysis. Finally, the dynamics of each spin group are inferred from the inherent relaxation characterization. The low hydration NaDNA structure is such that the exchange between the protons on the water and those on the NaDNA is limited, a priori, to dipolar mixing. The results of the exchange analysis indicate that the dipolar mixing between water and NaDNA protons is faster than the spin diffusion within the NaDNA proton group itself. The spin-diffusion on the macromolecule is the bottleneck for the exchange between the water protons and the NaDNA protons. The water protons serve as the relaxation sink both at high fields and in the rotating frame for the total NaDNA-water spin bath. The inherent relaxation of the water is characteristic of water undergoing anisotropic motion with a fast reorientational correlation time about one axis (5 X 10(-10) less than or equal to tau r less than or equal to 8 X 10(-9)S) which is about three orders of magnitude slower than that of water in the bulk; and a slow tumbling correlation time for this axis (1.5 x 10(-7) less than or equal to tau t less than or equal to 8 x 10(-7)S) which is two orders of magnitude slower yet. 相似文献
4.
S-3',4'-anhydroadenosyl-l-methionine is an analogue of the S-adenosyl-l-methionine coenzyme. Here we report on a rapid solvent exchange of the methylene protons at the 5'-position of this analogue. The rate of H/D exchange was measured by nuclear magnetic resonance spectroscopy under buffered conditions in deuterium oxide. The reaction is specific base catalyzed and displays a second-order rate constant of 2 x 10(4) M(-1) s(-1), which corresponds to a rate enhancement of 10(12) compared to solvent exchange of alpha-methylene protons in acyclic, aliphatic sulfonium ions. No other carbon bonded hydrogens in the molecule exchange with solvent under the experimental conditions. Allylic stabilization of a carbanionic-like transition state for the solvent exchange process can account for these results. Solvent exchange under these mild conditions provides a simple way to prepare a 5'-2H-labeled form of the coenzyme analogue. 相似文献
5.
S Roy C Dibello H Taniuchi 《International journal of peptide and protein research》1986,27(2):165-174
RNase-(1-118) containing native disulfide bonds is similar in fold to native RNase A but not of lowest Gibbs energy as compared with the isomers containing non-native disulfide bonds. The present n.m.r. studies have indicated a dramatic increase in the exchange rate of all of the 'protected' amide protons of RNase-(1-118) over RNase A. A calculation shows a large increase in the rate of 'opening' of the structure. The exchange rate of the protected amide protons of RNase-(1-120) is slower than RNase-(1-118) but much faster than RNase A. Binding with a synthetic complementing fragment (114-124) markedly reduces the exchange rate of 20 to 25 amide protons of RNase-(1-118). It has previously been shown that binding with a complementing fragment of RNase-(1-118) generates a lowest Gibbs energy state. Thus, using available thermodynamic information for interpretation, we suggest that a) removal of six carboxy terminal residues of RNase A would disrupt coupling between these residues and those distant in the structure (loss of extra stabilizing energy), b) this would, in turn, alter the enthalpy-entropy compensation in such a way that the magnitude of Gibbs energy change favoring folding is significantly reduced without a large change of fold and c) in this activated state the molecule would be highly motile. 相似文献
6.
A theory of radiationless transitions of protons of the hydrogen bonds between the paired DNA bases is constructed. The lifetime of excited h-b-1 bond in guanine-cytosine pair is found to be 4.6 x 10(-9) sec. The dependence of frequencies, amplitudes and normal coordinates of heavy atoms (N, O) on the state of H-bond (ground or excited) are taken into account. 相似文献
7.
8.
