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Insights into the domains required for dimerization and assembly of human B crystallin

¹ Biomolecular Structure and Design,
² Department of Biological Structure, and
³ Department of Ophthalmology, University of Washington, Seattle, Washington 98195-7420, USA

(RECEIVED October 1, 2004; FINAL REVISION November 29, 2004; ACCEPTED November 30, 2004)

Protein pin array technology was used to identify subunit–subunit interaction sites in the small heat shock protein (sHSP) B crystallin. Subunit–subunit interaction sites were defined as consensus sequences that interacted with both human A crystallin and B crystallin. The human B crystallin protein pin array consisted of contiguous and overlapping peptides, eight amino acids in length, immobilized on pins that were in a 96-well ELISA plate format. The interaction of B crystallin peptides with physiological partner proteins, A crystallin and B crystallin, was detected using antibodies and recorded using spectrophotometric absorbance. Five peptide sequences including ₃₇LFPTSTSLSPFYLRPPSF₅₄ in the N terminus, ₇₅FSVNLDVK₈₂ (3), ₁₃₁LTITSSLS₁₃₈ (8) and ₁₄₁GVLTVNGP₁₄₈ (9) that form strands in the conserved crystallin core domain, and ₁₅₅PERTIPITREEK₁₆₆ in the C-terminal extension were identified as subunit–subunit interaction sites in human B crystallin using the novel protein pin array assay. The subunit–subunit interaction sites were mapped to a three-dimensional (3D) homology model of wild-type human B crystallin that was based on the crystal structure of wheat sHSP16.9 and Methanococcus jannaschi sHSP16.5 (Mj sHSP16.5). The subunit–subunit interaction sites identified and mapped onto the homology model were solvent-exposed and had variable secondary structures ranging from strands to random coils and short helices. The subunit–subunit interaction sites formed a pattern of hydrophobic patches on the 3D surface of human B crystallin.

Keywords: B crystallin; lens; chaperone; small heat shock protein; assembly; molecular modeling

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041152805.

Reprint requests to: John I. Clark, Department of Biological Structure, HSB G514, Box 357420, University of Washington, Seattle, WA 98195-7420, USA; e-mail: clarkji{at}u.washington.edu; fax: (206) 543-1524.

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