INTERACTION OF PROFILIN WITH G-ACTIN AND POLY(L-PROLINE)

The interaction of bovine spleen profilin with ATP- and ADP-G-actin and poly(L-proline) has been studied by spectrofluorimetry, analytical ultracentrifugation, and rapid kinetics in low ionic strength buffer. Profilin binding to G-actin is accompanied by a large quenching of tryptophan fluorescence, allowing the measurement of an equilibrium dissociation constant of 0.1-0.2 mu M for the 1:1 profilin-actin complex, in which metal ion and nucleotide are bound. Fluorescence quenching monitored the bimolecular reaction between G-actin and profilin, from which association and dissociation rate constants of 45 mu M(-1) s(-1) and 10 s(-1) at 20 degrees C could be derived. The tryptophan(s) which are quenched in the profilin-actin complex are no longer accessible to solvent, which points to W356 in actin as a likely candidate, consistent with the 3D structure of the crystalline profilin-actin complex [Schutt, C. E., Myslik, J. C., Rozycki, M. D., Goonesekere, N. C. W., and Lindberg, U. (1993) Nature 365, 810-816]. Upon binding poly(L-proline), the fluorescence of both tyrosines and tryptophans of profilin is enhanced 2.2-fold. A minimum of 10 prolines [three turns of poly(L-proline) helix II] is necessary to obtain binding (K-D = 50 mu M), the optimum size being larger than 10. Binding of poly(L-proline) is extremely fast, with k(+) > 200 mu M(-1) s(-1) at 10 degrees C. Neither the affinity nor the rate of association of G-actin to profilin, nor the promotion of actin assembly by profilin in the presence of thymosin beta(4) [Pantaloni, D., and Carlier, M.-F., (1993) Cell 75, 1007-1014], are affected by poly(L-proline). These results are discussed in relation with profilin structure and function in vivo.