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 microM 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 microM-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., & 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 (KD = 50 microM), the optimum size being larger than 10. Binding of poly(L-proline) is extremely fast, with k+ > 200 microM-1 s-1 at 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Laboratoire d'Enzymologie, CNRS, Gif-sur-Yvette, France.
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I
Perelroizen
J B
JB
Marchand
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L
Blanchoin
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D
Didry
M F
MF
Carlier
D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D002417:Cattle; D003285:Contractile Proteins; D007700:Kinetics; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D009994:Osmolar Concentration; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D051304:Profilins; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013154:Spleen / Q000737:chemistry; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry; D014461:Ultracentrifugation
D000199:Actins; D002413:Cations, Divalent; D003285:Contractile Proteins; D008840:Microfilament Proteins; D010455:Peptides; D051304:Profilins; C011083:polyproline; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D002118:Calcium