Conformational flexibility of domain III of annexin V at membrane water interfaces

The conformational dynamics of domain III in annexin V bound to negatively charged phospholipid vesicles of 1-palmitoyl-2-oleoyl-sn-glycerophosphochol ine and 1-palmitoyl-2-oleoyl-sn-glycerophosphoserine or incorporated into r everse micelles of water/sodium bis(2-ethylhexyl) sulfosuccinate in isoocta ne, used to mimic the phospholipid/water interface, was studied by steady-s tate and time-resolved fluorescence of its single tryptophan residue (W187) . Upon interaction with sonicated phospholipid vesicles in the presence of calcium, or upon incorporation into reverse micelles without calcium, a pro gressive 12-14 nm red shift of the fluorescence emission spectrum of W187 i s observed. The indole environment becomes therefore more polar than in the unbound protein. Three major lifetime populations describe the fluorescenc e intensity decays of W187 in both systems. A long-lived excited-state popu lation characterizes the membrane-bound state of the protein. The existence of local conformers with different subnanosecond mobility is suggested by specific association between lifetimes and correlation times both for the p rotein in buffer and in interaction with the membrane surface. The interact ion of the protein with the membrane surface preserves the existence of a r apid unhindered rotational motion, which is coupled with all three lifetime s. The longest lifetime is coupled to restricted motions in subnanosecond a nd nanosecond time scales. The overall amplitude of rotation of the indole ring is increased in the membrane-bound conformation of the protein. In rev erse micelles, the local dynamics reported by W187 is also considerably inc reased whereas the overall folding of the protein remains unaffected. The s ame conformational change of domain III can therefore be provoked by differ ent conditions: calcium binding at high concentration, mild acidic pH [Sopk ova, J., Vincent, M., Takahashi, M., Lewit-Bentley, A., and Gallay, J. (199 8) Biochemistry 37, 11952-11970] and the interaction of the protein with th e membrane surface. The high flexibility of domain III in the membrane-boun d protein suggests that this domain may not be crucial for the interaction of the protein with the membrane, in contrast with previous models. Our dat a are compatible with atomic force microscopy results which suggest that do main III of annexin V does not interact strongly with the membrane surface [Reviakine, I., Bergma-Schutter, W., and Brisson, A. (1998) J. Struct. Biol . 121, 356-361].