Biophysical studies of macromolecules and molecular assemblies

大分子和分子组装体的生物物理研究

基本信息

批准号：
10669720
负责人：
STEVEN G. BOXER
金额：
$ 67.05万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-01 至 2026-06-30
项目状态：
未结题

项目摘要

Project summary/abstract This MIRA renewal grant proposal briefly summarizes the accomplishments over the past 4 years of support and outlines plans for continued support. The theme that unifies this research is the development and application of new physical methods that can impact the quantitative analysis of complex biological systems. The freedom to develop and broaden our research provided by the MIRA support has led to a significant evolution of the emphasis of part of our work on infectious diseases. Specifically, we will focus on the biomedically critical need to understand the origin(s) of antibiotic resistance using the TEM -lactamases as an initial target. Likewise, our efforts to develop novel ways to organize and manipulate biological membranes now focus on the mechanism of viral membrane fusion. While these two areas had completely separate origins in the parent R01’s that were merged in the MIRA, they have both provided rich areas for new and impactful research. My lab develops spectroscopic methods for probing protein-exerted electric fields which we use to obtain quantitative information on how electric fields contribute to catalysis at the active sites of enzymes. We led the development of vibrational Stark effect spectroscopy as a general approach to map these fields. Using this approach, we can, for the first time, quantify the electrostatic contribution to the catalytic proficiency of enzymes. Moving beyond ideal model enzymes, we will use this approach to provide a deeper understanding of the mechanism(s) by which TEM--lactamases evolve to cope with man-made antibiotics. By studying the connection between evolution and electric fields, we hope to develop general design principles for these enzymes and discover the physical origins of antibiotic resistance. We discovered that “split” GFPs can be photo-dissociated, and we study the underlying mechanism of this unusual process for optogenetic applications. This deeper view of strand photo-dissociation along with our work elucidating factors that control bond-specific photo-isomerization pathways are connected to our work on protein electrostatics and will provide a framework for understanding GFP’s electro-optic properties. Our lab pioneered the development of model membrane architectures, along with imaging and analytical methods that probe fundamental aspects of biological membrane organization and dynamics. Our current focus is the application of these architectures and novel single particle assays to characterize the elementary steps by which enveloped viruses, such as influenza A, fuse to target membranes. In parallel, we characterize the organization of lipids with high lateral resolution using imaging mass spectrometry. Recently we showed that atom recombination can be used to identify which lipids and proteins are in very close proximity (< 3nm) in biological membranes. This new approach addresses major challenges in membrane biophysics and structural biology where local organization is key to emergent function.

项目概要/摘要 MIRA 续签拨款提案简要总结了过去 4 年的成就支持并概述了持续支持的计划统一这项研究的主题是发展。以及新物理方法的应用，可以影响复杂生物的定量分析 MIRA 支持提供的开发和扩大研究的自由导致了我们在传染病方面的部分工作重点发生了重大变化。使用 TEM -内酰胺酶了解抗生素耐药性起源的生物医学关键需求同样，我们努力开发组织和操纵生物的新方法。膜现在的重点是病毒膜融合的机制，而这两个领域已经完全。母体 R01 的不同起源被合并到 MIRA 中，它们都为新的、有影响力的研究。我的实验室开发了用于探测蛋白质施加电场的光谱方法，我们用它来获得有关定量电场如何促进酶活性位点催化的信息。我们领导了振动斯塔克效应光谱的开发，作为绘制这些场的通用方法。使用这种方法，我们第一次可以量化静电对催化的贡献超越理想的酶模型，我们将使用这种方法提供更深入的知识。了解 TEM-β-内酰胺酶进化以应对人造抗生素的机制。通过研究进化与电场之间的联系，我们希望开发通用设计这些酶的原理并发现抗生素耐药性的物理根源。我们发现“分裂”的 GFP 可以光解离，并研究了其潜在机制这种不寻常的光遗传学应用过程以及对链光解离的更深入的了解。我们的工作阐明了控制键特异性光异构化途径的因素，这与我们的研究有关研究蛋白质静电学，将为理解 GFP 的电光特性提供一个框架。我们的实验室率先开发了模型膜结构以及成像和分析技术我们当前探索生物膜组织和动力学基本方面的方法。重点是应用这些架构和新颖的单粒子测定来表征包膜病毒（例如甲型流感病毒）与目标膜融合的基本步骤。我们使用成像质谱法以高横向分辨率表征了脂质的组织。最近我们表明，原子重组可用于识别哪些脂质和蛋白质处于非常好的状态。这种新方法解决了生物膜中的近距离（< 3nm）。膜生物物理学和结构生物学，其中局部组织是新兴功能的关键。