A comprehensive analysis of multifield N-15 relaxation parameters in proteins: Determination of N-15 chemical shift anisotropies

This study deals with the exploitation of the three classical N-15 relaxati on parameters (the longitudinal relaxation rate, R-1, the transverse relaxa tion rate, R-2, and the H-1-N-15 cross-relaxation rate, sigma (NH)) measure d at several magnetic fields in uniformly N-15-labeled proteins. Spectral d ensities involved in R-1, R-2 and sigma (NH) are analyzed according to the functional form A + B/(1 + omega (2)tau (2)(x)), where tau (s) is the corre lation time associated with slow motions sensed by the NH vector at the lev el of the residue to which it belongs. The coefficient B provides a realist ic view of the backbone dynamics, whereas A is associated with fast local m otions. According to the "model free approach", B can be identified with 2 tau S-s(2) where S is the generalized order parameter. The correlation time tau (s) is determined from the field dependency of the relaxation paramete rs while A and B are determined through linear equations. This simple data processing is needed for obtaining realistic error bars based on a statisti cal approach. This proved to be the key point for validating an extended an alysis aiming at the determination of nitrogen chemical shift anisotropy. T he protein C12A-p8(MTCP1) has been chosen as a model for this study. It wil l be shown that all data (obtained at five magnetic field strengths corresp onding to proton resonance of 400, 500, 600, 700, and 800 MHz) are very con sistently fitted provided that a specific effective correlation time associ ated with slow motions is defined for each residue. This is assessed by sma ll deviations between experimental and recalculated values, which, in all c ases, remain within experimental uncertainty. This strategy makes needless elaborate approaches based on the combination of several slow motions or th eir possible anisotropy. Within the core of the protein tau (s) fluctuates in a relatively narrow range (with a mean value of 6.15 ns and a root-mean- square deviation of 0.36 ns) while it is considerably reduced at the protei n extremities (down to similar to3 ns). To a certain extent, these fluctuat ions are correlated with the protein structure. A is not obtained with suff icient accuracy to be valuably discussed. Conversely, order parameters deri ved from B exhibit a significant correlation with the protein structure. Fi nally, the multi-field analysis of the evolution of longitudinal and transv erse relaxation rates has been refined by allowing the N-15 chemical shift anisotropy (csa) to vary residue by residue. Within uncertainties (derived here on a statistical basis) an almost constant value is obtained. This str ongly indicates an absence of correlation between the experimental value of this parameter obtained for a given residue in the protein, the nature of this residue, and the possible involvement of this residue in a structured area of the protein.