The dynamic behavior of annexin V as a function of calcium ion binding: a circular dichroism, UV absorption, and steady-state and time-resolved fluorescence study.

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.