Diskin Ron, Engelberg David, Livnah Oded
The Wolfson Centre for Applied Structural Biology, The Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel.
Acta Crystallogr D Biol Crystallogr. 2007 Feb;63(Pt 2):260-5. doi: 10.1107/S0907444906042910. Epub 2007 Jan 16.
p38 MAP kinases are central signalling molecules that mediate cellular responses to numerous environmental conditions and signalling molecules. Their proper function is required for many processes, including stress response, apoptosis, differentiation, growth and even learning and memory. Abnormal activity of p38 MAP kinases is associated with the aetiology of many diseases, making understanding their activation mechanisms highly critical. In this respect, mechanistic insights may be derived from structural studies of recently developed intrinsically active p38alpha mutants. Unlike wild-type p38alpha, which routinely crystallized, the active mutants caused severe difficulties during the crystallization process. The main hindrance was found to be protein heterogeneity, which was meticulously resolved by genetically modifying the recombinant protein and optimizing the expression and purification protocols. The success in obtaining crystallizable proteins strongly emphasizes that in certain cases, high-throughput techniques (crystallization robots) together with low-throughput approaches, with careful monitoring and analysis of the results, are essential.
p38丝裂原活化蛋白激酶是核心信号分子，介导细胞对多种环境条件和信号分子的反应。许多过程都需要它们的正常功能，包括应激反应、细胞凋亡、分化、生长，甚至学习和记忆。p38丝裂原活化蛋白激酶的异常活性与许多疾病的病因有关，因此了解它们的激活机制至关重要。在这方面，可从最近开发的内在活性p38α突变体的结构研究中获得机制性见解。与常规结晶的野生型p38α不同，活性突变体在结晶过程中造成了严重困难。主要障碍是蛋白质异质性，通过对重组蛋白进行基因改造并优化表达和纯化方案，这一问题得到了精心解决。获得可结晶蛋白的成功有力地强调，在某些情况下，高通量技术（结晶机器人）与低通量方法相结合，并仔细监测和分析结果，是必不可少的。
