Perelroizen I, Marchand J B, Blanchoin L, Didry D, Carlier M F
Laboratoire d'Enzymologie, CNRS, Gif-sur-Yvette, France.
Biochemistry. 1994 Jul 19;33(28):8472-8. doi: 10.1021/bi00194a011.
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)
利用荧光光谱法、分析超速离心法以及在低离子强度缓冲液中进行的快速动力学方法，研究了牛脾肌动蛋白单体结合蛋白与ATP-和ADP-G-肌动蛋白以及聚（L-脯氨酸）之间的相互作用。肌动蛋白单体结合蛋白与G-肌动蛋白的结合伴随着色氨酸荧光的大幅淬灭，由此测得1:1肌动蛋白单体结合蛋白-肌动蛋白复合物（其中金属离子和核苷酸已结合）的平衡解离常数为0.1 - 0.2 microM。荧光淬灭监测了G-肌动蛋白与肌动蛋白单体结合蛋白之间的双分子反应，据此可得出在20℃时缔合速率常数为45 microM⁻¹ s⁻¹，解离速率常数为10 s⁻¹。在肌动蛋白单体结合蛋白-肌动蛋白复合物中被淬灭的色氨酸不再能接触到溶剂，这表明肌动蛋白中的W356可能是候选位点，这与结晶肌动蛋白单体结合蛋白-肌动蛋白复合物的三维结构一致[舒特，C. E.，米斯利克，J. C.，罗齐茨基，M. D.，古内塞克雷，N. C. W.，& 林德伯格，U.（1993年）《自然》365，810 - 816]。在结合聚（L-脯氨酸）后，肌动蛋白单体结合蛋白的酪氨酸和色氨酸的荧光增强了2.2倍。至少需要10个脯氨酸（聚（L-脯氨酸）螺旋II的三圈）才能实现结合（KD = 50 microM），最佳大小大于10。聚（L-脯氨酸）的结合极其迅速，在10℃时k⁺ > 200 microM⁻¹ s⁻¹。（摘要截短于250字）
