Biophys J, November 2002, p. 2733-2741, Vol. 83, No. 5

Conformational Dynamics of the SH1-SH2 Helix in the Transition States of Myosin Subfragment-1

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

The -helix containing the thiols, SH1 (Cys-707) and SH2 (Cys-697), has been proposed to be one of the structural elements responsible for the transduction of conformational changes in the myosin head (subfragment-1 (S1)). Previous studies, using a method that isolated and measured the rate of the SH1-SH2 cross-linking step, showed that this helix undergoes ligand-induced conformational changes. However, because of long incubation times required for the formation of the transition state complexes (S1.ADP.BeF_x, S1.ADP.AlF₄, and S1.ADP.V_i), this method could not be used to determine the cross-linking rate constants for such states. In this study, kinetic data from the SH1-SH2 cross-linking reaction were analyzed by computational methods to extract rate constants for the two-step mechanism. For S1.ADP.BeF_x, the results obtained were similar to those for S1.ATPS. For reactions involving S1.ADP.AlF₄ and S1.ADP.V_i, the first step (SH1 modification) is rate limiting; consequently, only lower limits could be established for the rate constants of the cross-linking step. Nevertheless, these results show that the cross-linking rate constants in the transition state complexes are increased at least 20-fold for all the reagents, including the shortest one, compared with nucleotide-free S1. Thus, the SH1-SH2 helix appears to be destabilized in the post-hydrolysis state.

Biophys J, November 2002, p. 2733-2741, Vol. 83, No. 5
© 2002 by the Biophysical Society   0006-3495/02/11/2733/09  $2.00