DYNAMICS OF METHYL-GROUPS IN PROTEINS AS STUDIED BY PROTON-DETECTED C-13 NMR-SPECTROSCOPY - APPLICATION TO THE LEUCINE RESIDUES OF STAPHYLOCOCCAL NUCLEASE

This paper describes the application of recently developed nuclear magnetic resonance (NMR) pulse sequences to obtain information about the internal dynamics of isotopically enriched hydrophobic side chains in proteins. The two-dimensional spectra provided by the pulse sequences enable one to make accurate measurements of nuclear Overhauser effects (NOE) and longitudinal (T1) and transverse (T2) relaxation times of enriched methyl carbons in proteins. Herein, these techniques are used to investigate the internal dynamics of the 11 leucine side chains of staphylococcal nuclease (SNase), a small enzyme having M(r) = 16.8K, in the absence and presence of ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+. We report the synthesis of [5,5'-C-13(2)]leucine, the preparation of SNase containing the labeled leucine, the sequential assignment of the leucine methyl carbons and protons in the liganded and unliganded proteins, and the measurement of the C-13 T1, T2, and NOE values for the SNase leucine methyl carbons. Analysis of the relaxation parameters using the formalism of Lipari and Szabo shows that the internal motions of the leucine methyl carbons are characterized by effective correlation times tau(f) (5-80 ps) and tau(s) (<2 ns). The fast motion is identified with the rapid rotation of the methyl group about the C(gamma)-C(delta) bond axis, while the slow motion is associated with reorientation of the C(gamma)-C(delta) bond axis itself. The mean squared order parameters associated with the latter motion, S(s)2, lie in the range 0.34-0.92. The values of S(s)2 correlate reasonably well with the temperature factors of the leucine methyl carbons obtained from the crystal structures, but some are smaller than anticipated on the basis of the fact that nearly all leucine methyl carbons are buried and have temperature factors no larger than that of the leucine backbone atoms. Five leucine residues in liganded SNase and eight in unliganded SNase have values of S(s)2 less than 0.71. These order parameters correspond to large amplitude motions (angular excursions of 27-67-degrees) of the C(gamma)-C(delta) bond axis. These results indicate that, in solution, the internal motions of the leucine side chains of SNase are significantly larger than suggested by the X-ray structures or by qualitative analysis of NOESY spectra. Comparison of S(s)2 values obtained from liganded and unliganded SNase reveals a strong correlation between DELTA-S(s)2 and distance between the leucine methyl carbon and the ligands. A significant increase in S(s)2 upon ligand binding occurs exclusively in those leucine side chains in the vicinity (<10 angstrom) of either the Ca2+ atom or the pdTp heavy atoms, indicating a localized stiffening of the protein structure in regions near the ligand-binding sites.