Walsh S T, Lee A L, DeGrado W F, Wand A J
The Johnson Research Foundation and Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia 19104-6059, USA.
Biochemistry. 2001 Aug 14;40(32):9560-9. doi: 10.1021/bi0105274.
Understanding how the amino acid sequence of a polypeptide chain specifies a unique, functional three-dimensional structure remains an important goal, especially in the context of the emerging discipline of de novo protein design. Alpha3D is a single chain protein of 73 amino acids resulting from a de novo design effort. Previous solution nuclear magnetic resonance studies of alpha3D confirm that the protein adopts the designed structure of a three-helix bundle. Furthermore, alpha3D has been previously shown to possess all of the major thermodynamic and structural characteristics of natural proteins, though it shares no sequence homology to any protein sequence in the database. In this work, the backbone and side-chain dynamics of alpha3D were investigated using 15N, 13C, and 2H nuclear magnetic resonance relaxation methods with the aim of assessing the character of the internal motions of this native-like protein of de novo design. At the backbone level, both 15N and 13C(alpha) relaxation studies indicate highly restrictive motion on the picosecond to nanosecond time scale in the alpha-helical regions of alpha3D, with increasing mobility at the ends of the alpha-helices and in the two loop regions. This is largely consistent with what is seen in proteins of natural origin. Overall, the view provided by both 2H and 13C methyl relaxation methods suggest that the side chains of alpha3D are more dynamic compared to natural proteins. Regions of relative flexibility bound clusters of rigid methyl-bearing side-chain groups that are interspersed with aromatic and beta-branched amino acids. The time scale of motions associated with methyl-bearing side chains of alpha3D are significantly longer than that seen in natural proteins. These results indicate that the strategies underlying the design of alpha3D have largely, but not completely, captured both the structural and dynamic character of natural proteins.
理解多肽链的氨基酸序列如何决定一个独特的、功能性的三维结构仍然是一个重要目标，特别是在新兴的从头蛋白质设计学科背景下。Alpha3D是通过从头设计得到的由73个氨基酸组成的单链蛋白质。先前对Alpha3D进行的溶液核磁共振研究证实，该蛋白质采用了设计的三螺旋束结构。此外，先前已表明Alpha3D具有天然蛋白质的所有主要热力学和结构特征，尽管它与数据库中的任何蛋白质序列都没有序列同源性。在这项工作中，使用15N、13C和2H核磁共振弛豫方法研究了Alpha3D的主链和侧链动力学，目的是评估这种从头设计的类天然蛋白质内部运动的特征。在主链水平上，15N和13C（α）弛豫研究均表明，在Alpha3D的α螺旋区域中，皮秒到纳秒时间尺度上的运动受到高度限制，在α螺旋末端和两个环区域的流动性增加。这在很大程度上与天然来源蛋白质中的情况一致。总体而言，2H和13C甲基弛豫方法提供的观点表明，与天然蛋白质相比，Alpha3D的侧链更具动态性。相对灵活的区域结合了刚性的含甲基侧链基团簇，这些基团散布着芳香族和β分支氨基酸。与Alpha3D含甲基侧链相关的运动时间尺度明显长于天然蛋白质中的情况。这些结果表明，Alpha3D设计背后的策略在很大程度上，但并非完全，捕捉到了天然蛋白质的结构和动态特征。
