Determining the Effects of Sequence Variation on SARS-CoV-2 Spike Protein through High-Resolution Characterization of Protein Energy Landscapes

通过蛋白质能量景观的高分辨率表征确定序列变异对 SARS-CoV-2 刺突蛋白的影响

• 批准号: 2322801
• 负责人: Judith Klinman
• 金额: $ 168.19万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2322801&HistoricalAwards=false
• 关键词: Determining; Effects; Sequence; Variation; SARS

Every protein is defined by a unique sequence of amino acids that, together with the environment, dictates its function. Today, thanks to advances in machine learning, genomic data bases, and the large number of known structures, we have the ability to accurately predict a structural model for the folded state of a protein given only this amino-acid sequence. But the sequence encodes more than just the structure; the protein is an ensemble, constantly sampling other conformations. Small changes in sequence, through mutation or evolution, rarely change the folded structure, yet they can have dramatic effects on behavior and function. The conformational diversity and the dynamics associated with this diversity play an important role; this ensemble view of proteins explains how small genetic changes can alter phenotype and drive evolution. Therefore, in spite of the recent advances in predicting structure, we still cannot predict the behavior of a protein from sequence. In order to harvest all of the information encoded in the genome, it is imperative that we understand this relationship between protein sequence and the entire ensemble (referred to as the energy landscape). A critical example of this is the variants of the SARS-CoV-2 spike protein that are continuing to arise in the population — the lack of a molecular understanding of the effects of these changes has impeded our understanding of the effects on the viral lifecycle and the evolutionary pressures on the virus. The proposal has broader impacts beyond the proposed research outcomes. The PI will continue to grow her career development program, establishing a dedicated post-doctoral affairs program for the 300 postdoctoral fellows at Berkeley (Department of Molecular and Cell Biology). The PI and her lab will provide access to HDX-MS instrumentation and expertise to the local scientific community and the methodological developments will enhance the resolution of HDX-MS studies beyond the SARS-CoV-2 spike protein. The objective of this award is to develop and use new hydrogen exchange approaches (HDX-MS) that will allow facile and rapid characterization of a protein’s energy landscape and determine the effects of sequence variation. Amide Hydrogen-Deuterium Exchange is uniquely suited to characterizing a protein’s energy landscape; it is ‘blind’ to the folded-state and sensitive to high-energy conformations and fluctuations. By deconvoluting the information in the time-dependent changes of the isotope envelope of a protein, the Principal Investigator (PI) will define a rate profile (fingerprint) - a set of individual amide exchange rates accounting for the entire sequence of a protein. This fingerprint is a unique reflection of the energy landscape for a protein under the given conditions. Then, using a combination of intact protein and peptide-level HDX-MS, the investigators will carry out experiments to characterize and quantify the distribution of conformations accessible from the native conformation. The PI and her team will use these new developments together with conventional HDX-MS to look at the effect of sequence variants in the SARS-CoV-2 spike protein and the isolated receptor binding domain to directly compare the changes in its ensemble of conformations to observed changes in fitness and function. The results will provide a deeper understanding of the relationship between SARS-CoV-2 variants and their effects on the virus and its lifecycle. The methodological developments will enhance the resolution of HDX-MS studies and the PI and her lab will provide access to HDX-MS instrumentation and expertise to the local scientific community.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

每种蛋白质都由独特的氨基酸序列定义，这些氨基酸序列与环境一起决定其功能。如今，由于机器学习、基因组数据库和大量已知结构的进步，我们有能力准确预测。仅给出该氨基酸序列的蛋白质折叠状态的结构模型，但该序列编码的不仅仅是结构；它是一个整体，通过突变或进化不断采样序列的微小变化。改变折叠结构，但它们可以具有戏剧性对行为和功能的影响。构象多样性和与这种多样性相关的动力学发挥着重要作用；这种蛋白质的整体观点解释了微小的遗传变化如何改变表型并驱动进化。我们仍然无法从序列中预测蛋白质的行为。为了获取基因组中编码的所有信息，我们必须了解蛋白质序列与整个整体（称为能量景观）之间的关系。关键的例子是SARS-CoV-2 刺突蛋白在人群中持续出现——由于缺乏对这些变化影响的分子了解，阻碍了我们对病毒生命周期的影响和病毒进化压力的理解。除了拟议的研究成果之外，PI 将继续发展她的职业发展计划，为伯克利分校（分子和细胞生物学系）的 300 名博士后研究员建立专门的博士后事务计划。访问HDX-MS 仪器和当地科学界的专业知识以及方法的发展将提高 HDX-MS 研究的分辨率，超越 SARS-CoV-2 刺突蛋白。该奖项的目标是开发和使用新的氢交换方法 (HDX)。 -MS）可以轻松快速地表征蛋白质的能量景观并确定序列变异的影响，它特别适合表征蛋白质的能量景观；折叠状态并对高能构象和波动敏感 通过对蛋白质同位素包膜随时间变化的信息进行解卷积，首席研究员 (PI) 将定义速率曲线（指纹）——一组个体。该指纹是蛋白质在给定条件下能量分布的独特反映，然后，研究人员将结合完整蛋白质和肽级 HDX-MS 进行分析。进行实验来表征PI 和她的团队将使用这些新进展与传统的 HDX-MS 来研究 SARS-CoV-2 刺突蛋白和分离的受体结合中序列变异的影响。域直接比较其构象整体的变化与观察到的适应性和功能的变化，结果将有助于更深入地了解 SARS-CoV-2 变体及其对病毒及其生命周期的影响之间的关系。提高 HDX-MS 研究的分辨率PI 和她的实验室将为当地科学界提供 HDX-MS 仪器和专业知识。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。