Hydrogen exchange in thermally denatured ribonuclease A

Hydrogen exchange has been used to test for the presence of nonrandom structure in thermally denatured ribonuclease A (RNase A). Quenched-flow methods and 2D 1H NMR spectroscopy were used to measure exchange rates for 36 backbone amide protons (NHs) at 65 degrees C and at pH* (uncorrected pH measured in D2O) values ranging from 1.5 to 3.8. The results show that exchange is approximately that predicted for a disordered polypeptide [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158]; we thus are unable to detect any stable hydrogen-bonded structure in thermally denatured RNase A. Two observations suggest, however, that the predicted rates should be viewed with some caution. First, we discovered that one of the approximations made by Molday et al. (1972), that exchange for valine NHs is similar to that for alanine NHs, had to be modified; the exchange rates for valine NHs are about 4-fold slower. Second, the pH minima for exchange tend to fall at lower pH values than predicted, by as much as 0.45 pH units. These results are in accord with those of Roder and co-workers for bovine pancreatic trypsin inhibitor [see Table I in Roder, H., Wagner, G., & Wüthrich, K. (1985) Biochemistry 24, 7407-7411]. The origin of the disagreement between predicted and observed pH minima is unknown but may be the high net positive charge on these proteins at low pH. In common with some other thermally unfolded proteins, heat-denatured ribonuclease A shows a significant circular dichroism spectrum in the far-ultraviolet region [Labhardt, A. M. (1982) J. Mol. Biol. 157, 331-355].(ABSTRACT TRUNCATED AT 250 WORDS)     

Backbone dynamics of a model membrane protein: measurement of individual amide hydrogen-exchange rates in detergent-solubilized M13 coat protein using 13C NMR hydrogen/deuterium isotope shifts

Hydrogen-exchange rates have been measured for individual assigned amide protons in M13 coat protein, a 50-residue integral membrane protein, using a 13C nuclear magnetic resonance (NMR) equilibrium isotope shift technique. The locations of the more rapidly exchanging amides have been determined. In D2O solutions, a peptide carbonyl resonance undergoes a small upfield isotope shift (0.08-0.09 ppm) from its position in H2O solutions; in 1:1 H2O/D2O mixtures, the carbonyl line shape is determined by the exchange rate at the adjacent nitrogen atom. M13 coat protein was labeled biosynthetically with 13C at the peptide carbonyls of alanine, glycine, phenylalanine, proline, and lysine, and the exchange rates of 12 assigned amide protons in the hydrophilic regions were measured as a function of pH by using the isotope shift method. This equilibrium technique is sensitive to the more rapidly exchanging protons which are difficult to measure by classical exchange-out experiments. In proteins, structural factors, notably H bonding, can decrease the exchange rate of an amide proton by many orders of magnitude from that observed in the freely exposed amides of model peptides such as poly(DL-alanine). With corrections for sequence-related inductive effects [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158], the retardation of amide exchange in sodium dodecyl sulfate solubilized coat protein has been calculated with respect to poly(DL-alanine). The most rapidly exchanging protons, which are retarded very little or not at all, are shown to occur at the N- and C-termini of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Back Hydrogen Exchange Procedure via the Acid-Unfolded State for a Large Protein

A deuterated protein sample is required for nuclear magnetic resonance (NMR) measurements of a large protein because severe signal broadenings occur because of the high molecular weight. The deuterated sample expressed in (2)H(2)O should subsequently be subjected to a back hydrogen exchange at amide groups. To perform the back exchange, the protein molecule is unfolded or destabilized so that internal residues become accessible to the solvent. However, the refolding yield from the destabilized or unfolded state of a large protein is usually low, leading to a dilemma in NMR measurements of large proteins. In our previous paper [Suzuki, M., et al. (2011) Biochemistry50, 10390-10398], we suggested that an acid-denatured microbial transglutaminase (MTG) consisting of 331 amino acid residues can be recovered effectively under low-salt conditions, escaping from the aggregation-prone intermediate. Here, we demonstrate that proMTG, the pro form of MTG consisting of 376 amino acid residues, can be refolded perfectly from the acid-unfolded state under low-salt conditions, as confirmed by circular dichroism and NMR spectroscopies. By performing the same procedure with a deuterated proMTG expressed in (2)H(2)O, we observed complete back exchanges for internal residues by NMR spectroscopy. Our procedure has potential applications to the back hydrogen exchange of large proteins for NMR measurements.     

