This proposal is to characterize the solution structure and dynamics of polyubiquitin (polyUb) chains, a universal proteolytic signal in eukaryotes. The ubiquitin-proteasome proteolytic pathway is the principal regulatory mechanism for the turnover of short-lived proteins that influences a variety of vital cellular events. Understanding how polyUb chains are recognized by the 26S proteasome is central to understanding of the mechanisms of regulation. Although significant evidence suggests that the specific recognition of different polyUb chains is based on conformational determinants, as yet there is no clear view of the conformational properties of these molecules in solution. Obtaining such information is absolutely necessary in order to develop a molecular understanding of how different chains are able to act as specific signals. We will use NMR approaches to determine the three-dimensional conformation of the tetra-Ub chain free in solution and bound to a peptide mimic of the proteasome. This will be performed using long-range, orientational constraints derived from spin-relaxation and residual dipolar couplings measurements. These novel NMR approaches will be used in combination with the conventional methods based on NOES and mapping of the interdomain or intermolecular interface based on chemical shift perturbations and solvent accessibility measurements. For these studies, we will use segmentally isotope-labeled chains assembled from bacterially-expressed Ub monomers (unlabeled and uniformly labeled) using in vitro conjugation reaction. We will measure protein dynamics in the dual Ub and tetra-Ub, in comparison with that of the monomeric Ub, in order to determine the contribution from interdomain motions and characterize the conformational flexibility of polyUb chains.
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