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 vinculi n as fusion proteins with schistosomal glutathione S-transferase, we d etermined 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 previou sly detected by viscometry and electron microscopy [Johnson, R. P. & C raig, S. W. (1995) Nature 373, 261-264]. This is also supported by dyn amic 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 fusio n proteins with F-actin indicates that the F-actin binding site(s) on vinculin are located between residues 884-1012. No binding of pure glu tathione S-transferase or its fusion protein with vinculin peptide 101 2-1066 with F-actin was detected by either method.