The use of spin desalting columns in DMSO‐quenched H/D‐exchange NMR experiments

Dimethylsulfoxide (DMSO)‐quenched hydrogen/deuterium (H/D)‐exchange is a powerful method to characterize the H/D‐exchange behaviors of proteins and protein assemblies, and it is potentially useful for investigating non‐protected fast‐exchanging amide protons in the unfolded state. However, the method has not been used for studies on fully unfolded proteins in a concentrated denaturant or protein solutions at high salt concentrations. In all of the current DMSO‐quenched H/D‐exchange studies of proteins so far reported, lyophilization was used to remove D₂O from the protein solution, and the lyophilized protein was dissolved in the DMSO solution to quench the H/D exchange reactions and to measure the amide proton signals by two‐dimensional nuclear magnetic resonance (2D NMR) spectra. The denaturants or salts remaining after lyophilization thus prevent the measurement of good NMR spectra. In this article, we report that the use of spin desalting columns is a very effective alternative to lyophilization for the medium exchange from the D₂O buffer to the DMSO solution. We show that the medium exchange by a spin desalting column takes only about 10 min in contrast to an overnight length of time required for lyophilization, and that the use of spin desalting columns has made it possible to monitor the H/D‐exchange behavior of a fully unfolded protein in a concentrated denaturant. We report the results of unfolded ubiquitin in 6.0M guanidinium chloride.     

Assignment of the 1H and 15N NMR spectra of Rhodobacter capsulatus ferrocytochrome c2

The peptide resonances of the 1H and 15N nuclear magnetic resonance spectra of ferrocytochrome c2 from Rhodobacter capsulatus are sequentially assigned by a combination of 2D 1H-1H and 1H-15N spectroscopy, the latter performed on 15N-enriched protein. Short-range nuclear Overhauser effect (NOE) data show alpha-helices from residues 3-17, 55-65, 69-88, and 103-115. Within the latter two alpha-helices, there are three single 3(10) turns, 70-72, 76-78, and 107-109. In addition alpha H-NHi+1 and alpha H-NHi+2 NOEs indicate that the N-terminal helix (3-17) is distorted. Compared to horse or tuna cytochrome c and cytochrome c2 of Rhodospirillium rubrum, there is a 6-residue insertion at residues 23-29 in R. capsulatus cytochrome c2. The NOE data show that this insertion forms a loop, probably an omega loop. 1H-15N heteronuclear multiple quantum correlation experiments are used to follow NH exchange over a period of 40 h. As the 2D spectra are acquired in short time periods (30 min), rates for intermediate exchanging protons can be measured. Comparison of the NH exchange data for the N-terminal helix of cytochrome c2 of R. capsulatus with the highly homologous horse heart cytochrome c [Wand, A. J., Roder, H., & Englander, S. W. (1986) Biochemistry 25, 1107-1114] shows that this helix is less stable in cytochrome c2.     

A Fourier transform infrared investigation of the structural differences between ribonuclease A and ribonuclease S

