Deuterium spin probes of side-chain dynamics in proteins. 2. Spectral density mapping and identification of nanosecond time-scale side-chain motions

In the previous paper in this issue we have demonstrated that it is possible to measure the five different relaxation rates of a deuteron in (13)CH(2)D methyl groups of (13)C-labeled, fractionally deuterated proteins. The extensive set of data acquired in these experiments provides an opportunity to investigate side-chain dynamics in proteins at a level of detail that heretofore was not possible. The data, acquired on the B1 domain of peptostreptococcal protein L, include 16 (9) relaxation measurements at 4 (2) different magnetic field strengths, 25 degrees C (5 degrees C). These data are shown to be self-consistent and are analyzed using a spectral density mapping procedure which allows extraction of values of the spectral density function at a number of frequencies with no assumptions about the underlying dynamics. Dynamics data from 31 of 35 methyls in the protein for which data could be obtained were well-fitted using the two-parameter Lipari-Szabo model (Lipari, G.; Szabo, A. J. Am. Chem. Soc. 1982, 104, 4546). The data from the remaining 4 methyls can be fitted using a three-parameter version of the Lipari-Szabo model that takes into account, in a simple manner, additional nanosecond time-scale local dynamics. This interpretation is supported by analysis of a molecular dynamics trajectory where spectral density profiles calculated for side-chain methyl sites reflect the influence of slower (nanosecond) time-scale motions involving jumps between rotameric wells. A discussion of the minimum number of relaxation measurements that are necessary to extract the full complement of dynamics information is presented along with an interpretation of the extracted dynamics parameters.     

Determination of methyl 13C-15N dipolar couplings in peptides and proteins by three-dimensional and four-dimensional magic-angle spinning solid-state NMR spectroscopy

We describe three- and four-dimensional semiconstant-time transferred echo double resonance (SCT-TEDOR) magic-angle spinning solid-state nuclear magnetic resonance (NMR) experiments for the simultaneous measurement of multiple long-range (15)N-(13)C(methyl) dipolar couplings in uniformly (13)C, (15)N-enriched peptides and proteins with high resolution and sensitivity. The methods take advantage of (13)C spin topologies characteristic of the side-chain methyl groups in amino acids alanine, isoleucine, leucine, methionine, threonine, and valine to encode up to three distinct frequencies ((15)N-(13)C(methyl) dipolar coupling, (15)N chemical shift, and (13)C(methyl) chemical shift) within a single SCT evolution period of initial duration approximately 1(1)J(CC) (where (1)J(CC) approximately 35 Hz, is the one-bond (13)C(methyl)-(13)C J-coupling) while concurrently suppressing the modulation of NMR coherences due to (13)C-(13)C and (15)N-(13)C J-couplings and transverse relaxation. The SCT-TEDOR schemes offer several important advantages over previous methods of this type. First, significant (approximately twofold to threefold) gains in experimental sensitivity can be realized for weak (15)N-(13)C(methyl) dipolar couplings (corresponding to structurally interesting, approximately 3.5 A or longer, distances) and typical (13)C(methyl) transverse relaxation rates. Second, the entire SCT evolution period can be used for (13)C(methyl) and/or (15)N frequency encoding, leading to increased spectral resolution with minimal additional coherence decay. Third, the experiments are inherently "methyl selective," which results in simplified NMR spectra and obviates the use of frequency-selective pulses or other spectral filtering techniques. Finally, the (15)N-(13)C cross-peak buildup trajectories are purely dipolar in nature (i.e., not influenced by J-couplings or relaxation), which enables the straightforward extraction of (15)N-(13)C(methyl) distances using an analytical model. The SCT-TEDOR experiments are demonstrated on a uniformly (13)C, (15)N-labeled peptide, N-acetyl-valine, and a 56 amino acid protein, B1 immunoglobulin-binding domain of protein G (GB1), where the measured (15)N-(13)C(methyl) dipolar couplings provide site-specific information about side-chain dihedral angles and the packing of protein molecules in the crystal lattice.     

