INTERNAL DYNAMICS OF HUMAN UBIQUITIN REVEALED BY C-13-RELAXATION STUDIES OF RANDOMLY FRACTIONALLY LABELED PROTEIN

The use of random, fractional C-13-enrichment combined with low pass f iltration has allowed the determination of NMR relaxation parameters a t an unprecedented number of sites within recombinant human ubiquitin. Essentially complete H-1, C-13, and N-15 resonance assignments for th e protein are reported. Carbon spin lattice and heteronuclear NOE rela xation data have been analyzed in the context of the Lipari-Szabo ''mo del free'' formalism. The generalized order parameters for 56 main cha in alpha C-H vectors have been determined and are found to correspond to the highly restricted motion seen in previous studies of the motion of amide N-H vectors. In distinct contrast, the analysis presented he re indicates an unexpected range of dynamics within the interior of th e protein. The generalized order parameters of 45 methyl groups of hum an ubiquitin have been determined. The methyl groups of Thr and Ala re sidues show generalized order parameters ranging from the Woessner lim it (0.111) to below 0.01. Generalized order parameters for all methyl groups of the seven isoleucine residues were determined. With one exce ption, the generalized order parameters of the gamma methyls were equa l to or greater than the corresponding delta methyls, indicating highe r mobility away from the main chain. Generalized order parameters for 11 methyl groups of leucine residues were also determined. In six of t he seven cases where the generalized order parameters of both prochira l methyl groups were determined, the pro-R methyl consistently shows f our valines were also determined. There is no apparent correlation of methyl group prochirality with the value of the generalized order para meter. These data have several implications and generally indicate tha t the interior of the protein is heterogeneously dynamic.