Backbone dynamics of a model membrane protein: carbon-13 NMR spectroscopy of alanine methyl groups in detergent-solubilized M13 coat protein

Abstract

The filamentous coliphage M13 possesses multiple copies of a 50-residue coat protein which is inserted into the inner membrane of Escherichia coli during infection. 13C nuclear magnetic resonance (NMR) spectroscopy has been used to probe the structure and dynamics of M13 coat protein solubilized in detergent micelles. A comparison of backbone dynamics within the hydrophobic core region and the hydrophilic terminal domains was obtained by biosynthetic incorporation of [3-13C]alanine. Alanine is distributed throughout the protein and accounts for 10 residues (i.e., 20% of the total). Similar 13C NMR spectra of the protein have been obtained in two anionic detergents, sodium deoxycholate and sodium dodecyl sulfate, although the structures and physical properties of these solubilizing agents are quite different. The N-terminal alanine residues, assigned by pH titration, and the penultimate residue, assigned by carboxypeptidase A digestion, give rise to analogous peaks in both detergent systems. The pKa of Ala-1 (approximately 8.8) and the relaxation parameters of individual carbon atoms (T1, T2, and the nuclear Overhauser enhancement) are also generally similar, suggesting a similarity in the overall protein structure. Relaxation data have been analyzed according to the model-free approach of Lipari and Szabo [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4559]. The overall correlation times were obtained by fitting the three experimental relaxation values for a given well-resolved single carbon atom to obtain a unique value for the generalized order parameter, S2, and the effective correlation time, tau e. The former parameter reflects the spatial restriction of motion, and the latter, the rate.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…Provided that spins I 1 and I 2 have non-degenerate chemical shifts, the two tensor components that remain are 2 1z (2 z) I S z of T̃2 ,0 and of T̃2 ,±1 , giving rise to the and spectral density terms. 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.…”

Angular variances for internal bond rotations of side chains in GXG-based tripeptides derived from13C-NMR relaxation measurements: Implications to protein folding

“…(3)] with D Ϫq ϭ D q . The ␤ is the angle formed between the Z axis of the rotational diffusion tensor and the C ␣ -C ␤ bond, and d (2) are second-order Wigner rotation matrices. The a , a , b , and b are time-independent polar angles for a and b motional vectors in a molecular frame where the Z axis is directed along the C ␣ -C ␤ bond.…”

“…The ( j ) 1 are correlation times for methyl group rotations determined by using the model of anisotropic diffusion with multiple internal rotations. The ( j ) 2 and (E j ) 2 are correlation times and potential energy barriers for methyl group rotations determined by using a two-parameter model expressed in Eq. (17).…”

“…Rotations about specific side-chain bonds, for example, are often more sensitive to changes in protein conformation and folding than are backbone bond rotations. [1][2][3][4][5][6][7][8][9][10][11][12] In folded proteins and peptides, backbone bond rotations that occur primarily via rotational fluctuations within potential wells are often easier to model, whereas side chains can generally probe greater conformational space and also undergo jumps among various rotomer states. From studies of the temperature dependence of nmrderived motional parameters, it is apparent that in partially folded peptides, 11,12 amplitudes of internal rotations about backbone , bonds vary little with temperature, whereas in folded structures, correlations between various i (t) and k (t) bond rotations can be substantial and can change dramatically with temperature.…”

Global and internal molecular dynamics of poly(acrylamide-co-allyl 2-acetamido-2-deoxy-D-glucopyranoside) glycopolymers from 13C NMR relaxation studies

“…Since analyses have proven useful for the analysis of relaxation data in recent years, a certain preference has been given to the utilization of the "modelfree" approach of Lipari and Szabo (16) (equivalent in spectral density form to the "two-step" model suggested by Wennerstrom et al (17)). A number of authors have applied this model-free approach to the molecular dynamics of a number of biologically related systems (18)(19)(20)(21). In particular, and in relation to this work, the model has been adapted to examine both the internal and overall motions of a number of saccharides (22)(23)(24).…”