Sopkova J, Gallay J, Vincent M, Pancoska P, Lewit-Bentley A
LURE, Centre Universitaire Paris-Sud, Orsay, France.
Biochemistry. 1994 Apr 19;33(15):4490-9. doi: 10.1021/bi00181a008.
The binding of calcium ions to annexin V in the absence of phospholipids has been studied by UV-difference spectroscopy, circular dichroism, and steady-state and time-resolved fluorescence. In the absence of calcium, the unique tryptophan 187, located in domain III of annexin V, is surrounded by a strongly hydrophobic environment, as indicated by its "blue" fluorescence emission maximum (325 nm). This corresponds well with the description of the structure determined by X-ray crystallography of several crystal forms. The Trp187 time-resolved fluorescence decay shows the existence of a fast (picosecond) excited-state reaction which can involve the formation of an H-bond between the indole NH group and the proximate epsilon-OH and/or alpha-carbonyl groups of Thr224. Titration with calcium tends to stabilize the overall structure, as shown by circular dichroism, while leading to large modifications of the local structure around Trp187 making it accessible to the solvent as shown by UV-difference spectra, circular dichroism spectra, and the displacement of its fluorescence emission maximum at saturating concentrations of calcium (350 nm). A rapid (picosecond) formation of an excited-state complex, probably involving one or a few water molecules of the solvation shell, is observed. These observations correlate well with the conformational change observed in crystal structures obtained in high calcium concentrations, involving the removal of Trp187 from the buried position to the surface of the molecule [Sopkova, J., Renouard, M., & Lewit-Bentley, A. (1993) J. Mol. Biol. 234, 816-825; Concha, N. O., Head, J. F., Kaetzel, M. A., Dedman, J. R., & Seaton, B. A. (1993) Science 261, 1321-1324]. In the solvent-exposed conformation, the indole ring becomes mobile in the subnanosecond and nanosecond time range. This conformational change and the increase in local flexibility can be important for the accommodation of the protein on the surface of phospholipid membranes.
通过紫外差光谱、圆二色光谱以及稳态和时间分辨荧光技术，研究了在无磷脂情况下钙离子与膜联蛋白V的结合。在无钙条件下，膜联蛋白V结构域III中的独特色氨酸187被强疏水环境包围，这由其“蓝色”荧光发射最大值（325nm）表明。这与几种晶体形式的X射线晶体学确定的结构描述非常吻合。色氨酸187的时间分辨荧光衰减显示存在快速（皮秒级）激发态反应，该反应可能涉及吲哚NH基团与苏氨酸224的近端ε-OH和/或α-羰基之间形成氢键。如圆二色光谱所示，用钙滴定倾向于稳定整体结构，同时导致色氨酸187周围局部结构的大量改变，如紫外差光谱、圆二色光谱以及在钙饱和浓度下其荧光发射最大值的位移（350nm）所示，使其可被溶剂接触。观察到快速（皮秒级）形成激发态复合物，可能涉及溶剂化壳中的一个或几个水分子。这些观察结果与在高钙浓度下获得的晶体结构中观察到的构象变化密切相关，该变化涉及色氨酸187从埋藏位置移至分子表面[Sopkova, J., Renouard, M., & Lewit - Bentley, A. (1993) J. Mol. Biol. 234, 816 - 825; Concha, N. O., Head, J. F., Kaetzel, M. A., Dedman, J. R., & Seaton, B. A. (1993) Science 261, 1321 - 1324]。在溶剂暴露的构象中，吲哚环在亚纳秒和纳秒时间范围内变得可移动。这种构象变化和局部灵活性的增加对于蛋白质在磷脂膜表面的容纳可能很重要。