A 2H NMR relaxation experiment for the measurement of the time scale of methyl side-chain dynamics in large proteins

An NMR experiment is presented for the measurement of the time scale of methyl side-chain dynamics in proteins that are labeled with methyl groups of the (13)CHD(2) variety. The measurement is accomplished by selecting a magnetization mode that to excellent approximation relaxes in a single-exponential manner with a T(1)-like rate. The combination of R(1)((13)CHD(2)) and R(2)((13)CHD(2)) (2)H relaxation rates facilitates the extraction of motional parameters from (13)CHD(2)-labeled proteins exclusively. The utility of the methodology is demonstrated with applications to proteins with tumbling times ranging from 2 ns (protein L, 7.5 kDa, 45 degrees C) to 54 ns (malate synthase G, 82 kDa, 37 degrees C); dynamics parameters are shown to be in excellent agreement with those obtained in (2)H NMR studies of other methyl isotopomers. A consistency relationship is found to exist between R(1)((13)CHD(2)) and the relaxation rates of pure longitudinal and quadrupolar order modes in (13)CH(2)D-labeled methyl groups, and experimental rates measured for a number of proteins are shown to be in excellent agreement with expectations based on theory. The present methodology extends the applicability of (2)H relaxation methods for the quantification of side-chain dynamics in high molecular weight proteins.     

Slow internal dynamics in proteins: application of NMR relaxation dispersion spectroscopy to methyl groups in a cavity mutant of T4 lysozyme

Recently developed carbon transverse relaxation dispersion experiments (Skrynnikov, N. R.; et al. J. Am. Chem. Soc. 2001, 123, 4556-4566) were applied to the study of millisecond to microsecond time scale motions in a cavity mutant of T4 lysozyme (L99A) using methyl groups as probes of dynamics. Protein expressed in E. coli cells with (13)CH(3)-pyruvate as the sole carbon source contained high levels of (13)C enrichment at a total of 80 Val gamma, Leu delta, Ile gamma (2), Ala beta, and Met epsilon methyl positions with little extraneous incorporation. Data for 72 methyl groups were available for analysis. Dispersion profiles with large amplitudes were measured for many of these residues and were well fit to a two-state exchange model. The interconversion rates and populations of the states, obtained from fitting relaxation dispersion profiles of each individual probe, were remarkably homogeneous and data for nearly all methyl groups in the protein could be collectively fit to a single cooperative conformational transition. The present study demonstrates the general applicability of methyl relaxation dispersion measurements for the investigation of millisecond time scale protein motions at a large number of side-chain positions. Potential artifacts associated with the experiments are described and methods to minimize their effects presented. These experiments should be particularly well suited for probing dynamics in high molecular weight systems due to the favorable NMR spectroscopic properties of methyl groups.     

Side chain dynamics in unfolded protein states: an NMR based 2H spin relaxation study of delta131delta

NMR relaxation data on disordered proteins can provide insight into both structural and dynamic properties of these molecules. Because of chemical shift degeneracy in correlation spectra, detailed site-specific analyses of side chain dynamics have not been possible. Here, we present new experiments for the measurement of side chain dynamics in methyl-containing residues in unfolded protein states. The pulse schemes are similar to recently proposed methods for measuring deuterium spin relaxation rates in (13)CH(2)D methyl groups in folded proteins.(1) However, because resolution in (1)H-(13)C correlation maps of unfolded proteins is limiting, relaxation data are recorded as a series of (1)H-(15)N spectra. The methodology is illustrated with an application to the study of side chain dynamics in delta131delta, a large disordered fragment of staphylococcal nuclease containing residues 1-3 and 13-140 of the wide-type protein. A good correlation between the order parameters of the symmetry axes of the methyl groups and the backbone (1)H-(15)N bond vectors of the same residue is observed. Simulations establish that such a correlation is only possible if the unfolded state is comprised of an ensemble of structures which are not equiprobable. A motional model, which combines wobbling-in-a-cone and Gaussian axial fluctuations, is proposed to estimate chi(1) torsion angle fluctuations, sigma(chi)()1, of Val and Thr residues on the basis of the backbone and side chain order parameters. Values of sigma(chi)()1 are approximately 10 degrees larger than what has previously been observed in folded proteins. Of interest, the value of sigma(chi)()1 for Val 104 is considerably smaller than for other Val or Thr residues, suggesting that it may be part of a hydrophobic cluster. Notably large (15)N transverse relaxation rates are observed in this region. To our knowledge, this is the first time that side chain dynamics in an unfolded state have been studied in detail by NMR.     

