title: Conformational dynamics of the SH1-SH2 helix in the transition states of myosin subfragment-1.
abstract: The alpha-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.AlF(4)-, 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.ATPgammaS. For reactions involving S1.ADP.AlF(4)- 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.
authors.affiliation: Department of Chemistry and Biochemistry, Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
authors.forename: Lisa K
authors.initials: LK
authors.lastname: Nitao
authors.forename: Todd O
authors.initials: TO
authors.lastname: Yeates
authors.forename: Emil
authors.initials: E
authors.lastname: Reisler
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D003198:Computer Simulation; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D006868:Hydrolysis; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry*; D009218:Myosins / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012984:Software; D013696:Temperature; D013997:Time Factors; D018909:src Homology Domains
chemical_list: D015879:Myosin Subfragments; D004228:Dithionitrobenzoic Acid; D000251:Adenosine Triphosphatases; D009218:Myosins