Structural Properties of Initial States in Protein Folding from NMR Alignment Data

最近的研究表明，在极端变性条件下的蛋白质在浸泡在液晶或凝胶的不对称环境中时，相对于核磁共振磁场保持净排列。这些基质的不对称形状对溶质分子的重定向施加了微弱的空间限制。由此产生的时间和集合平均排列导致核磁共振偶极耦合不完全抵消。偶极耦合报告了相对于分子排列框架的单个键向量的取向。该项目的目标是进一步了解变性蛋白质的净排列结构基础，并利用偶极偶联编码的排列信息来表征变性状态。这些目标将通过两个模型系统来解决。第一种由CspA和LysN两种蛋白组成，它们具有天然的五链β -桶结构基序，但没有可检测到的序列相似性。偶极偶联将用于研究在两种蛋白质的变性状态之间天然结构的保守程度。第二个模型系统由两个采用规则扩展的α -螺旋结构的多肽组成：s肽，对应于蛋白质核糖核酸酶A的第一个α -螺旋；和GCN4-p螺旋盘状二聚体。偶极偶联将用于确定在两个多肽的变性形式的α -螺旋构象的持续长度。这项工作的主要动机是为了更好地了解蛋白质折叠的结构基础。蛋白质折叠仍然是当今生物学中最困难和最有趣的问题之一，对结构基因组学的进展至关重要。核磁共振排列数据提供了变性状态下结构的新实验窗口，将允许更准确地模拟蛋白质折叠和负责蛋白质展开稳定性的因素。蛋白质折叠的建模需要变性状态的信息，因为这些初始状态的结构可以支持或反对功能性天然状态的折叠。变性状态的结构特征还影响变性蛋白质聚集反应的机制，这可能通过不完全形成的新生结构的非法重组发生。对变性蛋白质中持续存在的结构构建块的描述提供了对蛋白质结构进化机制的见解，这些信息反过来可能对蛋白质设计有用。该项目为学生提供了丰富的、多学科的蛋白质折叠方法的培训机会，该方法结合了蛋白质化学、最先进的核磁共振实验和结构建模。

Recent work has shown that proteins under extreme denaturing conditions retain a net alignment with respect to an NMR magnetic field when immersed in the asymmetric environment of liquid crystals or gels. The asymmetric shapes of these matrices impose weak steric restrictions on the molecular reorientation of solutes. The resulting time- and ensemble-averaged alignment leads to incomplete cancellation of NMR dipolar couplings. Dipolar couplings report on the orientation of individual bond vectors with respect to the molecular alignment frame. The objectives of this project are to further the understanding of the structural basis for net alignment in denatured proteins and to use the alignment information encoded in dipolar couplings towards a structural characterization of denatured states. The objectives will be addressed with two model systems. The first consists of the two proteins CspA and LysN, which share a native five-stranded beta-barrel structural motif but no detectable sequence similarity. Dipolar couplings will be used to investigate the extent to which native-like structure is conserved between the denatured states of the two proteins. The second model system is comprised of two polypeptides that adopt regular extended alpha-helical structures: the S-peptide, which corresponds to the first alpha-helix of the protein ribonuclease A; and the GCN4-p alpha-helical coiled-coil dimer. Dipolar couplings will be used to determine the persistence length of alpha-helical conformations in the denatured forms of the two polypeptides.The principal motivation of this work is to obtain an improved understanding of the structural basis of protein folding. Protein folding remains one of the most difficult and intriguing problems in biology today, and is essential for progress in structural genomics. The new experimental window on structure in denatured states afforded by NMR alignment data, will allow more accurate modeling of protein folding and of the factors responsible for the stability of proteins to unfolding. Modeling of protein folding requires information on denatured states in as much as structure in these initial states can favor or oppose folding to the functional native state. The structural characterization of denatured states additionally impacts mechanisms for denatured protein aggregation reactions, which can occur through illegitimate recombination of incompletely formed nascent structures. The delineation of structural building blocks that persist in denatured proteins provides insights into the mechanisms by which protein structures evolve, information that in turn could be useful in protein design. This project offers the opportunity for students to train in a rich, multidisciplinary approach to protein folding that combines protein chemistry, state of the art NMR experiments, and structure modeling.