An improved picture of methyl dynamics in proteins from slowly relaxing local structure analysis of 2H spin relaxation

Protein dynamics is intimately related to biological function. Core dynamics is usually studied with 2H spin relaxation of the 13CDH2 group, analyzed traditionally with the model-free (MF) approach. We showed recently that MF is oversimplified in several respects. This includes the assumption that the local motion of the dynamic probe and the global motion of the protein are decoupled, the local geometry is simple, and the local ordering is axially symmetric. Because of these simplifications MF has yielded a puzzling picture where the methyl rotation axis is moving rapidly with amplitudes ranging from nearly complete disorder to nearly complete order in tightly packed protein cores. Our conclusions emerged from applying to methyl dynamics in proteins the slowly relaxing local structure (SRLS) approach of Polimeno and Freed (Polimeno, A.; Freed, J. H. J. Phys. Chem. 1995, 99, 10995-11006.), which can be considered the generalization of MF, with all the simplifications mentioned above removed. The SRLS picture derived here for the B1 immunoglobulin binding domain of peptostreptococcal protein L, studied over the temperature range of 15-45 degrees C, is fundamentally different from the MF picture. Thus, methyl dynamics is characterized structurally by rhombic local potentials with varying symmetries and dynamically by tenfold slower rates of local motion. On average, potential rhombicity decreases, mode-coupling increases, and the rate of local motion increases with increasing temperature. The average activation energy for local motion is 2.0 +/- 0.2 kcal/mol. Mode-coupling affects the analysis even at 15 degrees C. The accuracy of the results is improved by including in the experimental data set relaxation rates associated with rank 2 coherences.     

Three-dimensional 13C-detected CH3-TOCSY using selectively protonated proteins: facile methyl resonance assignment and protein structure determination

Recent advances in instrumentation and isotope labeling methodology allow proteins up to 100 kDa in size to be studied in detail using NMR spectroscopy. Using 2H/13C/15N enrichment and selective methyl protonation, we show that newly developed 13C direct detection methods can be used to rapidly yield proton and carbon resonance assignments for the methyl groups of Val, Leu, and Ile residues. We present a highly sensitive 13C-detected CH3-TOCSY experiment that, in combination with standard 1H-detected backbone experiments, allows the full assignment of side chain resonances in methyl-protonated residues. Selective methyl protonation, originally developed by Kay and co-workers (Rosen, M. K.; Gardner, K. H.; Willis, R. C.; Parris, W. E.; Pawson, T.; Kay, L. E. J. Mol. Biol. 1996, 263, 627-636; Gardner, K. G.; Kay, L. E. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 357-406; Goto, N. K.; Kay, L. E. Curr. Opin. Struct. Biol. 2000, 10, 585-592), improves the nuclear relaxation behavior of larger proteins compared to their fully protonated counterparts, allows significant simplification of spectra, and facilitates NOE assignments. Here, we demonstrate the usefulness of the 13C-detected CH3-TOCSY experiment through studies of (i) a medium-sized protein (CbpA-R1; 14 kDa) with a repetitive primary sequence that yields highly degenerate NMR spectra, and (ii) a larger, bimolecular protein complex (p21-KID/Cdk2; 45 kDa) at low concentration in a high ionic strength solution. Through the analysis of NOEs involving amide and Ile, Leu, and Val methyl protons, we determined the global fold of CbpA-R1, a bacterial protein that mediates the pathogenic effects of Streptococcus pneumoniae, demonstrating that this approach can significantly reduce the time required to determine protein structures by NMR.     

Heteronuclear NMR spectroscopy for lysine NH(3) groups in proteins: unique effect of water exchange on (15)N transverse relaxation

