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 phospholipi ds has been studied by UV-difference spectroscopy, circular dichroism, and steady-state and time-resolved fluorescence. In the absence of ca lcium, the unique tryptophan 187, located in domain III of annexin V, is surrounded by a strongly hydrophobic environment, as indicated by i ts ''blue'' fluorescence emission maximum (325 nm). This corresponds w ell with the description of the structure determined by X-ray crystall ography of several crystal forms. The Trp187 time-resolved fluorescenc e decay shows the existence of a fast (picosecond) excited-state react ion 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 Th r224. 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 solve nt as shown by UV-difference spectra, circular dichroism spectra, and the displacement of its fluorescence emission maximum at saturating co ncentrations of calcium (350 nm). A rapid (picosecond) formation of an excited-state complex, probably involving one or a few water molecule s of the solvation shell, is observed. These observations correlate we ll with the conformational change observed in crystal structures obtai ned in high calcium concentrations, involving the removal of Trp187 fr om the buried position to the surface of the molecule Sopkova, J., Re nouard, M., & Lewit-Bentley, A. (1993) J. Mol. Biol. 234, 816-825; Con cha, 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 ti me range. This conformational change and the increase in local flexibi lity can be important for the accommodation of the protein on the surf ace of phospholipid membranes.