Amide proton exchange in proteins by EX1 kinetics: studies of the basic pancreatic trypsin inhibitor at variable p2H and temperature 总被引:8,自引:0,他引:8
With the use of one-dimensional 1H nuclear magnetic resonance, two-dimensional correlated spectroscopy, and two-dimensional nuclear Overhauser enhancement spectroscopy, the exchange mechanisms for numerous individual amide protons in the basic pancreatic trypsin inhibitor (BPTI) were investigated over a wide range of p2H and temperature. Correlated exchange under an EX1 regime was observed only for the most slowly exchanging protons in the central hydrogen bonds of the antiparallel beta-sheet and only over a narrow range of temperature and p2H, i.e., above ca. 55 degrees C and between p2H 7 and 9, where the opening rates of the structure fluctuations which promote the exchange of these protons are of the order 0.1 min-1. At p2H below 7, the exchange of this most stable group of protons is uncorrelated and is governed by an EX2 mechanism. At p2H above 9, the exchange is also uncorrelated and occurs via either EX2 or EX1 processes promoted by strictly local structure fluctuations. For all other backbone amide protons in BPTI, the exchange was found to be uncorrelated and by an EX2 mechanism under all conditions of p2H and temperature where quantitative measurements could be obtained with the methods used, i.e., for kex approximately less than 5 min-1. From these observations with BPTI it can be concluded that the amide proton exchange in globular proteins is quite generally via EX2 processes, with rare exceptions for measurements with extremely stable protons at high temperature and basic p2H. This emphasizes the need for further development of suitable concepts for the structural interpretation of EX2 amide proton exchange [Wagner, G. (1983) Q. Rev. Biophys. 16, 1-57; Wagner, G., Stassinopoulou, C. I., & Wüthrich, K. (1984) Eur. J. Biochem. 145, 431-436] and for more detailed investigations of the intrinsic exchange rates for solvent-exposed amide protons in the "open" states of a protein [Roder, H., Wagner, G., & Wüthrich, K. (1985) Biochemistry (following paper in this issue)]. 相似文献
9.
Iron loss in vitro by the iron scavenger bovine lactoferrin was investigated in acidic media in the presence of three different monoanions (NO(3)(-), Cl(-) and Br(-)) and one dianion (SO(4)(2-)). Holo and monoferric C-site lactoferrins lose iron in acidic media (pH< or =3.5) by a four-step mechanism. The first two steps describe modifications in the conformation affecting the whole protein, which occur also with apolactoferrin. These two processes are independent of iron load and are followed by a third step consisting of the gain of two protons. This third step is kinetically controlled by the interaction with two Cl(-), Br(-) and NO(3)(-) or one SO(4)(2-). In the fourth step, iron loss is under the kinetic control of a slow gain of two protons; third-order rate-constants k(2), 4.3(+/-0.2)x10(3), 3.4(+/-0.5)x10(3), 3.3(+/-0.5)x10(3) and 1.5(+/-0.5)x10(3) M(-2) s(-1) when the protein is in interaction with SO(4)(2-), NO(3)(-), Cl(-) or Br(-), respectively. This step is accompanied by the loss of the interaction with the anions; equilibrium constant K(2), 20+/-5 mM, 1.0(+/-0.2)x10(-1), 1.5(+/-0.5)x10(-1) and 1.0(+/-0.3)x10(-1) M(2), for SO(4)(-), NO(3)(-), Cl(-) and Br(-), respectively. This mechanism is very different from that determined in mildly acidic media at low ionic strength (micro<0.5) for the iron transport proteins, serum transferrin and ovotransferrin, with which no prior change in conformation or interaction with anions is required. These differences may result from the fact that in the transport proteins, the interdomain hydrogen bonds that consolidate the closed conformation of the iron-binding cleft occur between amino acid side-chain residues that can protonate in mildly acidic media. With bovine lactoferrin, most of the interdomain hydrogen bonds involved in the C-site and one of those involved in the N-site occur between amino acid side-chain residues that cannot protonate. The breaking of the interdomain H-bond upon protonation can trigger the opening of the iron cleft, facilitating iron loss in serum transferrin and ovotransferrin. This situation is, however, different in lactoferrin, where iron loss requires a prior change in conformation. This can explain why lactoferrin does not lose its iron load in acidic media and why it is not involved in iron transport in acidic endosomes. 相似文献
10.
S W Fesik J R Luly H H Stein N BaMaung 《Biochemical and biophysical research communications》1987,147(3):892-898
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. 相似文献
11.