Fourier transform infrared spectroscopy was used to investigate the small conformational differences which exist between ribonuclease A and ribonuclease S in aqueous systems. Deconvolution and derivative methods were used to observe the overlapping components of the amide I and II bands. These proteins give identical spectra in H2O and after complete exchange in 2H2O. However structural differences are revealed by monitoring the rate of 1H-2H exchange by Fourier transform infrared spectroscopy. At equivalent times of exposure in 2H2O buffer ribonuclease S undergoes greater isotopic exchange than ribonuclease A. Thus complete exchange takes place for ribonuclease S but not ribonuclease A after incubation at room temperature for 8 days. Complete 1H-2H exchange of ribonuclease A was achieved by incubation at 62 degrees C for 30 min. The available X-ray data and comparison with the infrared spectra of other soluble proteins was used to assign the components of the amide I and II bands to various secondary structures. In particular, band shifts observed during the later stages of exchange are associated with slowly exchanging residues in beta-strand and alpha-helical regions. The higher rate of exchange for ribonuclease S is associated with a greater conformational flexibility and a more open structure. The results show that it is necessary to be cautious in making band assignments based on exchange methods unless the extent of exchange is known. Furthermore, it is seen that the combination of Fourier transform infrared spectroscopy and hydrogen-deuterium exchange is a powerful technique for revealing small differences in protein secondary structure.     

NMR study of the exchange rates of allosterically responsive labile protons in the heme pockets of hemoglobin A.

1H NMR spectroscopy has been used to measure the proximal histidyl labile ring proton (NH) rates of exchange with bulk solvent in the individual subunits of hemoglobin (Hb) A. These protons displayed a substantial decrease in their exchange rates in comparison with related monomeric proteins and exhibited sensitivity to the quarternary state. With the beta subunit NH, the exchange behaviour was similar to an allosterically responsive subset of protons, which have been identified using 1H-3H methods (Englander, J.J., R. Rogero, and S. W. Englander, 1983, J. Mol. Biol. 169:325-344). Assuming similar exchange mechanisms for the two subunits, the NMR data suggested a more flexible alpha than beta subunit in Hb A.     

Oxidation state dependence of proton exchange near the iron-sulfur centers in ferredoxins and high-potential iron-sulfur proteins

For both the [2Fe-2S] and the [4Fe-4S] ferredoxins, dialysis against 2H2O prior to single electron reduction leads to the appearance of a deuterium modulation pattern in the electron spin echo decay envelope indicative of deuteron-proton exchange very near the paramagnetic center. In contrast, if the ferredoxin is exposed to 2H2O after its reduction in H2O, far less deuterium exchange near the metal center takes place. Thus, proton exchange with solvent is in part dependent on the redox state of the protein. For high potential iron-sulfur proteins, this type of proton-deuteron exchange near the metal center does not occur unless the protein is partially unfolded in dimethylsulfoxide in 2H2O.     

Insights into conformation and dynamics of protein GB1 during folding and unfolding by NMR

Understanding protein stability requires characterization of structural determinants of the folded and unfolded states. Many proteins are capable of populating partially folded states under specific solution conditions. Occasionally, coexistence of the folded and an unfolded state under non- or mildly denaturing conditions can be observed by NMR, allowing us to structurally probe these states under identical conditions. Here we report on a destabilized mutant of the B1 domain of protein G (GB1) whose equilibrium unfolding was systematically investigated. Backbone amide residual dipolar couplings (RDCs), the tryptophan Nepsilon-H resonance and the amide nitrogen transverse relaxation rates (R2s) for varying pH values and different temperatures were measured. The backbone amide RDCs indicate that prior to complete unfolding, two melting hot spots are formed at the turn around T11, L12 and K13 and the N terminus of the helix at A24 and T25. The RDCs for the low pH, thermally unfolded state of GB1 are very small and do not indicate the presence of any native-like structure. Amide nitrogen transverse relaxation rates for GB1 in the folded state at different temperatures exhibit large contributions from exchange processes and the associated dynamics display considerable heterogeneity. Our data provide clear evidence for intermediate conformations and multi-state equilibrium un/folding for this GB1 variant.     

Redox-dependent dynamics of putidaredoxin characterized by amide proton exchange.

