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Unfolding crystallins: The destabilizing role of a -hairpin cysteine in B2-crystallin by simulation and experiment

School of Crystallography, Birkbeck College, London, WC1E 7HX, United Kingdom

(RECEIVED November 10, 2004; FINAL REVISION February 9, 2005; ACCEPTED February 16, 2005)

The thermodynamic and kinetic stabilities of the eye lens family of -crystallins are important factors in the etiology of senile cataract. They control the chance of proteins unfolding, which can lead to aggregation and loss of transparency. B2-Crystallin orthologs are of low stability and comprise two typical -crystallin domains, although, uniquely, the N-terminal domain has a cysteine in one of the conserved folded -hairpins. Using high-temperature (500 K) molecular dynamics simulations with explicit solvent on the N-terminal domain of rodent B2-crystallin, we have identified in silico local flexibility in this folded -hairpin. We have shown in vitro using two-domain human B2-crystallin that replacement of this cysteine with a more usual aromatic residue (phenylalanine) results in a gain in conformational stability and a reduction in the rate of unfolding. We have used principal components analysis to visualize and cluster the coordinates from eight separate simulated unfolding trajectories of both the wild-type and the C50F mutant N-terminal domains. These data, representing fluctuations around the native well, show that although the mutant and wild-type appear to behave similarly over the early time period, the wild type appears to explore a different region of conformational space. It is proposed that the advantage of having this low-stability cysteine may be correlated with a subunit-exchange mechanism that allows B2-crystallin to interact with a range of other -crystallin subunits.

Keywords: age-related cataract; -crystallin fold; essential dynamics; principal components analysis; protein molecular dynamics simulation; protein stability; protein unfolding rates

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041227805.

Reprint requests to: Christine Slingsby, School of Crystallography, Birkbeck College, Malet Street, London, WC1E 7HX, UK; e-mail: c.slingsby{at}mail.cryst.bbk.ac.uk; fax: +44-20-7631-6832.

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