The anisotropy of rapid fluctuations of the peptide planes in ubiquitin is explored by combined 15N and 13C‘ nuclear spin relaxation measurements and molecular dynamics (MD) computer simulation. T 1, T 2, and NOE data were collected at B 0-field strenghts corresponding to 400 and 600 MHz proton resonance. A 1.5-ns simulation of ubiquitin in an explicit water environment was performed using CHARMM 24. The simulation suggests that, for 76% of the peptide planes, the relaxation-active motion of the backbone 15N and 13C‘ spins is dominated by anisotropic Gaussian axial fluctuations of the peptide planes about three orthogonal axes. The dominant fluctuation axes are nearly parallel to the − axes. The remaining peptide planes belong to more flexible regions of the backbone and cannot be described by this type of motion alone. Based on the results of the computer simulation, an analytical 3D GAF motional model (Bremi, T.; Brüschweiler, R. J. Am. Chem. Soc. 1997, 119, 6672−6673) was applied to the experimental relaxation data. The fluctuation amplitudes of the peptide planes show a significant anisotropy of the internal motion. This analysis demonstrates that a combined interpretation of 15N and 13C‘ relaxation data by a model derived from a computer simulation may provide detailed insight into the fast time-scale backbone dynamics that goes beyond the results of a standard model-free analysis.

No abstract

A new protocol for the interpretation of NMR relaxation data in terms of intramolecular motion is described. At first, a long molecular dynamics simulation of the system is generated and analyzed with respect to nuclear spin relaxation-active motional modes. In a second step, an analytical model is conceived on the basis of the computational results. Finally, the model parameters are determined numerically by a least-squares fit to the experimental NMR data. This protocol was applied to the phenylalanine side-chain dynamics in the cyclic decapeptide antamanide. A 100 ns Langevin dynamics simulation was analyzed in terms of dihedral angle fluctuations, correlation functions, and potentials of mean force. For χ1 and χ2, motion in a harmonic potential is observed which is interrupted by occasional jumps between different rotamers over relatively high barriers. This behavior leads to an analytical Gaussian axial fluctuation and jump model, which is an extension of the previously proposed GAF model (Brüschweiler, R.; Wright, P. E. J. Am. Chem. Soc. 1994, 116, 8426).

High-resolution heteronuclear NMR correlation experiments and strategies are proposed for the assignment of fully 13C/15N-labelled polypeptides in the solid state. By the combination of intra-residue and inter-residue 13C-15N correlation experiments with 13C-13C spin-diffusion studies, it becomes feasible to partially assign backbone and side-chain resonance in solid proteins. The performance of sequences using 15N instead of 13C detection is evaluated regarding sensitivity and resolution for a labelled dipeptide (L-Val-L-Phe). The techniques are used for a partial assignment of the 15N and 13C resonances in human ubiquitin.

No abstract