Hydrogen exchange of individual amide protons in the F helix of cyanometmyoglobin

Hydrogen exchange of the individual amide protons of alanine-90 (F5), glutamine-91 (F6), serine-92 (F7), and histidine-93 (F8) residues in cyanometmyoglobin of sperm whale has been studied by 1H nuclear magnetic resonance spectroscopy at 360 MHz. The amide proton resonance of F5, F6, and F7 have been assigned by use of the selective nuclear Overhauser effect between the consecutive amide protons. At pH 6.8, and in the temperature range of 5-20 degrees C, these protons show a 10(4)-fold retardation compared to the rates in free peptides. Apparent activation enthalpies for hydrogen exchange of F5, F6, and F8 protons are 18.5 +/- 0.4, 9.5 +/- 0.3, and 18.5 +/- 0.3 kcal/mol, respectively. Some implications of these results on the nature of the opening processes involved in hydrogen exchange are considered.     

Protection of amide protons in folding intermediates of ribonuclease A measured by pH-pulse exchange curves

pH-pulse exchange curves have been measured for samples taken during the folding of ribonuclease A. The curve gives the number of protected amide protons remaining after a 10-s pulse of exchange at pHs from 6.0 to 9.5, at 10 degrees C. Amide proton exchange is base catalyzed, and the rate of exchange increases 3000-fold between pH 6.0 and pH 9.5. The pH at which exchange occurs depends on the degree of protection against exchange provided by structure. Pulse exchange curves have been measured for samples taken at three times during folding, and these are compared to the pulse exchange curves of N, the native protein, of U, the unfolded protein in 4 M guanidinium chloride, and of IN, the native-like intermediate obtained by the prefolding method of Schmid. The results are used to determine whether folding intermediates are present that can be distinguished from N and U and to measure the average degree of protection of the protected protons in folding intermediates. The amide (peptide NH) protons of unfolded ribonuclease A were prelabeled with 3H by a previous procedure that labels only the slow-folding species. Folding was initiated at pH 4.0, 10 degrees C, where amide proton exchange is slower than the folding of the slow-folding species. Samples were taken at 0-, 10-, and 20-s folding, and their pH-pulse exchange curves were measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amide proton exchange used to monitor the formation of a stable alpha-helix by residues 3 to 13 during folding of ribonuclease S

We make use of the known exchange rates of individual amide proton in the S-peptide moiety of ribonuclease S (RNAase S) to determine when during folding the alpha-helix formed by residues 3 to 13 becomes stable. The method is based on pulse-labeling with [3H]H2O during the folding followed by an exchange-out step after folding that removes 3H from all amide protons of the S-peptide except from residues 7 to 14, after which S-peptide is separated rapidly from S-protein by high performance liquid chromatography. The slow-folding species of unfolded RNAase S are studied. Folding takes place in strongly native conditions (pH 6.0, 10 degrees C). The seven H-bonded amide protons of the 3-13 helix become stable to exchange at a late stage in folding at the same time as the tertiary structure of RNAase S is formed, as monitored by tyrosine absorbance. At this stage in folding, the isomerization reaction that creates the major slow-folding species has not yet been reversed. Our result for the 3-13 helix is consistent with the finding of Labhardt (1984), who has studied the kinetics of folding of RNAase S at 32 degrees C by fast circular dichroism. He finds the dichroic change expected for formation of the 3-13 helix occurring when the tertiary structure is formed. Protected amide protons are found in the S-protein moiety earlier in folding. Formation or stabilization of this folding intermediate depends upon S-peptide: the intermediate is not observed when S-protein folds alone, and folding of S-protein is twice as slow in the absence of S-peptide. Although S-peptide combines with S-protein early in folding and is needed to stabilize an S-protein folding intermediate, the S-peptide helix does not itself become stable until the tertiary structure of RNAase S is formed.     

Proline for alanine substitutions in the C-peptide helix of ribonuclease A

The effect on overall alpha-helix content of substituting proline for alanine has been determined at 5 positions (1, 2, 4, 5, and 13) of a 13-residue peptide related in sequence to residues 1-13 of ribonuclease A. The helix content falls off rapidly as proline is moved inward, and the proline residue effectively truncates the helix. No helix-stabilizing effect of proline is found at positions 2 or 4 within the first turn of the helix. Proline substitution at either end position (1, 13) has little effect on overall helix content, in agreement with an earlier study of glycine for alanine substitutions. The two end residues of the helix appear to be strongly frayed.     

Anomalous exchange kinetics of peptide amide protons in aqueous solutions

Reports concerning anomalous rates of exchange of some amides in oxytocin, alumichrome, and gramicidin S are reexamined through systematic analysis of the exchange data as a function of pH and primary structure. It is shown that such an analysis can provide useful information on secondary structure when the degree of hydrogen bonding to both the NH undergoing exchange and the neighboring carbonyl group are taken into consideration.     