The high-spin (S = 5/2) Fe(III) ion at the active site of recombinant human phenylalanine hydroxylase (PAH) has a paramagnetic effect on the longitudinal relaxation rate of water protons. This effect is proportional to the concentration of enzyme, with a paramagnetic molar-relaxivity value at 400 MHz and 25 degrees C of 1. 3 (+/- 0.03) x 10(3) s-1 M-1. The value of the Arrhenius activation energy (Ea) for the relaxation rate was -14.4 +/- 1.1 kJ/mol for the resting enzyme, indicating a fast exchange of water protons in the paramagnetic environment. The frequency dependence of the relaxation rate also supported this hypothesis. Thus, the recombinant human PAH appears to have a more solvent-accessible catalytic iron than the rat enzyme, in which the water coordinated to the metal is slowly exchanging with the solvent. These findings may be related to the level of basal activity before activation for these enzymes, which is higher for human than for rat PAH. In the presence of saturating (5 mM) concentrations of the substrate L-Phe, the paramagnetic molar relaxivity for human PAH decreased to 0.72 (+/- 0.05) x 10(3) s-1 M-1 with no significant change in the Ea. Effective correlation times (tauC) of 1.8 (+/- 0.3) x 10(-10) and 1.25 (+/- 0.2) x 10(-10) s-1 were calculated for the enzyme and the enzyme-substrate complex, respectively, and most likely represent the electron spin relaxation rate (tauS) for Fe(III) in each case. Together with the paramagnetic molar-relaxivity values, the tauC values were used to estimate Fe(III)-water distances. It seems that at least one of the three water molecules coordinated to the iron in the resting rat and human enzymes is displaced from coordination on the binding of L-Phe at the active site. 相似文献
12.
A stopped-flow H-D exchange kinetic study of spermine-polynucleotide interactions. 总被引:3,自引:3,他引:0
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The rates of H-D exchange for imino and amino protons in adenosine, calf thymus DNA, poly (dA-dT), poly(dG-dC), and poly (dG-me5dC) were determined using stopped flow kinetic methods in the presence of various concentrations of Tris, imidazole, Mg2+, and spermine in citrate buffer (pH 7, 25 degrees C). CD spectroscopic studies showed that all polynucleotides always remain in the B-form under these conditions. An increase in the concentration of Tris and imidazole from 5 mu M to 20 mM caused an increase in the rates of exchange of both fast-exchanging imino and slow-exchanging amino protons. The limiting rates of exchange at infinite concentrations of catalysts were found to be different for fast (31-57 sec-1) and slow (1-2 sec-1) exchanging protons. These results indicate that imino and amino protons of B-DNA exchange asymmetrically from two different open states as observed for Z-DNA. An increase in the concentration of spermine from a ratio of 1:50 to 1:2 of positive charge/phosphate decreased the rate of exchange of imino protons of calf-thymus DNA, poly(dG-dC), and poly(dG-me5dC), but increased the rate of exchange of the imino protons of poly(dA-dT) without affecting the exchange rate of the amino protons of any of the polynucleotides. These results are interpreted in terms of possible spermine-induced change of conformations of oligonucleotides of specific sequence that has been suggested by theoretical model building studies. 相似文献
13.
The relaxation rates of the carbon-bound protons and of the three assigned phosphorus resonances of propionyl-CoA were measured in solutions of free propionyl-CoA and of the transcarboxylase-propionyl-CoA complex. In free propionyl-CoA, analysis of the 1/T1 values of 15 protons at 100 and 220 MHz and of 1/T1 and 1/T2 of the three phosphorus atoms at 40.5 MHz indicated free rotation of the propionyl region (taur approximately 3 x 10(-11) sec) but hindered motion of the remainder of the molecule with correlation times of 1-3. 5 x 10(-10) sec, approaching the tumbling time of the entire molecule (taur - 6 x 10(-10) sec. The correlation times of the three phosphorus atoms were indistinguishable from those of their nearest neighbor protons. The effects of three homogeneous enzyme preparations with varying contents of Zn(II), Co(II), and Cu(II) on 1/T1 of 12 protons and 3 phosphorus atoms of prionyl-CoA were analyzed with the help of simultaneous equations to yield the individual contributions at the three metal sites. Only diamagnetic effects were detected on the relaxation rates of the three phosphorus atoms. From the diamagnetic effects it was calculated that the motions of the prionyl side chain and of the terminal pantetheine methylene protons were hindered on the enzyme by an order of magnitude (taur approximately 6 x 10(-10) sec) and that the phosphorus atoms were hindered by two orders of magnitude (taur approximately 1 x 10(-8) sec) over the taur values found in free propionyl-CoA, but that these taur values remained well below that of the entire protein molecule (taur =6 x 10(-7) sec)... 相似文献
14.
