1ab7

File:1ab7.gif

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NMR 15N RELAXATION AND STRUCTURAL STUDIES REVEAL CONFORMATIONAL EXCHANGE IN BARSTAR C40/82A, 30 STRUCTURES

OverviewOverview

Barstar an 89-residue protein consisting of four helices and a, three-stranded parallel beta-sheet, is the intracellular inhibitor of the, endoribonuclease barnase. Barstar C40/82A, a mutant in which the two, cysteine residues have been replaced by alanine, has been used as a pseudo, wild-type in folding studies and in the crystal structure of the, barnase:barstar C40/82A complex. We have determined a high resolution, solution structure of barstar C40/82A. The structures of barstar C40/82A, and the wild-type are superimposable. A comparison with the crystal, structure of the barnase:barstar C40/82A complex revealed subtle, differences in the regions involved in the binding of barstar to barnase., Side-chain rotations of residues Asn33, Asp35 and Asp39 and a movement of, the binding loop (Pro27-Glu32) towards the binding site of barnase, facilitate the formation of interface hydrogen bonds and aromatic contacts, in the complex. Extreme line broadening and missing signals in 1H-15N, correlation spectra indicate substantial conformational exchange for a, large subset of residues. 15N relaxation data at two magnetic field, strengths, 11.74 T and 14.10 T, were used to estimate exchange, contributions and to map the spectral density function at five, frequencies: 0, 50, 60, 450 and 540 MHz. Based on these results, model-free calculations with the inclusion of estimated exchange, contributions were used to derive order parameters and internal, correlation times. The validity of this approach has been investigated, with model-free calculations that incorporate longitudinal relaxation, rates and heteronuclear 1H-15N NOE data only at 11.74 T and 14.10 T. The, relaxation data suggest substantial conformational exchange in regions of, barstar C40/82A, including the binding loop, the second and the third, helices, and the second and the third strands. Amide proton exchange, experiments suggest a stable hydrogen bond network for all helices and, sheets except the third helix and the C-terminal of the second and the, third strands. The combined results indicate a rigid body movement of the, second helix and twisting motions of the beta-sheet of barstar, which, might be important for the interaction with barnase.

About this StructureAbout this Structure

1AB7 is a Single protein structure of sequence from Bacillus amyloliquefaciens. Structure known Active Site: BNB. Full crystallographic information is available from OCA.

ReferenceReference

NMR 15N relaxation and structural studies reveal slow conformational exchange in barstar C40/82A., Wong KB, Fersht AR, Freund SM, J Mol Biol. 1997 May 2;268(2):494-511. PMID:9159486

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