Dynamics of a de novo designed three-helix bundle protein studied by 15N, 13C, and 2H NMR relaxation methods.
Dynamics of a de novo designed three-helix bundle protein studied by 15N, 13C, and 2H NMR relaxation methods.
复制标题
通过 15N、13C 和 2H NMR 弛豫方法研究从头设计的三螺旋束蛋白的动力学。
作者：
Walsh,ST;Lee,AL;DeGrado,WF;Wand,AJ
理解多肽链的氨基酸序列如何指定一个独特的、功能性的三维结构仍然是一个重要的目标，特别是在从头蛋白质设计这一新兴学科的背景下。α3D是一种由73个氨基酸组成的单链蛋白质，由从头设计而成。此前对α3D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，α3D已被证明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，我们使用15N，13C和2H核磁共振弛豫方法研究了α3D的主链和侧链动力学，目的是评估这个从头设计的天然类蛋白质的内部运动特征。在主干水平上，15N和13Cα驰豫研究都表明，在α3D的α螺旋区域，在皮秒到纳秒的时间尺度上，运动具有高度的限制性，在α螺旋末端和两个环区的迁移率增加。这在很大程度上与天然来源的蛋白质中看到的一致。总体而言，2H和13C甲基松弛方法提供的观点表明，与天然蛋白质相比，α3D的侧链更具活力。相对柔韧性的区域结合了刚性甲基侧链基团簇，其中散布着芳香族和β支化的氨基酸。与α3D的甲基侧链相关的运动时间尺度明显长于天然蛋白质中的运动。这些结果表明，α3D设计的基本策略在很大程度上(但不是完全)捕捉了天然蛋白质的结构和动态特征。
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. α3D is a single chain protein of 73 amino acids resulting from a de novo design effort. Previous solution nuclear magnetic resonance studies of α3D confirm that the protein adopts the designed structure of a three-helix bundle. Furthermore, α3D 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 α3D were investigated using15N,13C, and2H 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, both15N and13Cαrelaxation studies indicate highly restrictive motion on the picosecond to nanosecond time scale in the α-helical regions of α3D, with increasing mobility at the ends of the α-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 both2H and13C methyl relaxation methods suggest that the side chains of α3D 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 β-branched amino acids. The time scale of motions associated with methyl-bearing side chains of α3D are significantly longer than that seen in natural proteins. These results indicate that the strategies underlying the design of α3D have largely, but not completely, captured both the structural and dynamic character of natural proteins.