Biomedical subjects

Lewis E Kay

Publications and source records attributed to Lewis E Kay.

79 records · Page 5Linked to original sources

PubMed2002

Deuterium spin probes of side-chain dynamics in proteins. 1. Measurement of five relaxation rates per deuteron in (13)C-labeled and fractionally (2)H-enriched proteins in solution.

New pulse sequences are presented for the measurement of the relaxation of deuterium double quantum, quadrupolar order, and transverse antiphase magnetization in (13)CH(2)D methyl groups of (15)N-, (13)C-labeled, fractionally deuterated proteins. Together with previously developed experiments for measuring deuterium longitudinal and transverse decay rates [Muhandiram, D. R.; Yamazaki, T.; Sykes, B. D.; Kay, L. E. J. Am. Chem. Soc. 1995, 117, 11536], these schemes allow measurement of the five unique decay constants of a single deuteron, providing an unprecedented opportunity to investigate side-chain dynamics in proteins. All five deuterium relaxation rates have been measured for deuterons in the methyl groups of the B1 immunoglobulin binding domain of peptostreptococcal protein L and the N-terminal SH3 domain from the protein drk. Since values of the spectral density function at only three different frequencies contribute to the five relaxation rates, the self-consistency of the relaxation data is readily established. Very good agreement is obtained between calculated parameters describing the amplitudes and time scales of motion when different subsets of the relaxation data are employed.

Bacterial Proteins↗

PubMed2002

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.

Bacterial Proteins↗

PubMed2002

Measurement of side-chain carboxyl pK(a) values of glutamate and aspartate residues in an unfolded protein by multinuclear NMR spectroscopy.

A triple-resonance NMR pulse scheme is presented for measuring aspartic and glutamic acid side-chain pK(a) values in unfolded protein states where chemical shift overlap is limiting. The experiment correlates side-chain carboxyl carbon chemical shifts of these residues with the backbone amide proton chemical shift of the following residue. The methodology is applied to an (15)N, (13)C labeled sample of the N-terminal SH3 domain of the Drosophila protein drk, which exists in equilibrium between folded (F(exch)) and unfolded (U(exch)) states under nondenaturing conditions. Residue-specific pK(a) values of side-chain carboxyl groups are presented for the first time for an unfolded protein (drk U(exch) state), determined from a pH titration. Results indicate that deviations from pK(a) values measured for model compounds are likely due to local effects, while long-range electrostatic interactions appear to be of minor importance for this protein.

Animals↗

PubMed2002

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.

Bacteriophage T4↗

PubMed2002

Distribution of molecular size within an unfolded state ensemble using small-angle X-ray scattering and pulse field gradient NMR techniques.

The size distribution of molecules within an unfolded state of the N-terminal SH3 domain of drk (drkN SH3) has been studied by small-angle X-ray scattering (SAXS) and pulsed-field-gradient NMR (PFG-NMR) methods. An empirical model to describe this distribution in the unfolded state ensemble has been proposed based on (i) the ensemble-averaged radius of gyration and hydrodynamic radius derived from the SAXS and PFG-NMR data, respectively, and (ii) a histogram of the size distribution of structures obtained from preliminary analyses of structural parameters recorded on the unfolded state. Results show that this unfolded state, U(exch), which exists in equilibrium with the folded state, F(exch), under non-denaturing conditions, is relatively compact, with the average size of conformers within the unfolded state ensemble only 30-40% larger than the folded state structure. In addition, the model predicts a significant overlap in the size range of structures comprising the U(exch) state with those in a denatured state obtained by addition of 2 M guanidinium chloride.

Animals↗

PubMed2002

A "three-pronged" binding mechanism for the SAP/SH2D1A SH2 domain: structural basis and relevance to the XLP syndrome.

The SH2 domain protein SAP/SH2D1A, encoded by the X-linked lymphoproliferative (XLP) syndrome gene, associates with the hematopoietic cell surface receptor SLAM in a phosphorylation-independent manner. By screening a repertoire of synthetic peptides, the specificity of SAP/SH2D1A has been mapped and a consensus sequence motif for binding identified, T/S-x-x-x-x-V/I, where x represents any amino acid. Remarkably, this motif contains neither a Tyr nor a pTyr residue, a hallmark of conventional SH2 domain-ligand interactions. The structures of the protein, determined by NMR, in complex with two distinct peptides provide direct evidence in support of a "three-pronged" binding mechanism for the SAP/SH2D1A SH2 domain in contrast to the "two-pronged" binding for conventional SH2 domains. Differences in the structures of the two complexes suggest considerable flexibility in the SH2 domain, as further confirmed and characterized by hydrogen exchange studies. The structures also explain binding defects observed in disease-causing SAP/SH2D1A mutants and suggest that phosphorylation-independent interactions mediated by SAP/SH2D1A likely play an important role in the pathogenesis of XLP.

Antigens, CD↗

PubMed2002

Effect of deuteration on some structural parameters of methyl groups in proteins as evaluated by residual dipolar couplings.

One bond methyl 1H-13C and 13Cmethyl-13C scalar and residual dipolar couplings have been measured at sites in an 15N, 13C, approximately 50% 2H labeled sample of the B1 immunoglobulin binding domain of peptostreptococcal protein L to investigate changes in the structure of methyl groups in response to deuterium substitution. Both one bond methyl 1H-13C and 13Cmethyl-13C scalar coupling constants have been found to decrease slightly with increasing deuterium content. Previous studies have shown that 1H-13C couplings in methyl groups are exquisitely sensitive to electronic structure, with decreases in coupling values as a function of deuteration consistent with a slight lengthening of the remaining H-C bonds. Changes in the HmethylCmethylC angle are found to be small, with average differences on the order of 0.3+/-0.1 degrees and 0.4+/-0.2 degrees between CH3, CH2D and CH3, CHD2 isotopomers, respectively. Knowledge of methyl geometry is a prerequisite for the extraction of accurate dynamics parameters from spin relaxation studies involving these groups.

Bacterial Proteins↗