Pfuhl M, Improta S, Politou A S, Pastore A
EMBL, Heidelberg, Germany.
J Mol Biol. 1997 Jan 17;265(2):242-56. doi: 10.1006/jmbi.1996.0725.
In the course of a structural study of titin, a giant modular protein from muscle, we have reported that N-terminal extension of immunoglobulin-like (Ig-like) domains from titin stabilizes this fold. In order to investigate the structural basis of such an effect, we have solved the structure of NEXTM5, which has six amino acids added to the sequence of M5, a domain for which full structure determination has been previously achieved. In the present work, the structures and the dynamics of M5 and NEXTM5 are compared in the light of data collected for these and other titin domains. In NEXTM5, three out of the six added residues are structured and pack against the nearby BC and FG loops. As a consequence, three new backbone hydrogen bonds are formed with the B strand, extending the A strand by two residues and decreasing the exposed surface area of the loops. Additional contacts which involve the side-chains give rise to a remarkable pH dependence of the stability. Interestingly, no correlation is observed on the NMR time-scale between the overall dynamics of the extended domain and its increased stability. The most noticeable differences between the two constructs are localised around the N terminus, which becomes more rigid upon extension. Since a similar pattern of contacts is observed for other domains of the immunoglobulin I-set, our results are of general relevance for this protein family. Our work might also inspire a more rational approach to the investigation of domain boundaries and their influence on module stability.
在对肌联蛋白（一种来自肌肉的巨大模块化蛋白质）进行结构研究的过程中，我们报告称，肌联蛋白中免疫球蛋白样（Ig样）结构域的N端延伸稳定了这种折叠结构。为了研究这种效应的结构基础，我们解析了NEXTM5的结构，它在M5序列上添加了六个氨基酸，M5是一个此前已完成完整结构测定的结构域。在本研究中，根据为这些以及其他肌联蛋白结构域收集的数据，对M5和NEXTM5的结构与动力学进行了比较。在NEXTM5中，添加的六个残基中有三个形成了结构，并与附近的BC环和FG环堆积在一起。结果，与B链形成了三个新的主链氢键，使A链延伸了两个残基，并减小了环的暴露表面积。涉及侧链的其他相互作用导致稳定性呈现出显著的pH依赖性。有趣的是，在核磁共振时间尺度上，未观察到延伸结构域的整体动力学与其增加的稳定性之间存在相关性。这两种构建体之间最明显的差异集中在N端周围，N端在延伸后变得更加刚性。由于在免疫球蛋白I-set的其他结构域中也观察到了类似的接触模式，我们的结果对这个蛋白质家族具有普遍意义。我们的工作也可能启发一种更合理的方法来研究结构域边界及其对模块稳定性的影响。