Ca2+ binding to calbindin D9k strongly affects backbone dynamics: measurements of exchange rates of individual amide protons using 1H NMR 总被引:1,自引:0,他引:1
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. 相似文献
15.
Dikanov SA Samoilova RI Kolling DR Holland JT Crofts AR 《The Journal of biological chemistry》2004,279(16):15814-15823
Exchangeable protons in the immediate neighborhood of the semiquinone (SQ) at the Qi-site of the bc1 complex (ubihydroquinone:cytochrome c oxidoreductase (EC 1.10.2.2)) from Rhodobacter sphaeroides have been characterized using electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation spectroscopy (HYSCORE) and visualized by substitution of H2O by 2H2O. Three exchangeable protons interact with the electron spin of the SQ. They possess different isotropic and anisotropic hyperfine couplings that allow a clear distinction between them. The strength of interactions indicates that the protons are involved in hydrogen bonds with SQ. The hyperfine couplings differ from values typical for in-plane hydrogen bonds previously observed in model experiments. It is suggested that the two stronger couplings involve formation of hydrogen bonds with carbonyl oxygens, which have a significant out-of-plane character due to the combined influence of bulky substituents and the protein environment. These two hydrogen bonds are most probably to side chains suggested from crystallographic structures (His-217 and Asp-252 in R. sphaeroides). Assignment of the third hydrogen bond is more ambiguous but may involve either a bond between Asn-221 and a methoxy O-atom or a bond to water. The structural and catalytic roles of the exchangeable protons are discussed in the context of three high resolution crystallographic structures for mitochondrial bc1 complexes. Potential H-bonds, including those to water molecules, form a network connecting the quinone (ubiquinone) occupant and its ligands to the propionates of heme bH and the external aqueous phase. They provide pathways for exchange of protons within the site and with the exteriors, needed to accommodate the different hydrogen bonding requirements of different quinone species during catalysis. 相似文献
16.
L Poppe J Dabrowski C W von der Lieth 《Biochemical and biophysical research communications》1991,174(3):1169-1175
The conformation of Forssman glycolipid, GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1ceramide, was analysed with the aid of the rotating frame NOE and Hartmann-Hahn spectroscopy. NOE contacts between C-, O-, and N-linked protons were used for distance mapping. The glycosidic bonds that are common to globotriaosylceramide and globoside showed a similar flexibility as found for these compounds [Poppe et al., (1990) Eur. J. Biochem. 189, 313-325; J. Am. Chem. Soc. 112, 7762-7771]. In contrast, the conformational mobility of the terminal GalNAc alpha 1-3GalNAc beta linkage appears to be restrained. A new approach, based on 2D exchange spectroscopy, was proposed for revealing of spatial proximities between exchangeable protons in Me2SO solution. 相似文献
17.
R Boelens P Gros R M Scheek J A Verpoorte R Kaptein 《Journal of biomolecular structure & dynamics》1985,3(2):269-280
Proton exchange in lac repressor headpiece was studied by COSY and 2D NOE spectroscopy. The exchange rates of amide protons, stabilized by the hydrogen bonds of the three alpha-helices of the headpiece, could be determined quantitatively. The exchange rates in these helices showed repetitive patterns of about three to four residues. A correlation with the position of the amide proton in the interior or the exterior of the alpha-helix of the protein was found. The exchange data strongly support the validity of the three-dimensional structure, as determined recently (Kaptein, R. et al., J. Mol. Biol. 182, 179-182 (1985)). 相似文献
18.
