Sequence Dependence of Polyproline II Helix Formation

聚脯氨酸 II 螺旋形成的序列依赖性

• 批准号: 0444049
• 负责人: Trevor Creamer
• 金额: $ 40.43万
• 依托单位: University of Kentucky Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0444049&HistoricalAwards=false
• 关键词: Sequence; Dependence; Polyproline; II; Helix

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.

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）螺旋构象的倾向的影响。据推测，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。这将通过确定周围的丙氨酸、谷氨酰胺、丝氨酸、亮氨酸和缬氨酸残基对中心客体残基构象性质的影响，以及确定静电在PII螺旋形成中的作用来实现。数据收集将使用圆二色光谱（CD）和核磁共振。CD数据将使用奇异值分解进行分析，这将提供PII螺旋含量和在所研究的每个肽中存在的其他二级结构的信息。核磁共振将用于确定一些待检测肽的客残基的平均构象性质。探索镧系化学移位试剂的使用，以减轻核磁共振实验中的共振重叠。在所有阶段收集的数据都将与圣路易斯华盛顿大学罗希特·帕普博士的计算结果进行比较。人们普遍认为，缺乏对蛋白质折叠状态的理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更加结构化。本项目执行过程中收集的数据将支持或反驳这一假设。此外，还将获得蛋白质未折叠状态中其他二级结构类型的数据。这些数据将提供蛋白质未折叠状态集合的全面图像的开端。此外，收集到的数据将有助于理解由PII螺旋介导的生物分子相互作用。在核磁共振中使用化学移位试剂的方案的发展将使小的、弱结构的肽的研究更容易处理，并将被证明具有广泛的普遍用途。PI计划让一名博士后学者和几名本科生参与这个项目的各个方面。在可能的情况下，本科生将从代表性不足的群体中招募。该博士后学者将接受光谱学和核磁共振光谱学的高级培训。本科生将接受基本的实验室训练、光谱学训练、科学伦理训练，以及数据分析与呈现训练。所有学员都将参与传播所产生的知识。项目负责人将把在这个项目中产生的知识整合到研究生水平的结构生物学课程中。此外，数据将发表在同行评议的期刊上，并在科学会议和研究机构上发表。