SUPRAMOLECULAR ASSEMBLIES |
Role of Actin DNase-I-Binding Loop in Myosin Subfragment 1-Induced Polymerization of G-actin. Implications to the Polymerization Mechanism
Barbara Wawro 1, Sofia Yu. Khaitlina 2, Agnieszka Galinska-Rakoczy 1 and Hanna Strzelecka-Golaszewska 1*
1 Dept. of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warsaw
2 Dept. of Cell Culture, Institute of Cytology, St Petersburg
* To whom correspondence should be addressed. E-mail: hannas{at}nencki.gov.pl.
Submitted on July 8, 2004
Revised on August 31, 2004
Accepted on 29 December 2004
 |
Abstract |
|---|
Previous studies demonstrated that proteolytic cleavage of actin between Gly42 and Val43 within its DNase-I-binding loop (D-loop) abolishes the ability of Ca-G-actin to spontaneously polymerize in the presence of KCl. Here we show that such modified actin is assembled into filaments, albeit at lower rate than unmodified actin, by myosin subfragment 1 (S1) carrying A1 essential light chain but not by S1(A2). S1 titration of pyrene-G-actin showed diminished affinity of cleaved actin to S1, this effect could be however compensated for by using S1 in excess. The most significant effect of cleavage, revealed by measuring the fluorescence of pyrene-actin and light-scattering intensities as a function of actin concentration, is strong inhibition of association of G-actin-S1 (GS) complexes into oligomers. Measurements of the fluorescence of dansyl cadaverine attached to Gln41 indicate substantial inhibition of the initial association of GS into longitudinal dimers. These data provide experimental evidence for critical role of D-loop conformation in stabilization of both longitudinal, subdomain 2/1 contacts and lateral, cross-strand actin-actin contacts in the nucleation reaction. Electron microscopic analysis of changes in filament length distribution during polymerization of actin by S1(A1) and S1(A2) suggests that the mechanism of S1-induced polymerization is not substantially different from the nucleation-elongation scheme of spontaneous actin polymerization.
Key Words:
G-actin-S1 interaction, Mechanism of actin polymerization, Myosin essential light chains
