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

FEBS Letters

Liang, Jack J.-N¹  Akhtar, Nila J¹  Sun, Tian-Xiao¹ 

[1] Center for Ophthalmic Research, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Avenue, Boston, MA 02115, USA

关键词: α-Crystallin;    αA-Crystallin mutant;    Trp fluorescence;    Fluorescence resonance energy transfer;    Intermolecular exchange;    Circular dichroism;   

DOI  :  10.1016/S0014-5793(98)00707-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A Trp-free αA-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 αA- and αA-crystallins as well as between αA- and αB-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 αA- or αB-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°C than at 25°C. The energy transfer efficiency was greater between homogeneous subunits (αA-αA) than between heterogeneous subunits (αA-αB). A previous exchange study with isoelectric focusing indicated a complete but slow exchange between αA and αB subunits. The present study showed that the exchange was a fast process, and the different energy transfer efficiencies between αA-αA and αA-αB indicated that αA- and αB-crystallins were not necessarily structurally equivalent.

【 授权许可】

Unknown