Whitley Matthew J, Zhang Jun, Lee Andrew L
Department of Biochemistry & Biophysics, School of Medicine, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Biochemistry. 2008 Aug 19;47(33):8566-76. doi: 10.1021/bi8007966. Epub 2008 Jul 26.
Protein dynamics is currently an area of intense research because of its importance as complementary information to the huge quantity of available data relating protein structure and function. Because it is usually the influence of dynamics on function that is studied, the physical determinants of the distribution of flexibility in proteins have not been explored as thoroughly. In the present NMR study, an expanded suite of five (2)H relaxation experiments was used to characterize the picosecond-to-nanosecond side-chain dynamics of chymotrypsin inhibitor 2 (CI2) and five hydrophobic core mutants, some of which are members of the folding nucleus. Because CI2 is a homologue of the serine protease inhibitor eglin c, which has already been extensively characterized in terms of its dynamics, it was possible to compare not only side-chain dynamics but also the responses of these dynamics to analogous mutations. Remarkably, each of the five core mutations in CI2 led to similar and reproducible increases in side-chain flexibility throughout the entire structure. Although the expanded suite of (2)H relaxation experiments does not affect model selection for the vast majority of residues, it did enable the detection of increasing levels of nanosecond-scale motions in CI2's reactive site binding loop as the L68 side chain was progressively shortened by mutation. Collectively, we observed that the CI2 mutants are more dynamically similar to each other than to the more rigid wild-type CI2, from which we propose that wild-type CI2 has been optimized to a specific level of rigidity which may aid in its function as a serine protease inhibitor. We also observed that the pattern of side-chain dynamics of CI2 is quantitatively similar to eglin c, but that this similarity is lost upon mutating both proteins at an equivalent position. Finally, (15)N relaxation was used to characterize the backbone dynamics of wild-type and mutant CI2. Interestingly, mutation at folding nucleus positions led to widespread increases in backbone flexibility, whereas non-folding-nucleus positions led to increases in flexibility in the C-terminal half of the protein only.
蛋白质动力学目前是一个研究热点领域，因为它作为补充信息，对于大量现有的有关蛋白质结构和功能的数据具有重要意义。由于通常研究的是动力学对功能的影响，所以蛋白质中柔韧性分布的物理决定因素尚未得到充分探索。在当前的核磁共振研究中，使用了一套扩展的五个（2）H弛豫实验来表征胰凝乳蛋白酶抑制剂2（CI2）和五个疏水核心突变体从皮秒到纳秒级的侧链动力学，其中一些突变体是折叠核心的成员。由于CI2是丝氨酸蛋白酶抑制剂依格林c的同源物，依格林c已经在动力学方面得到了广泛表征，因此不仅可以比较侧链动力学，还可以比较这些动力学对类似突变的响应。值得注意的是，CI2中的五个核心突变中的每一个都导致整个结构中侧链柔韧性出现相似且可重复的增加。尽管扩展的（2）H弛豫实验套件对绝大多数残基的模型选择没有影响，但随着L68侧链通过突变逐渐缩短，它确实能够检测到CI2反应位点结合环中纳秒级运动水平的增加。总体而言，我们观察到CI2突变体彼此之间的动力学比更刚性的野生型CI2更相似，由此我们提出野生型CI2已被优化到特定的刚性水平，这可能有助于其作为丝氨酸蛋白酶抑制剂的功能。我们还观察到CI2的侧链动力学模式在数量上与依格林c相似，但在等效位置对两种蛋白质进行突变后，这种相似性就消失了。最后，（15）N弛豫被用于表征野生型和突变型CI2的主链动力学。有趣的是，折叠核心位置的突变导致主链柔韧性普遍增加，而非折叠核心位置的突变仅导致蛋白质C端一半的柔韧性增加。