Conformational changes of pore helix coupled to gating of TRPV5 by protons

The transient receptor potential channel TRPV5 constitutes the apical entry pathway for transepithelial Ca2+ transport. We showed that TRPV5 was inhibited by both physiological intra- and extracellular acid pH. Inhibition of TRPV5 by internal protons was enhanced by extracellular acidification. Similarly, inhibition by external protons was enhanced by intracellular acidification. Mutation of either an extra- or an intracellular pH sensor blunted the cross-inhibition by internal and external protons. Both internal and external protons regulated the selectivity filter gate. Using the substituted cysteine accessibility method, we found that intracellular acidification of TRPV5 caused a conformational change of the pore helix consistent with clockwise rotation along its long axis. Thus, rotation of pore helix caused by internal protons facilitates closing of TRPV5 by external protons. This regulation by protons likely contributes to pathogenesis of disturbances of Ca2+ transport in many diseased states. Rotation of pore helix may be a common mechanism for cross-regulation of ion channels by extra- and intracellular signals.     

NMR signal assignments of amide protons in the alpha-helical domains of staphylococcal nuclease

We report complete assignments of the amide proton signals in the three long dNN connectivity sequences observed in the NOESY spectrum of deuteriated staphylococcal nuclease (Nase) complexed with thymidine 3',5'-bisphosphate (pdTp) and Ca2+, Mr 18K. The assignments are made by comparing NOESY spectra with 1H-15N and 1H-13C heteronuclear multiple-quantum shift correlation (HMQC) spectra of Nase samples containing 15N- and 13C-labeled amino acids. The assignments show that the residues which are linked by the dNN connectivity sequences are located in three alpha-helical domains of Nase. Our results indicate that by combining NOESY and HMQC spectra of appropriately labeled samples it should be possible to delineate and study alpha-helical domains in soluble proteins having molecular weights that are greater than 18K.     

Theoretical NMR study of the chemical exchange of amide protons of proteins

Summary A model is proposed to evaluate the rate of exchange between the amide protons of proteins and the solvent water molecules. Using this model we determined the extent of the error for the chemical exchange rate constant when cross relaxation was neglected; both selective inversion and saturation-transfer techniques were evaluated. Furthermore, the fluctuations in the NOE intensities were determined when the exchange rate was varied.     

Sensitivity-optimized experiment for the measurement of residual dipolar couplings between amide protons

High signal to noise is a necessity for the quantification of NMR spectral parameters to be translated into accurate and precise restraints on protein structure and dynamics. An important source of long-range structural information is obtained from ¹H–¹H residual dipolar couplings (RDCs) measured for weakly aligned molecules. For sensitivity reasons, such measurements are generally performed on highly deuterated protein samples. Here we show that high sensitivity is also obtained for protonated protein samples if the pulse schemes are optimized in terms of longitudinal relaxation efficiency and J-mismatch compensated coherence transfer. The new sensitivity-optimized quantitative J-correlation experiment yields important signal gains reaching factors of 1.5 to 8 for individual correlation peaks when compared to previously proposed pulse schemes. Paul Schanda and Ewen Lescop contributed equally to this work.     

Concentration-dependent hydrogen exchange kinetics of 3H-labeled S-peptide in ribonuclease S.

The hydrogen exchange kinetics of the S-peptide in ribonuclease S can be measured by first tritiating the S-peptide in the absence of S-protein and then allowing it to recombine rapidly with S-protein. Afterwards the exchange reactions of this specific segment of ribonuclease S can be studied. The exchange kinetics of bound S-peptide are complex, indicating that different protons exchange at markedly different rates. The terminal exchange reaction, involving at least five highly protected protons, has been studied as a function of pH.At low concentrations of ribonuclease S the exchange kinetics become concentration-dependent, owing to the dissociation of the S-peptide. Although the fraction of free S-peptide is always very small, its rate of exchange is several orders of magnitude faster than that of bound S-peptide, and the concentration dependence of the exchange kinetics is readily measurable. It provides a highly sensitive method for determining small dissociation constants (K_D). Values of K_D ranging from 10^?6m at pH 2.7, 0 °C, to 2 × 10^?10m at pH 7.0, 0 °C, are reported here. Our value for K_D at pH 7.0, 0 °C, confirms the data and extrapolation to 0 °C of Hearn et al. (1971).At high concentrations of ribonuclease S the terminal exchange reaction is independent of concentration. It probably results from a local unfolding reaction of the bound S-peptide. Above pH 4 the strong pH dependence of K_D closely resembles that of the apparent equilibrium constant for this local unfolding reaction. The latter may be one step in the dissociation process and we present such a model for ribonuclease S dissociation.Measurement of concentration-dependent exchange kinetics should provide a useful method of determining small dissociation constants in other systems: for example, in studies of protein-nucleic acid interactions.     

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

Summary 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., _0c~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.     

