Internal Dynamics of Human Ubiquitin Revealed by ¹³C-Relaxation Studies of Randomly Fractionally Labeled Protein

Abstract

The use of random, fractional 13C-enrichment combined with low pass filtration has allowed the determination of NMR relaxation parameters at an unprecedented number of sites within recombinant human ubiquitin. Essentially complete 1H, 13C, and 15N resonance assignments for the protein are reported. Carbon spin lattice and heteronuclear NOE relaxation data have been analyzed in the context of the Lipari-Szabo "model free" formalism. The generalized order parameters for 56 main chain 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 here indicates an unexpected range of dynamics within the interior of the protein. The generalized order parameters of 45 methyl groups of human ubiquitin have been determined. The methyl groups of Thr and Ala residues show generalized order parameters ranging from the Woessner limit (0.111) to below 0.01. Generalized order parameters for all methyl groups of the seven isoleucine residues were determined. With one exception, the generalized order parameters of the gamma methyls were equal to or greater than the corresponding delta methyls, indicating higher mobility away from the main chain. Generalized order parameters for 11 methyl groups of leucine residues were also determined. In six of the seven cases where the generalized order parameters of both prochiral methyl groups were determined, the pro-R methyl consistently shows a higher value than the pro-S methyl group. Generalized order parameters for seven methyl groups of four valines were also determined. There is no apparent correlation of methyl group prochirality with the value of the generalized order parameter. These data have several implications and generally indicate that the interior of the protein is heterogeneously dynamic.

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

“…The coefficients of and are obtained as follows: (34) Substitution of Equations (15) and (16) into the Equation (12a) results in the expression for : (35) The cross-correlation spectral density functions, , can be further simplified for the case of isotropic molecular tumbling. 28,29 In summary, we presented derivations of auto-and cross-correlated relaxation rates for several relaxation mechanisms.…”

Cold Denaturation of Encapsulated Ubiquitin

“…Summary of an intermediate state of cold-induced unfolding of encapsulated recombinant ubiquitin. The ribbon representations of the structure of encapsulated ubiquitin 13 are colorcoded according to whether a given amide N-H correlation in the 15 NHSQC spectrum is less than (red) or more than (green) a given fraction of its intensity at +20 °C. Panels A, B, and C are of ubiquitin encapsulated in 70% C 4 E 12 /25% AOT/5% DTAB at −20 °C with a equilibrium water loading of 7 and employ intensity criteria of 5%, 10% and 15% respectively.…”

De novo design of the hydrophobic core of ubiquitin

“…Secondly, ubiquitin is small (76 residues), extremely soluble, and gives well-dispersed NMR spectra, all of which should allow detailed structural characterization of variants by NMR. Third, there is a significant amount of structural (Di Stefan0 & Wand, 1987;Vijay-Kumar et al, 1987;Weber et al, 1987), dynamic (Schneider et al, 1992;Wand et al, 1996), thermodynamic (Wintrode et al, 1994), and kinetic folding data (Briggs & Roder, 1992;Khorasanizadeh et al, 1993;Khorasanizadeh et al, 1996) available for the WT protein, which will be useful for comparison with our designed proteins. In the present study, we designed and expressed several core variants of ubiquitin, and investigated how structure, stability, and conformational uniqueness is affected by alternative core packing arrangements.…”