Sequence Dependence of Polyproline II Helix Formation

该项目的总体目标是确定周围序列对蛋白质残基采用聚脯氨酸II（PII）螺旋构象倾向的影响。已经假设蛋白质未折叠状态具有显著的PII螺旋含量。本项目中概述的实验旨在验证这一假设，并提供有关其他二级结构形成的数据。这将通过确定丙氨酸，谷氨酰胺，丝氨酸，亮氨酸和缬氨酸残基的周围块的中心客体残基的构象性质后的影响，并通过确定PII螺旋形成中的静电的作用来实现。将使用圆二色谱（CD）和NMR收集数据。 将使用奇异值分解分析CD数据，这将提供有关PII螺旋含量和所研究的每种肽中是否存在其他二级结构的信息。NMR将用于确定一些待检测肽中客体残基的平均构象性质。将探索镧系元素化学位移试剂的使用，以减轻NMR实验中的共振重叠。在所有阶段，收集的数据都将与圣路易斯华盛顿大学的Rohit Pappu博士的计算结果进行比较。人们普遍认为，对蛋白质未折叠状态缺乏了解是理解蛋白质折叠过程的最大障碍。如果未折叠状态具有显著的PII螺旋含量的假设被证明是正确的，那么蛋白质未折叠状态比通常认为的结构化程度要高得多。在执行本项目过程中收集的数据将支持或反驳这一假设。此外，还将获得关于蛋白质未折叠状态下其他二级结构类型存在的数据。这些数据将提供一个全面的蛋白质未折叠状态的集合的开始。此外，收集的数据将被证明是有用的理解PII螺旋介导的生物分子相互作用。在NMR中使用化学位移试剂的协议的发展将使小的，弱结构的肽的研究更容易处理，并将被证明是广泛的一般用途。PI计划让一名博士后学者和几名本科生从事该项目的各个方面。 在可能的情况下，本科生将从代表性不足的群体中招募。博士后学者将接受光谱学和NMR光谱学的高级培训。本科生将接受基本的实验室培训，光谱学，科学伦理学以及数据分析和呈现方面的培训。所有受训人员都将参与传播所产生的知识。PI将把这个项目中产生的知识纳入结构生物学的研究生课程。此外，数据将在同行评审的期刊上发表，并在科学会议和研究机构上介绍。

The overall goal of this project is to determine the effects of surrounding sequence upon the propensity for protein residues to adopt the polyproline II (PII) helical conformation. It has been hypothesized that protein unfolded states possess significant PII helix content. The experiments outlined in this project are designed to test this hypothesis and to provide data concerning the formation of other secondary structures. This will be achieved by determining the effects of surrounding blocks of alanine, glutamine, serine, leucine and valine residues upon the conformational properties of central guest residues, and by determining the role of electrostatics in PII helix formation. Data will be collected using circular dichroism spectroscopy (CD) and NMR. The CD data will be analyzed using singular value decomposition, which will provide information on PII helix content and the presence of other secondary structures in each of the peptides studied. NMR will be used to determine average conformational properties of guest residues in some of the peptides to be examined. The use of lanthanide chemical shift reagents will be explored in order to alleviate resonance overlap in the NMR experiments. At all stages data collected will be compared to the calculations of Dr. Rohit Pappu at the Washington University in St. Louis.It is widely acknowledged that a lack of understanding of protein unfolded states is the single biggest impediment to understanding the protein folding process. Should the hypothesis that unfolded states possess significant PII helix content prove true, then protein unfolded states are significantly more structured than commonly believed. Data collected in the execution of this project will either support or refute this hypothesis. In addition, data will be obtained on the presence of other secondary structure types in protein unfolded states. These data will provide the beginnings of a comprehensive picture of the ensemble of protein unfolded states. In addition, data collected will prove useful in the understanding of biomolecular interactions mediated by PII helices. The development of protocols for the use of chemical shift reagents in NMR will make the study of small, weakly-structured peptides more tractable and will prove to be of wide general use. The PI plans to have a postdoctoral scholar and several undergraduate students work on aspects of this project. Where possible, undergraduates will be recruited from underrepresented groups. The postdoctoral scholar will receive advanced training in spectroscopy and NMR spectrometry. The undergraduate students will receive basic laboratory training, training in spectroscopy, scientific ethics, and in the analysis and presentation of data. All trainees will participate in the dissemination of knowledge generated. The PI will incorporate knowledge generated in this project into a graduate-level course on structural biology. In addition, data will be published in peer-reviewed journals, and presented at scientific meetings and research institutions.