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H.A. Sathish, H. Mchaourab; The Two-Mode Binding Mechanism of -Crystallin: Thermodynamic and Kinetic Studies . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2369.
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Purpose: The mechanism of chaperone activity of lens α-crystallin involves two-mode binding of substrate proteins. The two-modes have distinct energetic thresholds for activation and have been proposed to recognize and bind different substrate conformational states. In this study, fluorescence spectroscopy is used to gain insight into the conformation of a model substrate bound to αB-crystallin and its phosphorylation mimics . Methods: To investigate the recognition and binding steps of the α-crystallins, we have used site-directed mutagenesis to incrementally change the stability of T4 lysozyme (T4L). A set of T4L mutants was constructed that forms a ladder of stability spanning the 5 – 10 kcal/mol range of free energy of unfolding. Each of the T4L mutants contains a cysteine substitution at residue 151 to allow for the attachment of the fluorescent group monobromobimane. Results: The binding of bimane-labeled T4L mutants to αB-crystallin results in distinct changes in the fluorescent intensity and maximum emission wavelength. These changes depend on the molar ratio of T4L to αB-crystallin suggesting that αB-crystallin has two different binding sites and/or T4L binds in two distinct conformations. At ratios where binding occurs exclusively through the high affinity/low capacity mode, a sizeable blue shift in the emission maxima is observed accompanied by a small drop in the intensity. In contrast, analysis of the fluorescent characteristics of the bimane probe at ratios of T4L to αB-crystallin corresponding to binding at both modes reveals that a fraction of T4L, bound at the low affinity/high capacity sites, has a severely quenched fluorescence. Similar level of quenching can be induced by denaturant unfolding of bimane-labeled T4L, suggesting that binding at the high capacity mode involves substantially unfolded T4L conformations. Kinetic studies of complex formation yield two different time constants consistent with a bimodal binding mechanism. Conclusions: In addition to directly demonstrating that substrate binding occurs through two modes, the results suggest conformational preferences in the use of each mode. The low capacity mode appears to bind compact non-native states of the substrates, while the high capacity mode is selective for substantially unfolded states.
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