Multidimensional NMR methods were used to obtain 1H-15N correlations and 15N resonance assignments for amide and side-chain nitrogens of oxidized and reduced putidaredoxin (Pdx), the Fe2S2 ferredoxin, which acts as the physiological reductant of cytochrome P-450cam (CYP101). A model for the solution structure of oxidized Pdx has been determined recently using NMR methods (Pochapsky TC, Ye XM, Ratnaswamy G, Lyons TA, 1994, Biochemistry 33:6424-6432) and redox-dependent 1H NMR spectral features have been described (Pochapsky TC, Ratnaswamy G, Patera A, 1994, Biochemistry 33:6433-6441). 15N assignments were made with NOESY-(1H/15N) HMQC and TOCSY-(1H/15N) HSQC spectra obtained using samples of Pdx uniformly labeled with 15N. Local dynamics in both oxidation states of Pdx were then characterized by comparison of residue-specific amide proton exchange rates, which were measured by a combination of saturation transfer and H2O/D2O exchange methods at pH 6.4 and 7.4 (uncorrected for isotope effects). In general, where exchange rates for a given site exhibit significant oxidation-state dependence, the oxidized protein exchanges more rapidly than the reduced protein. The largest dependence of exchange rate upon oxidation state is found for residues near the metal center and in a region of compact structure that includes the loop-turn Val 74-Ser 82 and the C-terminal residues (Pro 102-Trp 106). The significance of these findings is discussed in light of the considerable dependence of the binding interaction between Pdx and CYP101 upon the oxidation state of Pdx.     

Amide proton exchange in proteins by EX1 kinetics: studies of the basic pancreatic trypsin inhibitor at variable p2H and temperature

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)].     

Position-dependent local motions in spin-labeled analogues of a short alpha-helical peptide determined by electron spin resonance.

We have used electron spin resonance and circular dichroism to examine and compare the dynamics in two analogues of the Ala-based 3K(I) peptide [Marqusee, S., Robbins, V.H., & Baldwin, R. L. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5286-5290], labeled at positions 4 and 8, throughout the alpha-helix----coil transition. In the middle of the thermal unfolding transition, our results demonstrate that the local mobility near the N-terminus is greater than at the center of the peptide. This provides evidence, from the perspective of dynamics, that the ends of Ala-based alpha-helices are frayed. We further find that the position dependence of the mobility for the thermally unfolded state differs from that of the denaturant unfolded state. Only the latter state exhibits the local dynamics expected for a genuine random coil.     

pH dependence of hydrogen exchange from backbone peptide amides in apamin

The kinetics of hydrogen exchange of the 11 most protected backbone amides of bee venom apamin have been measured between pH 1 and pH 8.5 by using time-resolved and saturation-transfer NMR spectroscopy. The five amides most protected from base-catalyzed exchange, those of residues 5 and 12-15, show highly correlated exchange behavior in the base-catalyzed regime. It is proposed that the intramolecular hydrogen bonds stabilizing these amides define a stable cooperative unit of secondary structure in apamin (a C-terminal helix and an N-terminal beta-turn). This conformational unit is further stabilized (by 5-6 kJ mol-1) on titration of the Glu-7 side-chain carboxyl group. The relative contributions of specific intramolecular interactions to this conformational stabilization are estimated. The pHminima in the pH-dependent single amide exchange curves are compared with values predicted by correcting for sequence-dependent contributions to amide exchange rates [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158]. The lack of correlation suggests that the "open" conformers from which amide exchange occurs are nonrandom. This conclusion is dependent on the assumption that acid-catalyzed exchange occurs via N-protonation so that residual conformational effects on exchange rates in the open conformers will affect acid- and base-catalyzed rates in approximately equal and opposite ways. A strong correlation between the measured pHminima and the amide proton chemical shifts is observed, however, and this may be most easily accommodated if acid-catalyzed exchange occurs by the imidic acid mechanism (via amide O-protonation).     

