Wh. Goldmann et al., ANALYSIS OF THE F-ACTIN BINDING FRAGMENTS OF VINCULIN USING STOPPED-FLOW AND DYNAMIC LIGHT-SCATTERING MEASUREMENTS, European journal of biochemistry, 254(2), 1998, pp. 413-419
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.