title: Binding of destabilized betaB2-crystallin mutants to alpha-crystallin: the role of a folding intermediate.
abstract: Age-related changes in protein-protein interactions in the lens play a critical role in the temporal evolution of its optical properties. In the relatively non-regenerating environment of the fiber cells, a critical determinant of these interactions is partial or global unfolding as a consequence of post-translational modifications or chemical damage to individual crystallins. One type of attractive force involves the recognition by alpha-crystallins of modified proteins prone to unfolding and aggregation. In this paper, we explore the energetic threshold and the structural determinants for the formation of a stable complex between alpha-crystallin and betaB2-crystallin as a consequence of destabilizing mutations in the latter. The mutations were designed in the framework of a folding model that proposes the equilibrium population of a monomeric intermediate. Binding to alpha-crystallin is detected through changes in the emission properties of a bimane fluorescent probe site-specifically introduced at a solvent exposed site in betaB2-crystallin. alpha-Crystallin binds the various betaB2-crystallin mutants, although with a significantly lower affinity relative to destabilized T4 lysozyme mutants. The extent of binding, while reflective of the overall destabilization, is determined by the dynamic population of a folding intermediate. The existence of the intermediate is inferred from the biphasic bimane emission unfolding curve of a mutant designed to disrupt interactions at the dimer interface. The results of this paper are consistent with a model in which the interaction of alpha-crystallins with substrates is not solely triggered by an energetic threshold but also by the population of excited states even under favorable folding conditions. The ability of alpha-crystallin to detect subtle changes in the population of betaB2-crystallin excited states supports a central role for this chaperone in delaying aggregation and scattering in the lens.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors.forename: Hasige A
authors.initials: HA
authors.lastname: Sathish
authors.forename: Hanane A
authors.initials: HA
authors.lastname: Koteiche
authors.forename: Hassane S
authors.initials: HS
authors.lastname: McHaourab
mesh_terms: D000375:Aging / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding / Q000502:physiology; D017510:Protein Folding; D013329:Structure-Activity Relationship; D038201:alpha-Crystallins / Q000737:chemistry / Q000378:metabolism*; D038206:beta-Crystallin B Chain / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D038201:alpha-Crystallins; D038206:beta-Crystallin B Chain; C113634:beta-crystallin B2