Myosin-induced changes in F-actin: fluorescence probing of subdomain 2 by dansyl ethylenediamine attached to Gln-41

Biophys J. 1996 Mar;70(3):1439-46. doi: 10.1016/S0006-3495(96)79703-5.

Abstract

Actin labeled at Gln-41 with dansyl ethylenediamine (DED) via transglutaminase reaction was used for monitoring the interaction of myosin subfragment 1 (S1) with the His-40-Gly-42 site in the 38-52 loop on F-actin. Proteolytic digestions of F-actin with subtilisin and trypsin, and acto-S1 ATPase measurements on heat-treated F-actin revealed that the labeling of Gln-41 had a stabilizing effect on subdomain 2 and the actin filaments. DED on Gln-41 had no effect on the values of K(m) and Vmax of the acto-S1 ATPase and the sliding velocities of actin filaments in the in vitro motility assays. This suggests either that S1 does not bind to the 40-42 site on actin or that such binding is not functionally important. The binding of monoclonal antidansyl IgG to DED-F-actin did not affect acto-S1 binding in the absence of nucleotides, indicating that the 40-42 site does not contribute much to rigor acto-S1 binding. Myosin-induced changes in subdomain 2 on actin were manifested through an increase in the fluorescence of DED-F-actin, a decrease in the accessibility of the probe to collisional quenchers, and a partial displacement of antidansyl IgG from actin by S1. It is proposed that these changes in the 38-52 loop on actin originate from S1 binding to other myosin recognition sites on actin.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Actins / chemistry*
  • Actins / metabolism
  • Animals
  • Binding Sites
  • Biophysical Phenomena
  • Biophysics
  • Dansyl Compounds
  • Ethylenediamines
  • Fluorescent Dyes
  • Glutamine / chemistry
  • In Vitro Techniques
  • Kinetics
  • Molecular Structure
  • Myosin Subfragments / chemistry
  • Myosin Subfragments / metabolism
  • Myosins / chemistry*
  • Myosins / metabolism
  • Protein Conformation
  • Rabbits

Substances

  • Actins
  • Dansyl Compounds
  • Ethylenediamines
  • Fluorescent Dyes
  • Myosin Subfragments
  • Glutamine
  • dansylethylenediamine
  • Myosins