Dynamics of methyl groups in proteins as studied by proton-detected carbon-13 NMR spectroscopy. Application to the leucine residues of staphylococcal nuclease

Abstract

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 Mr = 16.8K, in the absence and presence of ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+. We report the synthesis of [5,5'-13C2]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 13C 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 (less than 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, Ss2, lie in the range 0.34-0.92. The values of Ss2 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 Ss2 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 Ss2 values obtained from liganded and unliganded SNase reveals a strong correlation between delta Ss2 and distance between the leucine methyl carbon and the ligands.(ABSTRACT TRUNCATED AT 400 WORDS)

Insights into the local residual entropy of proteins provided by NMR relaxation

“…1). It is also interesting to note that the order parameter is essentially linear with entropy in the range of S 2 values commonly seen for methyl symmetry axes (see Nicholson et al, 1992;Muhandiram et al, 1995;Pascal et al, 1995;Kay et al, 1996;Wand et al, 1996). Finally, notwithstanding the potential uncertainty of the parameter C in the model, the heterogeneous distribution of dynamics seen in the interiors of several proteins corresponds to a significant range of contributions to their residual entropy.…”

“…Though comprehensive studies of the backbone dynamics of proteins based on analysis of I5N relaxation have appeared, it is only recently that general techniques have emerged to probe the fast dynamics of amino acid side chains. Earlier approaches employing 13C relaxation in selectively I3C enriched samples (e.g., Nicholson et al, 1992) have recently been supplemented by use of methods relying on general random fractional labeling (Wand et al, 1995). In parallel, techniques using deuterium relaxation in both selective (e.g.. Tamura et al, 1996) and randomly fractionally deuterated proteins (Muhandiram et al, 1995) have also been developed to investigate the fast dynamics of proteins.…”

Stereospecific gating of functional motions in Pin1

“…We used the Lipari-Szabo formalism (16)(17)(18) to extract motional parameters from the R 1 ð 2 DÞ and R 1ρ ð 2 DÞ values. The Lipari-Szabo parameters describe 13 C-2 D bond motions due to conformational dynamics via site-specific order parameters S 2 axis , and effective correlation times, τ e .…”

Characterization of the Fast Dynamics of Protein Amino Acid Side Chains Using NMR Relaxation in Solution

“…The term vanishes since S z commutes with T̃2 ,0 , while the contribution of can be calculated using standard procedures: (32) A symmetric pair of interactions between the and also contributes to cross-correlated relaxation rate, giving a factor of two in the final equation for . Using Equation 12a, we obtain the following expression for : (33) where d is defined in Equation 15.…”