title: Role of actin DNase-I-binding loop in myosin subfragment 1-induced polymerization of G-actin: implications for the mechanism of polymerization.
abstract: Proteolytic cleavage of actin between Gly(42) and Val(43) 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 a lower rate than unmodified actin, by myosin subfragment 1 (S1) carrying the A1 essential light chain but not by S1(A2). S1 titration of pyrene-G-actin showed a diminished affinity of cleaved actin for S1, but this could be compensated for by using S1 in excess. The most significant effect of the cleavage, revealed by measuring the fluorescence of pyrene-actin and light-scattering intensities as a function of actin concentration at saturating concentrations of S1, is strong inhibition of association of G-actin-S1 complexes into oligomers. Measurements of the fluorescence of dansyl cadaverine attached to Gln(41) indicate substantial inhibition of the initial association of G-actin-S1 into longitudinal dimers. The data provide experimental evidence for the critical role of D-loop conformation in both longitudinal and lateral, cross-strand actin-actin contact formation in the nucleation reaction. Electron microscopic analysis of the 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.
authors.affiliation: Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Wawro
authors.forename: Sofia Yu
authors.initials: SY
authors.lastname: Khaitlina
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Galińska-Rakoczy
authors.forename: Hanna
authors.initials: H
authors.lastname: Strzelecka-Gołaszewska
mesh_terms: D000199:Actins / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001703:Biophysics / Q000379:methods; D002103:Cadaverine / Q000031:analogs & derivatives* / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000737:chemistry*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005998:Glycine / Q000737:chemistry; D007700:Kinetics; D008027:Light; D008274:Magnesium / Q000737:chemistry; D008854:Microscopy, Electron; D015879:Myosin Subfragments / Q000737:chemistry; D010590:Phalloidine / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D011721:Pyrenes / Q000737:chemistry; D011817:Rabbits; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D014633:Valine / Q000737:chemistry
chemical_list: D000199:Actins; D015879:Myosin Subfragments; D011108:Polymers; D020033:Protein Isoforms; D011506:Proteins; D011721:Pyrenes; D010590:Phalloidine; D000255:Adenosine Triphosphate; C030984:pyrene; D003850:Deoxyribonuclease I; D014633:Valine; D008274:Magnesium; C008542:monodansylcadaverine; D002103:Cadaverine; D005998:Glycine