In this paper, we present a series of heteronuclear NMR experiments for the direct observation and characterization of lysine NH3 groups in proteins. In the context of the HoxD9 homeodomain bound specifically to DNA we were able to directly observe three cross-peaks, arising from lysine NH3 groups, with 15N chemical shifts around approximately 33 ppm at pH 5.8 and 35 degrees C. Measurement of water-exchange rates and various types of 15N transverse relaxation rates for these NH3 groups, reveals that rapid water exchange dominates the 15N relaxation for antiphase coherence with respect to 1H through scalar relaxation of the second kind. As a consequence of this phenomenon, 15N line shapes of NH3 signals in a conventional 1H-15N heteronuclear single quantum coherence (HSQC) correlation experiment are much broader than those of backbone amide groups. A 2D 1H-15N correlation experiment that exclusively observes in-phase 15N transverse coherence (termed HISQC for heteronuclear in-phase single quantum coherence spectroscopy) is independent of scalar relaxation in the t(1) (15N) time domain and as a result exhibits strikingly sharper 15N line shapes and higher intensities for NH3 cross-peaks than either HSQC or heteronuclear multiple quantum coherence (HMQC) correlation experiments. Coherence transfer through the relatively small J-coupling between 15Nzeta and 13Cepsilon (4.7-5.0 Hz) can be achieved with high efficiency by maintaining in-phase 15N coherence owing to its slow relaxation. With the use of a suite of triple resonance experiments based on the same design principles as the HISQC, all the NH3 cross-peaks observed in the HISQC spectrum could be assigned to lysines that directly interact with DNA phosphate groups. Selective observation of functional NH3 groups is feasible because of hydrogen bonding or salt bridges that protect them from rapid water exchange. Finally, we consider the potential use of lysine NH3 groups as an alternative probe for larger systems as illustrated by data obtained on the 128-kDa enzyme I dimer.     

Methyl rotation barriers in proteins from 2H relaxation data. Implications for protein structure

Side-chain 2H and backbone 15N relaxation data have been collected at multiple temperatures in the samples of the SH3 domain from alpha-spectrin. Combined analyses of the data allowed for determination of the temperature-dependent correlation times tauf characterizing fast methyl motion. Molecular dynamics simulations confirmed that tauf are dominated by methyl rotation; the corresponding activation energies approximate methyl rotation barriers. For 33 methyl groups in the alpha-spectrin SH3 domain the average barrier height was thus determined to be 2.8 +/- 0.9 kcal/mol. This value is deemed representative of the "fluid" hydrophobic protein core where some barriers are increased and others are lowered because of the contacts with surrounding atoms, but there is no local order that could produce systematically higher (lower) barriers. For comparison, the MD simulation predicts the average barrier of 3.1 kcal/mol (calculated via the potential of mean force) or 3.4-3.5 kcal/mol (rigid barriers after appropriate averaging over multiple MD snapshots). The latter result prompted us to investigate rigid methyl rotation barriers in a series of NMR structures from the Protein Databank. In most cases the barriers proved to be higher than expected, 4-6 kcal/mol. To a certain degree, this is caused by tight packing of the side chains in the NMR structures and stems from the structure calculation procedure where the coordinates are first annealed toward the temperature of 0 K and then subjected to energy minimization. In several cases the barriers >10 kcal/mol are indicative of van der Waals violations. The notable exceptions are (i) the structures solved using the GROMOS force field where tight methyl packing is avoided (3.0-3.6 kcal/mol) and (ii) the structure solved by means of the dynamic ensemble refinement method (Lindorff-Larsen, K.; Best, R. B.; DePristo, M. A.; Dobson, C. M.; Vendruscolo, M. Nature 2005, 433, 128) (3.5 kcal/mol). These results demonstrate that methyl rotation barriers, derived from the experiments that are traditionally associated with studies of protein dynamics, can be also used in the context of structural work. This is particularly interesting in view of the recent efforts to incorporate dynamics data in the process of protein structure elucidation.     

Relaxation rates of degenerate 1H transitions in methyl groups of proteins as reporters of side-chain dynamics

Experiments for quantifying the amplitudes of motion of methyl-containing side chains are presented that exploit the rich network of cross-correlated spin relaxation interactions between intra-methyl dipoles in highly deuterated, selectively 13CH2D- or 13CH3-labeled proteins. In particular, the experiments measure spin relaxation rates of degenerate 1H transitions in methyl groups that, for high-molecular-weight proteins, are very simply related to methyl three-fold symmetry axis order parameters. The methodology presented is applied to studies of dynamics in a pair of systems, including the 7.5-kDa protein L and the 82-kDa enzyme malate synthase G. Good agreement between 1H- and 2H-derived measures of side-chain order are obtained on highly deuterated proteins with correlation times exceeding approximately 10 ns (correlation coefficients greater than 0.95). Although 2H- and 13C-derived measures of side-chain dynamics are still preferred, the present work underscores the potential of using 1H relaxation for semiquantitative estimates of methyl side-chain flexibility, while the high level of consistency between the different spin probes of motion establishes the reliability of the dynamics parameters.     

