The ???? family of crystallins provide the high refractive index required by the lens, while the alpha crystallins, ?A? and ?B? help maintain their stability. ??crystallins confer stability by binding these client proteins and preventing their unfolding and aggregation under cellular stress conditions. This has even led to their designation as ?molecular sponges? that form ?chaperone complexes? with client proteins. ?A? and ?B?crystallins show 57% sequence identity and bind a number of clients, and only some clients are common to both chaperones. Chemical modifications such as Ser-phosphorylation or hydroimidazolone formation enhance client binding. Upon forming chaperone complexes alpha crystallins show increased binding to lipid membranes ? but it is not known how such binding affects membrane structure. We hypothesize that in aging-associated, and genetic cataracts, alpha crystallins complexed with ?client? proteins not only increasingly bind to membranes, but also alter their viscoelastic properties which could have a sustained, adverse effect on membrane function ? and hence ? on lens transparency. Therefore, an essential first step is determining the structural basis of complex formation and interaction with membranes. While several cryo- EM studies, aided by NMR and MS studies have provided high-resolution models of ?B?crystallin, these structures represent only a small percentage of the distribution of oligomers, and lack important features such as the placement of the N-terminal region, and several residues in the C-terminal region. More importantly, the structures of the chaperone complexes and bound client proteins are largely unknown. The???crystallins also bind small peptides like melittin (used to identify the putative alpha-crystallin domain), and metabolites such as ATP (which appears to enhance chaperone function), in addition to binding client proteins. Thermodynamic analysis of such small molecule binding will provide a quantitative understanding of the nature of binding which is currently missing.
The specific aims are: 1) Cryo-electron microscopy (cryo-EM) studies of recombinant human ??? ????????and ??? ????? homo-oligomers and their chaperone complexes with cataract-associated mutants of human ?-crystallins and insulin. 2) Examining the effect of chaperone complexes on cell membrane models, namely lipid monolayers and supported-bilayers. 3) Isothermal titration calorimetric (ITC) studies of melittin and ATP binding to HAA and HAB. The main techniques are: Cryo-EM for high-resolution structure determination; Isothermal titration calorimetry (ITC) for thermodynamic binding studies, Langmuir film balance for membrane-complex interactions, and Quartz crystal microbalance with dissipation (QCMD) to measure viscoelastic properties of model membranes.
The lens ??crystallins form large molecular complexes with proteins that they prevent from degradation. We subscribe to the hypothesis that over time, due to the binding of such complexes to the lens cell membrane, a barrier is formed (i.e. a barrier to normal metabolites) which leads to age-onset cataract. This disease, highly prevalent in the aging population, has no cure at present. We have proposed studies to understand the structure of the complexes, their binding to cell membrane models, and the effect of such binding on the integrity of the membrane, thereby testing the plausibility of this hypothesis.