Sathish Hasige A, Stein Richard A, Yang Guangyong, Mchaourab Hassane S
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
J Biol Chem. 2003 Nov 7;278(45):44214-21. doi: 10.1074/jbc.M307578200. Epub 2003 Aug 18.
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 bimane-labeled 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.
为了进一步深入了解小热休克蛋白（sHSP）伴侣活性的机制，我们研究了α-晶状体蛋白识别的中间状态的性质以及与之稳定结合的构象。模型底物由一组特征明确的、不稳定的T4溶菌酶（T4L）突变体组成，这些突变体已被证明以反映其解折叠自由能的方式与α-晶状体蛋白进行差异结合。基于位点特异性引入T4L中的荧光探针联硫双氯酚的构象敏感性，引入了一种检测复合物形成的新方法。联硫双氯酚标记的T4L的发射光谱揭示了结合时强度变化的两种不同模式，这取决于α-晶状体蛋白与T4L的摩尔比。这直接证明了α-晶状体蛋白结合过程的双模式性质。在广泛的T4L浓度范围内获得并分析的双相结合等温线表明，低亲和力结合的T4L中联硫双氯酚荧光大幅淬灭，这与变性剂诱导解折叠时观察到的淬灭水平相似。此外，在另一个溶剂暴露位点标记联硫双氯酚的T4L变体结合时发生的强度变化模式，建立了结合和解折叠中观察到的淬灭水平之间的直接相关性。这些结果可以用一个模型来解释，即α-晶状体蛋白结合T4L的至少两种构象不同的非天然状态，其中一种基本上是解折叠的，并且以低亲和力结合。与紧密状态的高亲和力结合模式可能与晶状体中的伴侣功能相关，在晶状体中蛋白质损伤不太可能导致整体解折叠。
