Evaluation of folding co-operativity of a chimeric protein based on the molecular recognition between polyproline ligands and SH3 domains
Evaluation of folding co-operativity of a chimeric protein based on the molecular recognition between polyproline ligands and SH3 domains
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DOI：
10.1093/protein/gzp041
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发表时间：
2009-10-01
影响因子：
2.4
通讯作者：
Martinez, Jose C.
中科院分区：
文献类型：
--
作者：
Candel, Adela M.;Cobos, Eva S.;Martinez, Jose C.
在以前的工作中，我们设计了一个嵌合蛋白，命名为SPCp 41，评估热力学的SH 3结构域和脯氨酸丰富的配体之间的相互作用，结合热去折叠测量和诱变。在这里，我们已经调查了充满活力的完整性的链延伸对应的配体序列的天然结构，因为相反的将产生的SH 3域的折叠机制的变化，可能会引起不希望的贡献的热力学参数。我们分析了标准条件（50 mM磷酸盐，pH 7）下的折叠-解折叠动力学。动力学演变是很好地描述了一个双指数，在主动力学阶段的顶部，一个低人口较慢的阶段出现的结果，顺反异构化的Pro39，脯氨酰异构酶的影响和突变分析所证明的。还有一个爆发阶段可能是由于一些螺旋合奏的生产性形成。主要的演变，占真正的折叠动力学的SPCp 41，可以被认为是一个两态过程，其中折叠过渡态产生基本上相同的图片所示的圆形排列S19-P20（设计的“核”）和配体将对接在两态过程的后期阶段。因此，所有的结论都支持SPC p41研究SH 3-配体相互作用的能量，动力学和结构方面的有效性。
In previous work, we designed a chimeric protein, named SPCp41, to evaluate the thermodynamics of the interaction between SH3 domains and proline-rich ligands by combining thermal unfolding measurements and mutagenesis. Here, we have investigated the energetic integrity of the chain extension corresponding to the ligand sequence into the native structure, since the opposite will produce changes in the folding mechanism of the SH3 domain that may give rise to undesirable contributions to the thermodynamic parameters. We have analysed the folding-unfolding kinetics under standard conditions (50 mM phosphate pH 7). Kinetic evolutions are well described by a bi-exponential where, on top of the main kinetic phase, a low-populated slower phase appears as a consequence of cis-trans isomerisation of Pro39, as demonstrated by the influence of prolyl isomerases and by mutational analysis. There is also a burst phase possibly due to a productive formation of some helical ensembles. The main evolution, accounting for the true folding kinetics of SPCp41, can be considered as a two-state process, where the folding transition state produces essentially the same picture shown by the circular permutant S19-P20s (the 'nucleus' of the design) and the ligand will dock at the latter stages of the two-state process. Thus, all conclusions argue in favour of the effectiveness of SPCp41 to study energetic, dynamic and structural aspects of SH3-ligand interactions.