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Fluorescence resonance energy transfer study of subunit exchange in human lens crystallins and congenital cataract crystallin mutants

Ophthalmic Research/Surgery, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA
Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02115, USA

(RECEIVED March 14, 2006; FINAL REVISION April 3, 2006; ACCEPTED April 3, 2006)

Lens -crystallin is an oligomeric protein with a molecular mass of 500–1000 kDa and a polydispersed assembly. It consists of two types of subunits, A and B, each with a molecular mass of 20 kDa. The subunits also form homo-oligomers in some other tissues and in vitro. Their quaternary structures, which are dynamic and characterized by subunit exchange, have been studied by many techniques, including fluorescence resonance energy transfer (FRET) and mass spectrometry analysis. The proposed mechanism of subunit exchange has been either by dissociation/association of monomeric subunits or by rapid equilibrium between oligomers and suboligomers. To explore the nature of subunit exchange further, we performed additional FRET measurements and analyses using a fluorescent dye-labeled W9F A-crystallin as the acceptor probe and Trp in other crystallins (wild-type and R116C A, wild-type and R120G B, wild-type and Q155* B2) as the donor probe and calculated the transfer efficiency, Förster distance, and average distance between two probes. The results indicate only slight decreased efficiency and increased distance between two probes for the R116C A and R120G B mutations despite conformational changes.

Keywords: crystallin; FRET; subunit exchange; protein–protein interaction; fluorescence; congenital cataract crystallin mutants

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062216006.

Abbreviations: Bis-ANS, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid; CD, circular dichroism; FRET, fluorescence resonance energy transfer; HMW, high-molecular-weight; IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid.

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