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Chemical Decoupling of ATPase Activation and Force Production from the Contractile Cycle in Myosin by Steric Hindrance of Lever-Arm Movement

Andras Muhlrad^*, Y. Michael Peyser^*, Mahta Nili, Katalin Ajtai, Emil Reisler and Thomas P. Burghardt

^* Department of Oral Biology, Hebrew University Hadassah School of Dental Medicine, Jerusalem, Israel 91120; Department of Physiology, University of California School of Medicine, Los Angeles, California 90095 USA; Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905 USA; and Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90095 USA

Correspondence: Address reprint requests to Thomas P. Burghardt. Tel.: 507-284-8120; Fax: 507-284-9349; E-mail: burghardt{at}mayo.edu.

The myosin motor protein generates force in muscle by hydrolyzing Adenosine 5'-triphosphate (ATP) while interacting transiently with actin. Structural evidence suggests the myosin globular head (subfragment 1 or S1) is articulated with semi-rigid catalytic and lever-arm domains joined by a flexible converter domain. According to the prevailing hypothesis for energy transduction, ATP binding and hydrolysis in the catalytic domain drives the relative movement of the lever arm. Actin binding and reversal of the lever-arm movement (power stroke) applies force to actin. These domains interface at the reactive lysine, Lys84, where trinitrophenylation (TNP-Lys84-S1) was observed in this work to block actin activation of myosin ATPase and in vitro sliding of actin over myosin. TNP-Lys84-S1's properties and interactions with actin were examined to determine how trinitrophenylation causes these effects. Weak and strong actin binding, the rate of mantADP release from actomyosin, and actomyosin dissociation by ATP were equivalent in TNP-Lys84-S1 and native S1. Molecular dynamics calculations indicate that lever-arm movement inhibition during ATP hydrolysis and the power stroke is caused by steric clashes between TNP and the converter or lever-arm domains. Together these findings suggest that TNP uncouples actin activation of myosin ATPase and the power stroke from other steps in the contraction cycle by inhibiting the converter and lever-arm domain movements.