Comparison of methyl rotation axis order parameters derived from model-free analyses of (2)H and (13)C longitudinal and transverse relaxation rates measured in the same protein sample.

Recombinant HIV-1 protease was obtained from bacteria grown on a 98% D(2)O medium containing 3-(13)C pyruvic acid as the sole source of (13)C and (1)H. The purified protein is highly deuterated at non-methyl carbons, but contains significant populations of (13)CHD(2) and (13)CH(2)D methyl isotopomers. This pattern of isotope labeling permitted measurements of (1)H and (13)C relaxation rates of (13)CHD(2) isotopomers and (2)H (D) relaxation rates of (13)CH(2)D isotopomers using a single sample. The order parameters S(axis)(2), which characterize the motions of the methyl rotation axes, were derived from model-free analyses of R(1) and R(2) data sets measured for (13)C and (2)H spins. Our primary goal was to compare the S(axis)(2) values derived from the two independent types of data sets to test our understanding of the relaxation mechanisms involved. However, S(axis)(2) values derived from the analyses depend strongly on the geometry of the methyl group, the sizes of the quadrupolar and dipolar couplings, and the effects of bond vibrations and librations on these couplings. Therefore uncertainties in these basic physical parameters complicate comparison of the order parameters. This problem was circumvented by using an experimental relationship, between the methyl quadrupolar, (13)C-(13)C and (13)C-(1)H dipolar couplings, derived from independent measurements of residual static couplings of weakly aligned proteins by Ottiger and Bax (J. Am. Chem. Soc. 1999, 121, 4690-4695) and Mittermaier and Kay (J. Am. Chem. Soc. 1999, 121, 10608-10613). This approach placed a tight experimental restraint on the values of the (2)H quadrupolar and (13)C-(1)H dipolar interactions and greatly facilitated the accurate comparison of the relative values of the order parameters. When applied to our data this approach yielded satisfactory agreement between the S(axis)(2) values derived from the (13)C and (2)H data sets.     

Probing side-chain dynamics in high molecular weight proteins by deuterium NMR spin relaxation: an application to an 82-kDa enzyme

New NMR experiments for the measurement of side-chain dynamics in high molecular weight ( approximately 100 kDa) proteins are presented. The experiments quantify (2)H spin relaxation rates in (13)CH(2)D or (13)CHD(2) methyl isotopomers and, for applications to large systems, offer significant gains both in sensitivity (2-3-fold) and resolution over previously published HSQC schemes. The methodology has been applied to investigate Ile dynamics in the 723-residue, single polypeptide chain enzyme, malate synthase G. Methyl-axis order parameters, S(axis), characterizing the amplitudes of motion of the methyl groups, have been derived from both (13)CH(2)D and (13)CHD(2) probes and are in excellent agreement. The distribution of order parameters is trimodal, reflecting the range of dynamics that are available to Ile residues. A reasonable correlation is noted between and inverse temperature factors from X-ray studies of the enzyme. The proposed methodology significantly extends the range of protein systems for which side-chain dynamics can be studied.     

Sequence-specific assignments of methyl groups in high-molecular weight proteins

A new 3D multiple-quantum (H)CCmHm-TOCSY experiment is proposed to assign methyl resonances in high-molecular weight proteins, on the basis of spectral patterns and prior backbone assignments. The favorable relaxation properties of the multiple-quantum coherences and the slow decays of in-phase methyl 13C magnetizations optimize performance of the proposed experiment for application to large proteins. The experiment has been demonstrated on an acyl carrier protein synthase (trimer, 42 kDa, overall correlation time of 26 ns) at 25 degrees C, and 63 out of 67 nonmethionine methyl groups have been assigned.     

Measurement of slow (micros-ms) time scale dynamics in protein side chains by (15)N relaxation dispersion NMR spectroscopy: application to Asn and Gln residues in a cavity mutant of T4 lysozyme.

