Analysis of the F-actin binding fragments of vinculin using stopped-flow and dynamic light-scattering measurements.
Using amino acids 884-1066 and 884-1012 expressed from chicken vinculin as fusion proteins with schistosomal glutathione S-transferase, we determined the binding kinetics of the protein fragments with F-actin. We established by the stopped-flow method a two-step binding process: an initial rapid reaction followed by a slower process. The latter is attributed to F-actin cross-linking and/or bundling, which was previously detected by viscometry and electron microscopy [Johnson, R. P. & Craig, S. W. (1995) Nature 373, 261-264]. This is also supported by dynamic light-scattering measurements, indicating dramatic changes in the internal actin filament dynamics, i.e. in bending undulations due to thermal noise. The similar size of the binding reaction for both fusion proteins with F-actin indicates that the F-actin binding site(s) on vinculin are located between residues 884-1012. No binding of pure glutathione S-transferase or its fusion protein with vinculin peptide 1012-1066 with F-actin was detected by either method.
Goldmann
W H
WH
Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA. goldman@helix.mgh.harvard.edu
Guttenberg
Z
Z
Tang
J X
JX
Kroy
K
K
Isenberg
G
G
Ezzell
R M
RM
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008954:Models, Biological; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012542:Scattering, Radiation; D012547:Schistosoma / Q000201:enzymology / Q000235:genetics; D016596:Vinculin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D016596:Vinculin; D005982:Glutathione Transferase