Subunit exchange of lens alpha-crystallin: a fluorescence energy transfer study with the fluorescent labeled alphaA-crystallin mutant W9F as a probe.
A Trp-free alphaA-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 alphaA- and alphaA-crystallins as well as between alphaA- and alphaB-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 alphaA- or alphaB-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 (alphaA-alphaA) than between heterogeneous subunits (alphaA-alphaB). A previous exchange study with isoelectric focusing indicated a complete but slow exchange between alphaA and alphaB subunits. The present study showed that the exchange was a fast process, and the different energy transfer efficiencies between alphaA-alphaA and alphaA-alphaB indicated that alphaA- and alphaB-crystallins were not necessarily structurally equivalent.
Center for Ophthalmic Research, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
T X
TX
Sun
N J
NJ
Akhtar
J J
JJ
Liang
D000817:Anilino Naphthalenesulfonates; D003459:Crystallins / Q000737:chemistry* / Q000235:genetics; D004735:Energy Transfer; D005456:Fluorescent Dyes; D006801:Humans; D009154:Mutation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D003459:Crystallins; D005456:Fluorescent Dyes; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D014364:Tryptophan