Myosin-induced changes in F-actin: fluorescence probing of subdomain 2 by dansyl ethylenediamine attached to Gln-41.
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.
Kim
E
E
Department of Chemistry and Biochemistry, UCLA 90095, USA.
Miller
C J
CJ
Motoki
M
M
Seguro
K
K
Muhlrad
A
A
Reisler
E
E
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D003619:Dansyl Compounds; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D005973:Glutamine / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D015394:Molecular Structure; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits
D000199:Actins; D003619:Dansyl Compounds; D005029:Ethylenediamines; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D005973:Glutamine; C032377:dansylethylenediamine; D009218:Myosins