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A study of the relaxation parameters of a 13C-enriched methylene carbon and a 13C-enriched perdeuteromethylene carbon attached to chymotrypsin.

ABSTRACT: L-1-Chloro-4-phenyl-3-tosylamido[1-13C]butan-2-one (Tos-[1-13C]Phe-CH2Cl) and Tos-[1-13C,2H2]Phe-CH2Cl were prepared and used to alkylate delta-chymotrypsin. The relaxation parameters of the 13C-n.m.r. signal resulting from the alkylation of histidine-57 in both enzyme-inhibitor complexes were determined at 1.88 T and 6.34 T as well as the spin-lattice relaxation times of the backbone alpha-carbon atoms of the unenriched Tos-Phe-CH2-delta-chymotrypsin complex. It is concluded that the species examined do not have significant internal librational motions and that the rotational correlation time of the monomeric enzyme-inhibitor complex is 16.0 +/- 3.2 ns. The signal from the 13C-enriched atom of Tos-[1-13C,2H2]Phe-CH2Cl is split into a quintet (JCD = 23 Hz) whereas in the Tos-[1-13C,2H2]Phe-CH2-delta-chymotrypsin complex the signal from the 13C-enriched inhibitor carbon atom is decoupled. This decoupled signal had linewidths of 16 +/- 3 Hz and 52 +/- 2 Hz at 1.88 T and 6.34 T respectively, whereas linewidths at 40 +/- 2 Hz and 53 +/- 4 Hz were obtained for the same signal in the Tos-[1-13C]Phe-CH2-delta-chymotrypsin complex at 1.88 T and 6.34 T respectively. Therefore whereas deuteration produces a 2.5-fold reduction in linewidth at 1.88 T there is no significant decrease in the linewidth at 6.34 T. This result is explained by using the rigid rotor model, which predicts that the quadrupolar spin-lattice relaxation rate will be faster at low field strengths, resulting in more efficient deuterium decoupling by scalar relaxation of the second kind at lower field strengths. It is also predicted that deuterium decoupling by scalar relaxation will become less efficient as rotational correlation times increase. The consequences of these predictions for the detection of 13C-enriched atomic probes of proteins are discussed. It is also shown that a spin-echo pulse sequence can be used to remove signals due to protonated carbon atoms without attenuating the signal due to deuterated carbon atoms.

SUBMITTER: Malthouse JP

PROVIDER: S-EPMC1130503 | biostudies-other | 1991 Dec

REPOSITORIES: biostudies-other

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Project description:The measurement of long-range distances remains a challenge in solid-state NMR structure determination of biological macromolecules. In 2D and 3D correlation spectra of uniformly (13)C-labeled biomolecules, inter-residue, inter-segmental, and intermolecular (13)C-(13)C cross peaks that provide important long-range distance constraints for three-dimensional structures often overlap with short-range cross peaks that only reflect the covalent structure of the molecule. It is therefore desirable to develop new approaches to obtain spectra containing only long-range cross peaks. Here we show that a relaxation-compensated modification of the commonly used 2D (1)H-driven spin diffusion (PDSD) experiment allows the clean detection of such long-range cross peaks. By adding a z-filter to keep the total z-period of the experiment constant, we compensate for (13)C T1 relaxation. As a result, the difference spectrum between a long- and a scaled short-mixing time spectrum show only long-range correlation signals. We show that one- and two-bond cross peaks equalize within a few tens of milliseconds. Within ~200 ms, the intensity equilibrates within an amino acid residue and a monosaccharide to a value that reflects the number of spins in the local network. With T1 relaxation compensation, at longer mixing times, inter-residue and inter-segmental cross peaks increase in intensity whereas intra-segmental cross-peak intensities remain unchanged relative to each other and can all be subtracted out. Without relaxation compensation, the difference 2D spectra exhibit both negative and positive intensities due to heterogeneous T1 relaxation in most biomolecules, which can cause peak cancellation. We demonstrate this relaxation-compensated difference PDSD approach on amino acids, monosaccharides, a crystalline model peptide, a membrane-bound peptide and a plant cell wall sample. The resulting difference spectra yield clean multi-bond, inter-residue and intermolecular correlation peaks, which are often difficult to resolve in the parent 2D spectra.

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A study of the relaxation parameters of a 13C-enriched methylene carbon and a 13C-enriched perdeuteromethylene carbon attached to chymotrypsin.

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