RUI: Characterization and modulation of SH3 domain binding pathway biophysics

这项研究的目的是了解如何以小的方式调整常见的蛋白质结合相互作用，以在不同的细胞环境中执行特定的功能。细胞内允许细胞过程发生的通信是通过蛋白质之间的相互作用来调节的。了解这些相互作用的细节，包括它们的强度和特异性，将使研究人员能够预测和修改许多类型的细胞行为。研究结果将为蛋白质结合相互作用在不同细胞环境中的功能提供更深入的见解，并有助于解释常见的相互作用如何专门执行许多不同的细胞功能。从事这个项目的本科生将有机会学习计算和实验生物物理技能，包括计算和实验数据如何对形成更完整的蛋白质相互作用模型的项目做出贡献。学生们还将与德克萨斯理工大学(Mike Latham)和利物浦大学(Elliott Stollar)的科学家密切合作，亲身体验合作对现代科学过程的重要性。为了让更多的学生体验本科研究，一门以研究为基础的实验室课程将让学生接触计算生物物理学和分子动力学模拟的技术。在本课程中，学生开发并实施一个有助于更大项目目标的研究项目。该项目还包括为理科专业的妇女和理科代表不足的群体开设一门只有一个学分的课程。这门课程将面向所有自然科学专业的学生，并将鼓励这个传统上女性代表不足的专业的学生考虑并努力解决科学中的身份认同问题。主题将包括妇女和少数民族在识别为科学家、刻板印象威胁和冒名顶替者综合症方面可能面临的挑战。这门课程将培养学生成为科学中代表性不足的群体主题的领导者，并包括一个服务学习项目。细胞信号相互作用通常涉及到内在无序的蛋白质区域与小区域的结合，但这些相互作用的结合路径通常没有被很好地理解。要充分了解这些无序的折叠和结合相互作用是如何促进功能的，需要知道如何通过调节相互作用的无序序列、结构域序列或溶剂环境在不同的环境中调节结合途径。这个项目将使用SH3结构域-肽的相互作用(Abp1p SH3结构域与Arka肽结合)作为模型系统，并采用分子动力学模拟和核磁共振光谱相结合的方法来解决这个问题。SH3结构域在真核生物中普遍存在，但SH3结构域家族包含许多影响功能的变异。这些变化会影响结合途径，进而影响生物物理性质，如结合亲和力和结合率。这项研究将调查SH3-肽相互作用的不同方面如何对结合途径和结合的生物物理性质做出贡献，最终定义一套有助于预测这种相互作用可能具有不同功能后果的方式的“规则”。结合假设从Arka和SH3结构域之间的初始相遇开始，然后是Arka片段1的快速初始结合，然后是更慢、更特异的片段2的结合。研究的目标1将确认两个Arka片段在结合途径中的作用，并确定每个片段如何影响亲和力、特异性和动力学。目标2将重点介绍静电在结合中的作用，以及它如何影响初始导向结合部位，以及它对特定结合正确的肽序列的重要性。目标3将重点介绍肽序列中的脯氨酸残基的重要性。通过在顺式和反式构象之间的转换以及采用刚性的多-脯氨酸II螺旋结构，脯氨酸可以唯一地影响构象。该项目将有助于促进我们对无序的蛋白质区域如何结合SH3结构域，以及不同SH3结构域和伙伴之间的多样性如何促进其功能分化的理解。了解这些生物物理属性是如何调整的，不仅将影响SH3信号转导领域，还将影响更广泛的无序肽结合领域，因为类似的机制可能存在于其他识别领域。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The goal of this research is to understand how common protein binding interactions can be tuned in small ways to perform specialized functions in different cellular contexts. The communication within cells that allows cellular processes to occur is mediated by interactions between proteins. Understanding the details of these interactions, including their strength and specificity, will allow researchers to predict and modify the many types of cell behavior. Results will provide deeper insights into how protein binding interactions function in different cellular contexts and help explain how a common interaction can specialize to perform many different cellular functions. Undergraduate students working on this project will have the opportunity to learn both computational and experimental biophysics skills, including how both computational and experimental data can contribute to a project to form a more complete model of protein interactions. Students will also work closely with scientists at Texas Tech University (Mike Latham) and the University of Liverpool (Elliott Stollar) and experience first-hand the importance of collaboration to the modern scientific process. To allow a larger number of students to have an experience with undergraduate research, a research-based lab course will expose students to techniques in computational biophysics and molecular dynamics simulations. In this course, students develop and carryout a research project contributing to the larger project goals. This project also includes the development of a one-credit course for science majors on women and underrepresented groups in science. This course will be geared toward all natural science majors, and will encourage students navigating a major where women are traditionally underrepresented to consider and grapple with ideas about identity in science. Topics will include the challenges that women and minorities may face regarding identifying as scientists, stereotype threat, and impostor syndrome. The course will prepare students to be leaders on the topic of underrepresented groups in science and include a service-learning project.Cellular signaling interactions often involve binding of intrinsically disordered protein regions to small domains, but the binding pathways for these interactions are often not well understood. Full understanding of how these disordered folding and binding interactions contribute to function requires knowing how the binding pathway can be tuned in different contexts by adjusting the disordered sequence, domain sequence, or solvent environment of the interaction. This project will address this question using an SH3 domain-peptide interaction (Abp1p SH3 domain binding to the ArkA peptide) as a model system and employing a combination of molecular dynamics simulations and NMR spectroscopy. SH3 domains are ubiquitous across eukaryotes, but the SH3 domain family contains many variations that impact function. These variations can influence the binding pathway, which in turn will affect biophysical properties such as binding affinity and association rates. This research will investigate how different aspects of the SH3-peptide interaction contribute to the binding pathway and biophysical properties of binding, ultimately defining a set of "rules" that help predict the ways that this interaction can be modulated with different functional consequences. Binding is hypothesized to begin with an initial encounter between ArkA and the SH3 domain, followed by fast initial binding of ArkA segment 1, and then slower, more specific binding of segment 2. Aim 1 of the research will confirm the role of the two ArkA segments in the binding pathway and determine how each affects affinity, specificity, and kinetics. Aim 2 will focus on the role of electrostatics in binding, and how it affects initial steering to the binding site as well its importance for specifically binding the correct peptide sequence. Aim 3 will focus on the importance of proline residues in the peptide sequence. Proline can uniquely affect conformation by switching between cis and trans conformational states and adopting a rigid poly-proline II helix structure. The project will help to advance our understanding of how disordered protein regions bind SH3 domains and how diversity between different SH3 domains and partners contributes to their functional differentiation. Understanding how these biophysical properties are tuned will not only impact the field of SH3 signaling, but also the wider field of disordered peptide binding, as similar mechanisms may exist for other recognition domains.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.