Alterations in chemical shifts and exchange broadening upon peptide boronic acid inhibitor binding to α-lytic protease |
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Authors: | Jonathan H Davis David A Agard Tracy M Handel Vladimir J Basus |
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Institution: | (1) Graduate Group in Biophysics, University of California, San Francisco, CA, 94143-0448, U.S.A;(2) Department of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143-0448, U.S.A;(3) Howard Hughes Medical Institute, University of California, San Francisco, CA, 94143-0724, U.S.A;(4) Department of Molecular and Cellular Biology, University of California, Berkeley, CA, 94720, U.S.A;(5) Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, 94143-0446, U.S.A |
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Abstract: | -Lytic protease, a bacterial serine protease of 198 aminoacids (19800 Da), has been used as a model system for studies of catalyticmechanism, structure–function relationships, and more recently forstudies of pro region-assisted protein folding. We have assigned thebackbones of the enzyme alone, and of its complex with the tetrahedraltransition state mimic N-tert-butyloxycarbonyl-Ala-Pro-boroVal, usingdouble- and triple-resonance 3D NMR spectroscopy on uniformly15N- and 13C/15N-labeled protein.Changes in backbone chemical shifts between the uncomplexed and inhibitedform of the protein are correlated with distance from the inhibitor, thedisplacement of backbone nitrogens, and change in hydrogen bond strengthupon inhibitor binding (derived from previously solved crystal structures).A comparison of the solution secondary structure of the uninhibited enzymewith that of the X-ray structure reveals no significant differences.Significant line broadening, indicating intermediate chemical exchange, wasobserved in many of the active site amides (including three broadened toinvisibility), and in a majority of cases the broadening was reversed uponaddition of the inhibitor. Implications and possible mechanisms of this linebroadening are discussed. |
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Keywords: | Serine protease 3D NMR Heteronuclear NMR |
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