| Abstract
| - Residual dipolar couplings (RDC) from partially aligned molecules provide long-range structuraldata and are thus particularly well adapted to rapid structure validation or protein fold recognition. Extensivemeasurements in two alignment media can also provide precise de novo structure from RDC alone. Wehave applied a novel combination of these approaches to the study of methionine sulfoxide reductase(MsrA) from Erwiniachrysanthemi, a 27 kDa enzyme essential for repairing oxidative stress damage. Thetertiary fold was initially validated by comparing backbone RDC to expected values from the crystal structureof the homologous MsrA from Escherichia coli. Good agreement was found throughout the chain, verifyingthe overall topology of the molecule, with the exception of the catalytically important peptide P196−L202,where strong and systematic RDC violation was observed. No evidence for local differential mobility in thisregion was detected, implying that the structure of the strand differs in the two molecules. We have thereforeapplied the de novo approach meccano to determine the conformation of this peptide using only RDC. Asingle conformation is found that is in agreement with all measured data. The aligned peptide can bedocked onto the expected covalence of the rest of the template molecule while respecting its strictly definedrelative orientation. In contrast to the structure of MsrA from E. coli, the reactive side chain of Cys200 isoriented toward the interior of the molecule and therefore closer to the catalytic Cys53, obviating the needfor previously proposed conformational reorganization prior to formation of this disulfide intermediate. Thisanalysis requires only backbone assignment and uses unambiguously assigned and readily measurablestructural data, thereby greatly economizing investigation time compared to established nuclear Overhausereffect- (nOe-) based structure calculation methods.
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