Subunit exchange of lens α-crystallin:: a fluorescence energy transfer study with the fluorescent labeled αA-crystallin mutant W9F as a probe

A Trp-free alpha A-crystallin mutant (W9F) was prepared by site-directed mutation. This mutant appears to be identical to the wild-type in terms of conformation (secondary and tertiary structures). W9F was labeled with a sulfhydryl-specific fluorescent probe, 2-(4'-maleimidylanilino) naphthalene-6-sulfonate (MIANS), and used in a subunit exchange between alpha A- and alpha A-crystallins as well as between alpha A- and alpha B-crystallins, studied by measurement of fluorescence resonance energy transfer, Energy transfer was observed between Trp (donor, with emission maximum at 336 nm) of wild-type alpha A- or alpha B-crystallin and MIANS (acceptor, with absorption maximum at 313 nm) of labeled W9F when subunit exchange occurred. Time-dependent decrease of Trp and increase of MIANS fluorescence were recorded. The exchange was faster at 37 degrees C than at 25 degrees C. The energy transfer efficiency was greater between homogeneous subunits (alpha A-alpha A) than between heterogeneous subunits (alpha A-alpha B). A previous exchange study with isoelectric focusing indicated a complete but slow exchange between alpha A and alpha B subunits, The present study showed that the exchange was a fast process, and the different energy transfer efficiencies between alpha A-alpha A and alpha A-alpha B indicated that alpha A- and alpha B-crystallins were not necessarily structurally equivalent. (C) 1998 Federation of European Biochemical Societies.