Koteiche Hanane A, McHaourab Hassane S
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA.
J Biol Chem. 2003 Mar 21;278(12):10361-7. doi: 10.1074/jbc.M211851200. Epub 2003 Jan 14.
The consequences of alphaB-crystallin phosphorylation on its chaperone activity were investigated using a detailed analysis of the recognition and binding of destabilized T4 lysozyme (T4L) mutants by alphaB-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. alphaB-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.
通过详细分析含丝氨酸到天冬氨酸取代组合的αB-晶状体蛋白磷酸化模拟物对不稳定T4溶菌酶（T4L）突变体的识别和结合，研究了αB-晶状体蛋白磷酸化对其伴侣活性的影响。选择T4L定点突变体以构成折叠状态下结构相似但稳定性逐渐降低的能量梯。αB-晶状体蛋白及其变体对T4L突变体的识别存在差异，对稳定性更低的突变体结合程度更高。此外，天冬氨酸取代导致与相同T4L突变体的结合程度增加，并出现双相结合等温线。后者表明存在两种结合模式，其特征为不同的亲和力和不同数量的结合位点。第二种模式的温度或pH激活也可诱导向双模式结合的转变。磷酸化、pH和温度效应之间的相似性表明存在共同的结构起源。磷酸化位点在N端结构域的位置以及该结构域在寡聚体结构核心中的假设埋藏，与小热休克蛋白在识别和结合过程中四级结构不稳定的关键作用一致。