Secondary amide cis peptide bonds are of even lower abundance than the cis tertiary amide bonds of prolines, yet they are of biochemical importance. Using 2D NMR exchange spectroscopy (EXSY) we investigated the formation of cis peptide bonds in several oligopeptides: Ac-G-G-G-NH(2) , Ac-I-G-G-NH(2) , Ac-I-G-G-N-NH(2) and its cyclic form: I-G-G-N in dimethylsulfoxide (DMSO). From the NMR studies, using the amide protons as monitors, an occurrence of 0.13-0.23% of cis bonds was obtained at 296 K. The rate constants for the trans to cis conversion determined from 2D EXSY spectroscopy were 4-9 × 10(-3) s(-1) . Multiple minor conformations were detected for most peptide bonds. From their thermodynamic and kinetic properties the cis isomers are distinguished from minor trans isomers that appear because of an adjacent cis peptide bond. Solvent and sequence effects were investigated utilizing N-methylacetamide (NMA) and various peptides, which revealed a unique enthalpy profile in DMSO. The cyclization of a tetrapeptide resulted in greatly lowered cis populations and slower isomerization rates compared to its linear counterpart, further highlighting the impact of structural constraints. 相似文献
19.
Porcine pancreatic phospholipase A2: sequence-specific 1H and 15N NMR assignments and secondary structure 总被引:1,自引:0,他引:1
The solution structure of porcine pancreatic phospholipase A2 (124 residues, 14 kDa) has been studied by two-dimensional homonuclear 1H and two- and three-dimensional heteronuclear 15N-1H nuclear magnetic resonance spectroscopy. Backbone assignments were made for 117 of the 124 amino acids. Short-range nuclear Overhauser effect (NOE) data show three alpha-helices from residues 1-13, 40-58, and 90-109, an antiparallel beta-sheet for residues 74-85, and a small antiparallel beta-sheet between residues 25-26 and 115-116. A 15N-1H heteronuclear multiple-quantum correlation experiment was used to monitor amide proton exchange over a period of 22 h. In total, 61 amide protons showed slow or intermediate exchange, 46 of which are located in the three large helices. Helix 90-109 was found to be considerably more stable than the other helices. For the beta-sheets, four hydrogen bonds could be identified. The secondary structure of porcine PLA in solution, as deduced from NMR, is basically the same as the structure of porcine PLA in the crystalline state. Differences were found in the following regions, however. Residues 1-6 in the first alpha-helix are less structured in solution than in the crystal structure. Whereas in the crystal structure residues 24-29 are involved both in a beta-sheet with residues 115-117 and in a hairpin turn, the expected hydrogen bonds between residues 24-117 and 25-29 do not show slow exchange behavior. This and the absence of several expected NOEs imply that this region has a less well defined structure in solution. Finally, the hydrogen bond between residues 78-81, which is part of a beta-sheet, does not show slow exchange behavior. 相似文献
20.
The temperature-dependence of a large number of NMR parameters describing hydrogen bond properties in the protein ubiquitin was followed over a range from 5 to 65 degrees C. The parameters comprise hydrogen bond (H-bond) scalar couplings, h3JNC', chemical shifts, amide proton exchange rates, 15N relaxation parameters as well as covalent 1JNC' and 1JNH couplings. A global weakening of the h3JNC' coupling with increasing temperature is accompanied by a global upfield shift of the amide protons and a decrease of the sequential 1JNC' couplings. If interpreted as a linear increase of the N...O distance, the change in h3JNC' corresponds to an average linear thermal expansion coefficient for the NH-->O hydrogen bonds of 1.7 x 10(-4)/K, which is in good agreement with overall volume expansion coefficients observed for proteins. A residue-specific analysis reveals that not all hydrogen bonds are affected to the same extent by the thermal expansion. The end of beta-sheet beta1/beta5 at hydrogen bond E64-->Q2 appears as the most thermolabile, whereas the adjacent hydrogen bond I3-->L15 connecting beta-strands beta1 and beta2 is even stabilized slightly at higher temperatures. Additional evidence for the stabilization of the beta1/beta2 beta-hairpin at higher temperatures is found in reduced hydrogen exchange rates for strand end residue V17. This reduction corresponds to a stabilizing change in free energy of 9.7 kJ/mol for the beta1/beta2 hairpin. The result can be linked to the finding that the beta1/beta2 hairpin behaves as an autonomously folding unit in the A-state of ubiquitin under changed solvent conditions. For several amide groups the temperature-dependencies of the amide exchange rates and H-bond scalar couplings are uncorrelated. Therefore, amide exchange rates are not a sole function of the hydrogen bond "strength" as given by the electronic overlap of donors and acceptors, but are clearly dependent on other blocking mechanisms. 相似文献