Exchange behavior of the H-bonded amide protons in the 3 to 13 helix of ribonuclease S

The preceding article shows that there are eight highly protected amide protons in the S-peptide moiety of RNAase S at pH 5, 0 degrees C. The residues with protected NH protons are 7 to 13, whose amide protons are H-bonded in the 3 to 13 alpha-helix, and Asp 14, whose NH proton is H-bonded to the CO group of Val47. We describe here the exchange behavior of these eight protected protons as a function of pH. Exchange rates of the individual NH protons are measured by 1H nuclear magnetic resonance in D2O. A procedure is used for specifically labeling with 1H only these eight NH protons. The resonance assignments of the eight protons are made chiefly by partial exchange, through correlating the resonance intensities in spectra taken when the peptide is bound and when it is dissociated from S-protein in 3.5 M-urea-d4, in D2O, pH 2.3, -4 degrees C. The two remaining assignments are made and some other assignments are checked by measurements of the nuclear Overhauser effect between adjacent NH protons of the alpha-helix. There is a transition in exchange behavior between pH 3, where the helix is weakly protected against exchange, and pH 5 where the helix is much more stable. At pH 3.1, 20 degrees C, exchange rates are uniform within the helix within a factor of two, after correction for different intrinsic exchange rates. The degree of protection within the helix is only 10 to 20-fold at this pH. At pH 5.1, 20 degrees C, the helix is more stable by two orders of magnitude and exchange occurs preferentially from the N-terminal end. At both pH values the NH proton of Asp 14, which is just outside the helix, is less protected by an order of magnitude than the adjacent NH protons inside the helix. Opening of the helix can be observed below pH 3.7 by changes in chemical shifts of the NH protons in the helix. At pH 2.4 the changes are 25% of those expected for complete opening. Helix opening is a fast reaction on the n.m.r. time scale (tau much less than 1 ms) unlike the generalized unfolding of RNAase S which is a slow reaction. Dissociation of S-peptide from S-protein in native RNAase S at pH 3.0 also is a slow reaction. Opening of the helix below pH 3.7 is a two-state reaction, as judged by comparing chemical shifts with exchange rates. The exchange rates at pH 3.1 are predicted correctly from the changes in chemical shift by assuming that helix opening is a two-state reaction. At pH values above 3.7, the nature of the helix opening reaction changes. These results indicate that at least one partially unfolded state of RNAase S is populated in the low pH unfolding transition.     

Backbone dynamics of apocytochrome b5 in its native, partially folded state

The backbone dynamics in the native state of apocytochrome b5 were studied using 15N nuclear magnetic spin relaxation measurements. The field (11.7 and 14.1 T) and temperature (10-25 degrees C) dependence of the relaxation parameters (R1, R2, and R1rho) and the 1H-15N NOE established that the protein undergoes multiple time scale internal motions related to the secondary structure. The relaxation data were analyzed with the reduced spectral density mapping approach and within the extended model-free framework. The apoprotein was confirmed to contain a disordered heme-binding loop of approximately 30 residues with dynamics on the sub-nanosecond time scale (0.6 < S2 < 0.7, 100 ps < taue < 500 ps). This loop is attached to a structured hydrophobic core, rigid on the picosecond time scale (S2 > 0.75, taue < 50 ps). The inability to fit the data for several residues with the model-free protocol revealed the presence of correlated motion. An exchange contribution was detected in the transverse relaxation rate (R2) of all residues. The differential temperature response of R2 along the backbone supported slower exchange rates for residues in the loop (tauex > 300 micros) than for the folded polypeptide chain (tauex < 150 micros). The distribution of the reduced spectral densities at the 1H and 15N frequencies followed the dynamic trend and predicted the slowing of the internal motions at 10 degrees C. Comparison of the dynamics with those of the holoprotein [Dangi, B., Sarma, S., Yan, C., Banville, D. L., and Guiles, R. D. (1998) Biochemistry 37, 8289-8302] demonstrated that binding of the heme alters the time scale of motions both in the heme-binding loop and in the structured hydrophobic core.     

