Biophysical studies of macromolecules and molecular assemblies

项目摘要/摘要 这份米拉续期赠款提案简要总结了过去4年的成就 支持并概述继续支持的计划。统一这项研究的主题是发展 以及能够影响复杂生物定量分析的新物理方法的应用 系统。由Mira支持提供的发展和扩大我们研究的自由导致了 我们关于传染病的部分工作的重点发生了重大变化。具体地说，我们将重点关注 利用透射电子显微镜-内酰胺酶了解抗生素耐药来源(S)的生物医学关键 作为最初的目标。同样，我们努力开发新的方法来组织和操纵生物 目前膜的研究重点是病毒膜融合的机制。而这两个地区已经完全 分离的起源于亲代R01的S，它们都为米拉提供了丰富的区域 新的和有影响力的研究。 我的实验室开发了探测蛋白质施加的电场的光谱方法，我们用它来 获取有关电场如何在酶的活性部位促进催化作用的定量信息。 我们领导了振动斯塔克效应光谱学的发展，作为绘制这些场的一般方法。 使用这种方法，我们可以第一次量化静电对催化剂的贡献。 对酶的熟练程度。超越理想的模型酶，我们将使用这种方法来提供更深层次的 了解透射电子显微镜--内酰胺酶进化以应对人造抗生素的机制(S)。 通过研究进化和电场之间的联系，我们希望能发展出总体设计 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”的绿色荧光蛋白可以被光解离，并且我们研究了 这一不寻常的过程适用于光遗传应用。这种关于链的光解离的更深层次的观点 我们的工作阐明了控制键特定的光异构化途径的因素与我们的 研究蛋白质静电学，并将为理解绿色荧光蛋白的电光性质提供一个框架。 我们的实验室率先开发了模型膜结构，以及成像和分析 探索生物膜组织和动力学的基本方面的方法。我们目前的情况 重点是应用这些体系结构和新的单颗粒分析来表征 被包裹的病毒，如甲型流感病毒与靶膜融合的基本步骤。同时， 我们使用成像质谱学来表征具有高横向分辨率的脂质的组织。 最近我们发现，原子重组可以用来识别哪些脂类和蛋白质是非常重要的。 生物膜中的近距离(&lt；3 nm)。这种新方法解决了以下方面的主要挑战 膜生物物理学和结构生物学，其中局部组织是紧急功能的关键。

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.