Protein Science, Vol 6, Issue 7 1529-1536, Copyright © 1997 by Cold Spring Harbor Laboratory Press

ARTICLE

Mutational analysis of hydrophobic domain interactions in {gamma}B-crystallin from bovine eye lens

S. PALME, C. SLINGSBY and R. JAENICKE
Institut fur Biophysik und Physikalische Biochemie, Universitat Regensburg, D-93040 Regensburg, Germany Department of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, United Kingdom

{gamma}B-crystallin is a monomeric member of the {beta}{gamma}-superfamily of vertebrate eye lens proteins. It consists of two similar domains with all-{beta} Greek key topology associating about an approximate two-fold axis. At pH 2, with urea as the denaturant, the domains show independent equilibrium unfolding transitions, suggesting different intrinsic stabilities. Denaturation experiments using recombinant one- or two-domain proteins showed that the N-terminal domain on its own exhibits unaltered intrinsic stability but contributes significantly to the stability of its C-terminal partner. It has been suggested that docking of the domains is determined by a hydrophobic interface that includes phenylalanine at position 56 of the N-terminal domain. In order to test this hypothesis, F56 was substituted by site-directed mutagenesis in both complete {gamma}B-crystallin and its isolated N-terminal domain. All mutations destabilize the N-terminal domain to about the same extent but affect the C-terminal domain in a different way. Replacement by the small alanine side chain or the charged aspartic acid residue results in a significant destabilization of the C-terminal domain, whereas the more bulky tryptophan residue causes only a moderate decrease in stability. In the mutants F56A and F56D, equilibrium unfolding transitions obtained by circular dichroism and intrinsic fluorescence differ, suggesting a more complex denaturation behavior than the one observed for {gamma}B wild type. These results confirm how mutations in one crystallin domain can affect the stability of another when they occur at the interface. The results strongly suggest that size, hydrophobicity, and optimal packing of amino acids involved in these interactions are critical for the stability of {gamma}B-crystallin.
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