SUBUNIT EXCHANGE OF LENS ALPHA-CRYSTALLIN - A FLUORESCENCE ENERGY-TRANSFER STUDY WITH THE FLUORESCENT-LABELED ALPHA-A-CRYSTALLIN MUTANT W9FAS A PROBE

A Trp-free alpha A-crystallin mutant (W9F) was prepared by site-direct ed mutation. This mutant appears to be identical to the wild-type in t erms of conformation (secondary and tertiary structures). W9F was labe led with a sulfhydryl-specific fluorescent probe, 2-(4'-maleimidylanil ino) naphthalene-6-sulfonate (MIANS), and used in a subunit exchange b etween alpha A- and alpha A-crystallins as well as between alpha A- an d alpha B-crystallins, studied by measurement of fluorescence resonanc e energy transfer, Energy transfer was observed between Trp (donor, wi th emission maximum at 336 nm) of wild-type alpha A- or alpha B-crysta llin and MIANS (acceptor, with absorption maximum at 313 nm) of labele d W9F when subunit exchange occurred. Time-dependent decrease of Trp a nd increase of MIANS fluorescence were recorded. The exchange was fast er at 37 degrees C than at 25 degrees C. The energy transfer efficienc y was greater between homogeneous subunits (alpha A-alpha A) than betw een heterogeneous subunits (alpha A-alpha B). A previous exchange stud y with isoelectric focusing indicated a complete but slow exchange bet ween alpha A and alpha B subunits, The present study showed that the e xchange was a fast process, and the different energy transfer efficien cies between alpha A-alpha A and alpha A-alpha B indicated that alpha A- and alpha B-crystallins were not necessarily structurally equivalen t. (C) 1998 Federation of European Biochemical Societies.