A new NMR experiment is presented for the measurement of micros-ms time scale dynamics of Asn and Gln side chains in proteins. Exchange contributions to the (15)N line widths of side chain residues are determined via a relaxation dispersion experiment in which the effective nitrogen transverse relaxation rate is measured as a function of the number of refocusing pulses in constant-time, variable spacing CPMG intervals. The evolution of magnetization from scalar couplings and dipole-dipole cross-correlations, which has limited studies of exchange in multi-spin systems in the past, does not affect the extraction of accurate exchange parameters from relaxation profiles of NH(2) groups obtained in the present experiment. The utility of the method is demonstrated with an application to a Leu --> Ala cavity mutant of T4 lysozyme, L99A. It is shown that many of the side chain amide groups of Asn and Gln residues in the C-terminal domain of the protein are affected by a chemical exchange process which may be important in facilitating the rapid binding of hydrophobic ligands to the cavity.     

Slow dynamics in folded and unfolded states of an SH3 domain.

(15)N relaxation dispersion experiments were applied to the isolated N-terminal SH3 domain of the Drosophila protein drk (drkN SH3) to study microsecond to second time scale exchange processes. The drkN SH3 domain exists in equilibrium between folded (F(exch)) and unfolded (U(exch)) states under nondenaturing conditions in a ratio of 2:1 at 20 degrees C, with an average exchange rate constant, k(ex), of 2.2 s(-1) (slow exchange on the NMR chemical shift time scale). Consequently a discrete set of resonances is observed for each state in NMR spectra. Within the U(exch) ensemble there is a contiguous stretch of residues undergoing conformational exchange on a micros/ms time scale, likely due to local, non-native hydrophobic collapse. For these residues both the F(exch) <--> U(exch) conformational exchange process and the micros/ms exchange event within the U(exch) state contribute to the (15)N line width and can be analyzed using CPMG-based (15)N relaxation dispersion measurements. The contribution of both processes to the apparent relaxation rate can be deconvoluted numerically by combining the experimental (15)N relaxation dispersion data with results from an (15)N longitudinal relaxation experiment that accurately quantifies exchange rates in slow exchanging systems (Farrow, N. A.; Zhang, O.; Forman-Kay, J. D.; Kay, L. E. J. Biomol. NMR 1994, 4, 727-734). A simple, generally applicable analytical expression for the dependence of the effective transverse relaxation rate constant on the pulse spacing in CPMG experiments has been derived for a two-state exchange process in the slow exchange limit, which can be used to fit the experimental data on the global folding/unfolding transition. The results illustrate that relaxation dispersion experiments provide an extremely sensitive tool to probe conformational exchange processes in unfolded states and to obtain information on the free energy landscape of such systems.     

Probing slow dynamics in high molecular weight proteins by methyl-TROSY NMR spectroscopy: application to a 723-residue enzyme

A new CPMG-based multiple quantum relaxation dispersion experiment is presented for measuring millisecond dynamic processes at side-chain methyl positions in high molecular weight proteins. The experiment benefits from a methyl-TROSY effect in which cancellation of intramethyl dipole fields occurs, leading to methyl (13)C-(1)H correlation spectra of high sensitivity and resolution (Tugarinov, V.; Hwang, P. M.; Ollerenshaw, J. E.; Kay, L. E. J. Am. Chem. Soc. 2003, 125, 10420-10428). The utility of the methodology is illustrated with an application to a highly deuterated, methyl-protonated sample of malate synthase G, an 82 kDa enzyme consisting of a single polypeptide chain. A comparison of the sensitivity obtained using the present approach relative to existing HSQC-type (13)C single quantum dispersion experiments shows a gain of a factor of 5.4 on average, significantly increasing the range of applications for this methodology.     

Measurement of (13)C(alpha)-(13)C(beta) dipolar couplings in (15)N,(13)C,(2)H-labeled proteins: application to domain orientation in maltose binding protein.

