The consequences of ␣B-crystallin phosphorylation on its chaperone activity were investigated using a detailed analysis of the recognition and binding of destabilized T4 lysozyme (T4L) mutants by ␣B-crystallin phosphorylation mimics containing combinations of serine to aspartate substitutions. The T4L site-directed mutants were selected to constitute an energetic ladder of progressively destabilized proteins having similar structures in the folded state. ␣B-crystallin and its variants differentially recognize the T4L mutants, binding the more destabilized ones to a larger extent. Furthermore, the aspartate substitutions result in an increase in the extent of binding to the same T4L mutant and in the appearance of biphasic binding isotherms. The latter indicates the presence of two modes of binding characterized by different affinities and different numbers of binding sites. The transition to two-mode binding can also be induced by temperature or pH activation of the second mode. The similarity between the phosphorylation, pH, and temperature effects suggests a common structural origin. The location of the phosphorylation sites in the N-terminal domain and the hypothesized burial of this domain in the core of the oligomeric structure are consistent with a critical role for the destabilization of the quaternary structure in the process of recognition and binding by small heat-shock proteins.Lens transparency is the consequence of a unique molecular architecture that involves the packing of three families of proteins, the crystallins, in a glass-like state (1-3). One of these families, consisting of the two highly homologous proteins ␣A-crystallin and ␣B-crystallin, belongs to the small heat-shock protein (sHSP) 1 superfamily of chaperones and shares the common structural and functional characteristics of the superfamily (4 -6). sHSP form oligomeric complexes that recognize and bind non-native protein states without the hydrolysis of ATP. The binding capacity is remarkably high with reported stoichiometries that approach one substrate of equal molecular mass per sHSP subunit. Current models of lens transparency hypothesize a critical role of the chaperone function of ␣-crystallins in delaying aggregation of damaged proteins and the onset of opacity in the fiber cells that lack the machineries necessary for protein turnover (3, 7).One of the two ␣-crystallin subunits, ␣B-crystallin, is also widely expressed in other tissues such as cardiac (8) and skeletal muscles and the brain (9, 10), where it appears to be involved in transduction pathways activated during growth and differentiation and in response to various forms of stress (11). Evidence for the participation of ␣B-crystallin and other mammalian sHSP in these pathways includes their phosphorylation by mitogen-activated protein kinase-activated protein kinases (12-15). ␣B-crystallin is phosphorylated at three serine residues during ischemia and in response to cytotoxic signals and mitogenic and inflammatory agents (16,17). Phosphorylation is presumably used to m...