Thermally induced hydrogen exchange processes in small proteins as seen by FTIR spectroscopy

Fourier-transform infrared (FTIR) spectroscopy has been used to study the thermally induced exchange characteristics of those backbone amide protons which persist H-D exchange at ambient conditions in ribonuclease A, in wild type ribonuclease T1 and some of its variants, and in the histone-like protein HBsu. The H-D exchange processes were induced by increasing the thermal energy of the protein solutions in two ways: (i) by linearly increasing the temperature, and (ii) by a temperature jump. To trace the H-D exchange in the proteins, various infrared absorption bands known to be sensitive to H-D exchange were used as specific monitors. Characteristic H-D exchange curves were obtained from which the endpoints (T_H/D) of H-D exchange could be determined. The H-D exchange curves, the T_H/D-values and the phase transition temperatures T_m were used to estimate the structural flexibility and stability of the given proteins. It is suggested that time-resolved FTIR spectroscopy can be used to determine global stability parameters of proteins.     

Mechanistic studies with general acyl-CoA dehydrogenase and butyryl-CoA dehydrogenase: evidence for the transfer of the beta-hydrogen to the flavin N(5)-position as a hydride

Butyryl-CoA dehydrogenase from Megasphera elsdenii catalyzes the exchange of the alpha- and beta-hydrogens of substrate with solvent [Gomes, B., Fendrich, G., & Abeles, R. H. (1981) Biochemistry 20, 1481-1490]. The stoichiometry of this exchange was determined by using 3H2O label as 1.94 +/- 0.1 per substrate molecule. The rate of 3H label incorporation into substrate under anaerobic conditions is monophasic, indicating that both the alpha- and beta-hydrogens exchange at the same rate. The exchange in 2H2O leads to incorporation of one 2H each into the alpha- and the beta-positions of butyryl-CoA, as determined by companion 1H NMR experiments and confirmed by mass spectroscopic analysis. In contrast, with general acyl-CoA dehydrogenase from pig kidney, only exchange of the alpha-hydrogen was found. The beta-hydrogen is the one that is transferred (reversibly) to the flavin 5-position during substrate dehydrogenation. This was demonstrated by reacting 5-3H- and 5-2H-reduced 5-deaza-FAD-general acyl-CoA dehydrogenase with crotonyl-CoA. Only one face of the reduced flavin analogue is capable of transferring hydrogen to substrate. The rate of this reaction is 11.1 s-1 for 5-deaza-FAD-enzyme and 2.2 s-1 for [5-2H]deaza-FAD-enzyme, yielding an isotope effect of 5. These values compare with a rate of 2.6 s-1 for the reaction of native reduced enzyme with crotonyl-CoA. The two reduced enzymes (normal vs. 5-deaza-FAD-enzyme) thus react at similar rates, indicating a similar mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies of the influence of dodecyl sulfate on the amide hydrogen exchange kinetics of a micelle-solubilized hydrophobic tripeptide

Backbone amide hydrogen exchange measurements are an important source of information about the internal dynamics of proteins. Before such measurements can be interpreted unambiguously, contributions to hydrogen exchange rates from the chemical and physical environment of the amides must be taken into account. Membrane proteins are often solubilized in detergents, yet there have not been any systematic investigations of the possible effects detergents may have on the amide hydrogen exchange rates of proteins. To address this question, we have measured individual backbone and carboxyl-terminal amide exchange rates for the amphipathic tripeptide Leu-Val-Ile-amide dissolved in water and dodecyl sulfate micelles. 1H NMR spectroscopy was used to measure exchange using the direct exchange-out into D2O technique at 5 degrees C and using an indirect steady-state saturation-transfer technique at 25 degrees C. The broadening effect of micelle-incorporated spin-labeled fatty acid (12-doxylstearate) on the 1H NMR spectra of both the detergent and the peptide resonances was used to demonstrate that the tripeptide is intimately associated with the micelle. The resonance from formate ion, which is excluded from the micelle, was unperturbed by the spin label. The detergent did not retard the exchange rates of either the primary (terminal) or secondary (backbone) amides of the tripeptide. This suggests that the micelle/peptide interaction does not restrict access of charged catalysts and water to these amides and shows that the peptide amides are not hydrogen bonded. However, the pH for the exchange minima of these amides in detergent was increased between 1.2 and 1.7 units compared to exchange in water.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and dynamics of a detergent-solubilized membrane protein: measurement of amide hydrogen exchange rates in M13 coat protein by 1H NMR spectroscopy

