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Experimental Investigation of the Seesaw Mechanism of the Relay Region That Moves the Myosin Lever Arm*S⃞

By Bálint Kintses, Zhenhui Yang and András Málnási-Csizmadia

Abstract

A seesaw-like movement of the relay region upon the recovery step of myosin was recently simulated in silico. In this model the relay helix tilts around its pivoting point formed by a phenylalanine cluster (Phe481, Phe482, and Phe652), which moves the lever arm of myosin. To study the effect of the elimination of the proposed pivoting point, these phenylalanines were mutated to alanines in two Dictyostelium myosin II motor domain constructs (MF481A, F482A and MF652A). The relay movement was followed by the fluorescence change of Trp501 located in the relay region. The steady-state and transient kinetic fluorescence experiments showed that the lack of the phenylalanine fulcrum perturbs the formation of the “up” lever arm state, and only moderate effects were found in the nucleotide binding, the formation of the “down” lever arm position, and the ATP hydrolysis steps. We conclude that the lack of the fulcrum decouples the distal part of the relay from the nucleotide binding site upon the recovery step. Our molecular dynamics simulations also showed that the conformation of the motor is not perturbed by the mutation in the down lever arm state, however, the lack of the pivoting point rearranges the dynamic pattern of the kink region of the relay helix

Topics: Protein Structure and Folding
Publisher: American Society for Biochemistry and Molecular Biology
OAI identifier: oai:pubmedcentral.nih.gov:2662230
Provided by: PubMed Central
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