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Biophys J, July 2000, p. 460-467, Vol. 79, No. 1

Is SH1-SH2-Cross-Linked Myosin Subfragment 1 a Structural Analog of the Weakly-Bound State of Myosin?

Andrey A. Bobkov* and Emil Reisler*

 *Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90095 USA

Myosin subfragment 1 (S1) with SH1 (Cys707) and SH2 (Cys697) groups cross-linked by p-phenylenedimaleimide (pPDM-S1) is thought to be an analog of the weakly bound states of myosin bound to actin. The structural properties of pPDM-S1 were compared in this study to those of S1·ADP·BeFx and S1·ADP·AlF4-, i.e., the established structural analogs of the myosin weakly bound states. To distinguish between the conformational effects of SH1-SH2 cross-linking and those due to their monofunctional modification, we used S1 with the SH1 and SH2 groups labeled with N-phenylmaleimide (NPM-S1) as a control in our experiments. The state of the nucleotide pocket was probed using a hydrophobic fluorescent dye, 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonylamido]phenylboronic acid (PPBA). Differential scanning calorimetry (DSC) was used to study the thermal stability of S1. By both methods the conformational state of pPDM-S1 was different from that of unmodified S1 in the S1·ADP·BeFx and S1·ADP·AlF4- complexes and closer to that of nucleotide-free S1. Moreover, BeFx and AlF4- binding failed to induce conformational changes in pPDM-S1 similar to those observed in unmodified S1. Surprisingly, when pPDM cross-linking was performed on S1·ADP·BeFx complex, ADP·BeFx protected to some extent the nucleotide pocket of S1 from the effects of pPDM modification. NPM-S1 behaved similarly to pPDM-S1 in our experiments. Overall, this work presents new evidence that the conformational state of pPDM-S1 is different from that of the weakly bound state analogs, S1·ADP·BeFx and S1·ADP·AlF4-. The similar structural effects of pPDM cross-linking of SH1 and SH2 groups and their monofunctional labeling with NPM are ascribed to the inhibitory effects of these modifications on the flexibility/mobility of the SH1-SH2 helix.

Biophys J, July 2000, p. 460-467, Vol. 79, No. 1
© 2000 by the Biophysical Society   0006-3495/00/07/460/08  $2.00


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