期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 278, 期 45, 页码 44214-44221出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M307578200
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资金
- NEI NIH HHS [EY-R0112683] Funding Source: Medline
To further develop the mechanistic understanding of small heat-shock protein ( sHSP) chaperone activity, we investigate the nature of the intermediate states recognized by alpha-crystallin and the conformations that are stably bound. The model substrates consist of a set of well characterized, destabilized T4 Lysozyme (T4L) mutants that have been shown to differentially bind alpha-crystallin in a manner that reflects their free-energy of unfolding. A new approach for the detection of complex formation is introduced based on the conformational sensitivity of the fluorescent probe bimane, site-specifically introduced in T4L. Emission spectra of bimanelabeled T4L reveal two distinct patterns of intensity changes upon binding that depend on the molar ratio of alpha-crystallin to T4L. This directly demonstrates the two-mode nature of the binding process by the alpha-crystallins. Biphasic binding isotherms, obtained and analyzed over a wide range of T4L concentrations, demonstrate a substantially quenched bimane fluorescence in the low affinity-bound T4L that is similar to the quenching level observed due to denaturant unfolding. Furthermore, the pattern of intensity changes that occur upon binding of a T4L variant, bimane-labeled at an alternative solvent-exposed site, establishes a direct correlation between the quenching level observed in binding and unfolding. The results can be interpreted in terms of a model where alpha-crystallin binds at least two conformationally distinct non-native states of T4L, one of which is substantially unfolded and is bound with low affinity. A high affinity binding mode to compact states may be relevant to chaperone function in the lens, where protein damage is unlikely to cause global unfolding.
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