Hydrogen--deuterium exchange kinetics of the amide protons of oxytocin studied by nuclear magnetic resonance

The contribution of intramolecular hydrogen bonding to the solution structure of oxytocin was evaluated by study of amide hydrogen exchange rates in D2O by Fourier transform 1H NMR spectroscopy. Resolution enhancement filtering was employed in the determination of individual pseudo-first-order rate constants. Apparent barriers to exchange of 0.5 and 0.6 kcal mol-1 were measured for Asn5 and Cys6 peptide NH, respectively. The slowing is best explained by steric hindrance to solvent access in the case of Asn5, while for the Cys6 participation in a weak intramolecular hydrogen bond is possible. Fourfold acceleration of base-catalyzed exchange was observed for Tyr2 NH; it is proposed that this is the result of electronic effects induced by hydrogen bonding of Cys1 C=0, either to Cys6 NH or to the N-terminal amino group. Exchange proceeds near the random coil limit for each of the remaining residues. Comparison with exchange data for the model tripeptide N-acetyl-L-prolyl-L-leucylglycinamide demonstrates no evidence of noncovalent association of the tocin ring with the tripeptide tail of the hormone.     

Hydrogen kinetics of peptide amide protons at the bovine pancreatic trypsin inhibitor protein-solvent interface

Hydrogen exchange rate constants of the 25 most rapidly exchanging peptide amide protons in bovine pancreatic trypsin inhibitor have been determined over a range of pH that spans pH min, the pH of minimum rate. Most of these are on the protein surface, exposed to solvent and not hydrogen bonded in the crystal structure. Contrary to commonly held assumptions, the exchange kinetics of surface NH groups are not equivalent to the kinetics of NH groups in peptides in the extended configuration. All surface NH groups exchange more slowly than NH groups in model peptides, with rate constants distributed over a range of more than two orders of magnitude. In addition, their pH min values vary widely. For most of the surface NH groups, pH min is lower than in model compounds and, for several, pH min is less than 1. These results indicate that the local environment of the surface peptide groups when the exchange event occurs is very different from that of extended peptides. Analysis based on consideration of an O-protonation mechanism for acid catalysis and of electrostatic effects on exchange kinetics further indicates (see the accompanying paper) that, in general, exchange of surface NH groups occurs from a conformation of the protein approximated by the crystal structure. The 1H-2H exchange rate constants were measured from 300 MHz nuclear magnetic resonance spectra in which assigned surface N1H resonances are resolved by the use of partially deuterated protein samples. A marked pH dependence of the chemical shifts observed in the pH range 1 to 4.5 for several surface NH groups reflects the titration of nearby carboxyl groups.     

A competing salt-bridge suppresses helix formation by the isolated C-peptide carboxylate of ribonuclease A

The C-peptide of ribonuclease A (residues 1 to 13) is obtained by cyanogen bromide cleavage at Met13, which converts methionine to a mixture of homoserine lactone (giving C-peptide lactone) and homoserine carboxylate (giving C-peptide carboxylate). The helix-forming properties of C-peptide lactone have been reported. The helix is formed intramolecularly in aqueous solution, is stabilized at low temperatures (0 to 20 °C) and also by a pH-dependent interaction between sidechains. The C-peptide lactone helix is about 1000-fold more stable than expected from “host-guest” data for helix formation in synthetic polypeptides.Here we report the failure of C-peptide carboxylate to form an α-helix in comparable conditions. Formation of a salt-bridge between the α-COO^? group and the imidazolium ring of His12⁺ appears to be responsible for the suppression of helix formation. The presence of the Hse13-COO^? … His12⁺ salt-bridge in C-peptide carboxylate is shown by ¹H nuclear magnetic resonance titration of the amide proton resonances of His12 and Hse13, and is expected from model peptide studies. The most probable reason why C-peptide carboxylate does not form an α-helix is that the Hse13-COO^? … His12⁺ salt-bridge competes successfully with a helix stabilizing salt-bridge (Glu9^? … His12⁺).S-peptide (residues 1 to 20 of ribonuclease A) does form an α-helix with properties similar to those of the C-peptide (lactone) helix, which shows that the lactone ring of C-peptide lactone is not needed for helix formation.These results support the hypothesis that a Glu9^? … His12⁺ salt-bridge stabilizes the C-peptide (lactone) helix, and they show that specific interactions between side-chains can be important in preventing as well as in promoting α-helix formation.     

Inferred motions of the S3a helix during voltage-dependent K+ channel gating

The gating of voltage-dependent potassium channels is controlled by conformational changes in voltage sensor domains. Previous studies have shown that the S1 and the S2 helices of the voltage sensor are static with respect to motion across the membrane, while the voltage sensor paddle consisting of the C-terminal half of S3 (S3b) and the charge-bearing S4 is mobile. The mobile component is attached to S1 and S2 via the S2-S3 turn and the N-terminal half of S3 (S3a). In this study, we analyze KvAP, an archaebacterial voltage-dependent potassium channel, to study the mobility with respect to translation across the membrane of S3a. We utilize an assay based on attachment of tethered biotin and its site-specific accessibility to avidin. Our results reveal that the S3a helix does not move appreciably across the membrane in association with gating. The static behavior of S3a constrains the conformations available to the voltage sensor when it closes and suggests that a set of negative countercharges within the membrane's inner leaflet remains intact in the closed conformation.