TROSY-based HN(CO)CA 2D and 3D pulse schemes are presented for measurement of (13)C(alpha)-(13)C(beta) dipolar couplings in high molecular weight (15)N,(13)C,(2)H-labeled proteins. In one approach, (13)C(alpha)-(13)C(beta) dipolar couplings are obtained directly from the time modulation of cross-peak intensities in a set of 2D (15)N-(1)HN correlated spectra recorded in both the presence and absence of aligning media. In a second approach 3D data sets are recorded with (13)C(alpha)-(13)C(beta) couplings encoded in a frequency dimension. The utility of the experiments is demonstrated with an application to an (15)N,(13)C,(2)H-labeled sample of the ligand free form of maltose binding protein. A comparison of experimental dipolar couplings with those predicted from the X-ray structure of the apo form of this two-domain protein establishes that the relative orientation of the domains in solution and in the crystal state are very similar. This is in contrast to the situation for maltose binding protein in complex with beta-cyclodextrin where the solution structure can be generated from the crystal state via a 11 degrees domain closure.     

Side chain orientation from methyl 1H-1H residual dipolar couplings measured in highly deuterated proteins

High-level deuteration is a prerequisite for the study of high molecular weight systems using liquid-state NMR. Here, we present new experiments for the measurement of proton-proton dipolar couplings in CH(2)D methyl groups of (13)C labeled, highly deuterated (70-80%) proteins. (1)H-(1)H residual dipolar couplings (RDCs) have been measured in two alignment media for 57 out of 70 possible methyl containing residues in the 167-residue flavodoxin-like domain of the E. coli sulfite reductase. These data yield information on the orientation of the methyl symmetry axis with respect to the molecular alignment frame. The alignment tensor characteristics were obtained very accurately from a set of backbone RDCs measured on the same protein sample. To demonstrate that accurate structural information is obtained from these data, the measured methyl RDCs for Valine residues are analyzed in terms of chi(1) torsion angles and stereospecific assignment of the prochiral methyl groups. On the basis of the previously determined backbone solution structure of this protein, the methyl RDC data proved sufficient to determine the chi(1) torsion angles in seven out of nine valines, assuming a single-rotamer model. Methyl RDCs are complementary to other NMR data, for example, methyl-methyl NOE, to determine side chain conformation in high molecular weight systems.     

Characterization of micros-ms dynamics of proteins using a combined analysis of 15N NMR relaxation and chemical shift: conformational exchange in plastocyanin induced by histidine protonations

An approach is presented that allows a detailed, quantitative characterization of conformational exchange processes in proteins on the micros-ms time scale. The approach relies on a combined analysis of NMR relaxation rates and chemical shift changes and requires that the chemical shift of the exchanging species can be determined independently of the relaxation rates. The applicability of the approach is demonstrated by a detailed analysis of the conformational exchange processes previously observed in the reduced form of the blue copper protein, plastocyanin from the cyanobacteria Anabaena variabilis (A.v. PCu) (Ma, L.; Hass, M. A. S.; Vierick, N.; Kristensen, S. M.; Ulstrup, J.; Led, J. J. Biochemistry 2003, 42, 320-330). The R1 and R2 relaxation rates of the backbone 15N nuclei were measured at a series of pH and temperatures on an 15N labeled sample of A.v. PCu, and the 15N chemical shifts were obtained from a series of HSQC spectra recorded in the pH range from 4 to 8. From the R1 and R2 relaxation rates, the contribution, Rex, to the transverse relaxation caused by the exchanges between the different allo-states of the protein were determined. Specifically, it is demonstrated that accurate Rex terms can be obtained from the R1 and R2 rates alone in the case of relatively rigid proteins with a small rotational anisotropy. The Rex terms belonging to the same exchange process were identified on the basis of their pH dependences. Subsequently the identifications were confirmed quantitatively by the correlation between the Rex terms and the corresponding chemical shift differences of the exchanging species. By this approach, the Rex terms of 15N nuclei belonging to contiguous regions in the protein could be assigned to the same exchange process. Furthermore, the analysis of the exchange terms shows that the observed micros-ms dynamics in A.v. PCu are caused primarily by the protonation/deprotonation of two histidine residues, His92 and His61, His92 being ligated to the Cu(I) ion. Also the exchange rate of the protonation/deprotonation process of His92 and its pH and temperature dependences were determined, revealing a reaction pathway that is more complex than a simple specific-acid/base catalysis. Finally, the approach allows a differentiation between two-site and multiple-site exchange processes, thus revealing that the protonation/deprotonation of His61 is at least a three-site exchange process. Overall, the approach makes it feasible to obtain exchange rates that are sufficiently accurate and versatile for studies of the kinetics and the mechanisms of local protein dynamics on the sub-millisecond time scale.