The coat protein of bacteriophage M13 is inserted into the inner membrane of Escherichia coli where it exists as an integral membrane protein during the reproductive cycle of the phage. The protein sequence consists of a highly hydrophobic 19-residue central segment flanked by an acidic 20-residue N-terminus and a basic 11-residue C-terminus. We have measured backbone amide hydrogen exchange of the protein solubilized in perdeuteriated sodium dodecyl sulfate using 1H nuclear magnetic resonance (NMR) spectroscopy. Direct proton exchange-out measurements in D2O at 24 degrees C were used to follow the exchange of the slowest amides in the protein. Multiple exponential fitting of the exchange data showed that these amides (29 +/- 3 at pH 4.5) exchanged in two kinetic sets with exchange rates [(1.2 +/- 0.4) x 10(-4) s-1 and (4.1 +/- 1.2) x 10(-7) s-1] that differed by more than 100-fold, the slower kinetic set being retarded 10(5)-fold relative to poly(DL-alanine). The exchange rate constant for the slowest set of amides exhibited an unusual pD dependence, being proportional to [OD-]1/2. It is shown that this is an artifact of the multiple exponential fitting of the data, and a new method of presentation of exchange data as a function of pD is introduced. Steady-state saturation-transfer techniques were also used to measure exchange. These methods showed that 15-20 amides in the protein are very stable at 55 degrees C and that about 30 amides have exchange rates retarded by at least 10(5)-fold at 24 degrees C. Saturation-transfer studies also showed that the pH dependence of exchange in the hydrophilic termini was unusual. This is explained as being due to long-range electrostatic effects arising both from the protein itself and also from the anionic detergent molecules. Hydrogen exchange studies on the products of proteinase K digestion of the protein localized the slowly exchanging amides to the hydrophobic core of the protein. Relaxation [Henry, G.D., Weiner, J.H., & Sykes, B.D. (1986) Biochemistry 25, 590-598] and solid-state NMR experiments [Leo, G.C., Colnago, L.A., Valentine, K.G., & Opella, S.J. (1987) Biochemistry 26, 854-862] have previously shown that the majority of the protein backbone is rigid on the picosecond to microsecond time scale, except for the extreme ends of the molecule which are mobile.(ABSTRACT TRUNCATED AT 400 WORDS)     

Measurement of 1H�C15N and 1H�C13C residual dipolar couplings in nucleic acids from TROSY intensities

Analogous to the recently introduced ARTSY method for measurement of one-bond (1)H-(15)N residual dipolar couplings (RDCs) in large perdeuterated proteins, we introduce methods for measurement of base (13)C-(1)H and (15)N-(1)H RDCs in protonated nucleic acids. Measurements are based on quantitative analysis of intensities in (1)H-(15)N and (13)C-(1)H TROSY-HSQC spectra, and are illustrated for a 71-nucleotide adenine riboswitch. Results compare favorably with those of conventional frequency-based measurements in terms of completeness and convenience of use. The ARTSY method derives the size of the coupling from the ratio of intensities observed in two TROSY-HSQC spectra recorded with different dephasing delays, thereby minimizing potential resonance overlap problems. Precision of the RDC measurements is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC reference spectrum, and is approximately given by 30/(S/N) Hz for (15)N-(1)H and 65/(S/N) Hz for (13)C-(1)H. The signal-to-noise ratio of both (1)H-(15)N and (1)H-(13)C spectra greatly benefits when water magnetization during the experiments is not perturbed, such that rapid magnetization transfer from bulk water to the nucleic acid, mediated by rapid amino and hydroxyl hydrogen exchange coupled with (1)H-(1)H NOE transfer, allows for fast repetition of the experiment. RDCs in the mutated helix 1 of the riboswitch are compatible with nucleotide-specifically modeled, idealized A-form geometry and a static orientation relative to the helix 2/3 pair, which differs by ca 6° relative to the X-ray structure of the native riboswitch.