Unfolding crystallins: the destabilizing role of a beta-hairpin cysteine in betaB2-crystallin by simulation and experiment.
The thermodynamic and kinetic stabilities of the eye lens family of betagamma-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. betaB2-Crystallin orthologs are of low stability and comprise two typical betagamma-crystallin domains, although, uniquely, the N-terminal domain has a cysteine in one of the conserved folded beta-hairpins. Using high-temperature (500 K) molecular dynamics simulations with explicit solvent on the N-terminal domain of rodent betaB2-crystallin, we have identified in silico local flexibility in this folded beta-hairpin. We have shown in vitro using two-domain human betaB2-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 betaB2-crystallin to interact with a range of other beta-crystallin subunits.
MacDonald
James T
JT
School of Crystallography, Birkbeck College, Malet Street, London, WC1E 7HX, UK.
Purkiss
Andrew G
AG
Smith
Myron A
MA
Evans
Paul
P
Goodfellow
Julia M
JM
Slingsby
Christine
C
D002942:Circular Dichroism; D003459:Crystallins / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation
D003459:Crystallins; D003545:Cysteine