PROTEIN NMR STRUCTURE AND DYNAMICS VIA ISOTOPIC LABELING

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DESCRIPTION (Adapted from abstract): For the first time efficient monitoring of dynamics is reported for essentially all proton-bearing carbons in a protein via NMR relaxation measurements. This biosynthetic labeling protocol generates each residue type is enriched with a 13C-12C-13C pattern out along each sidechain thus providing high levels of enrichment without one bond 13C-13C couplings and thus well suited for relaxation studies. A dynamics analysis formalism has been developed that robustly interprets protein internal motion in terms of an arbitrary multiexponential autocorrelation function. The resultant ability to characterize local correlated motion and its conformational entropy will be applied to the N-terminal domain of the calcium-dependent signal protein calmodulin and to the larger 23 kDa E. coli adenylate kinase. Quantitative analysis of the sidechain dynamics of calmodulin will substantially expand upon previous mainchain relaxation studies so as to provide a clearer understanding of how the cooperativity of calcium binding is related to the dynamics of the conformational transition, which yields the active signaling state. Binding of each of the two substrates ATP and AMP to adenylate kinase results in large scale cleft closures around the active site. The degree of dynamical crosstalk between these two cleft closure transitions is matter of active debate. Residues within the active site appear to substantially change their mobility upon substrate binding and strong dynamical coupling to the domain closure has been postulated. The proposed relaxation experiments will quantitatively address these key issues. The relaxation measurements will be complemented by residual dipolar coupling experiments to provide global orientational information to characterize the individual hinge fluctuations. The relaxation and dipolar coupling experiments will be facilitated by combining chiral deuteration with the alternate carbon enrichment of the glycerol carbon source in a perdeuterated background. This labeling approach gives rise to a large set of isolated 1H-13C spin pairs exhibiting excellent resolution and sensitivity suitable for both the relaxation and dipolar coupling experiments. A large number of stereoselective assignments are provided directly from the enrichment pattern. This labeling pattern is likewise well suited for more conventional NOE-based solution structure determinations and its range of utility will be demonstrated