Biophysical Studies of Macromolecules and Molecular Assemblies

项目概要/摘要 统一由MIRA资助的研究的主题是开发和应用 新的物理方法可以影响复杂生物系统的定量分析。自由 发展和扩大我们的研究提供的MIRA的支持导致了显着的演变， 强调我们在传染病方面的部分工作。具体来说，我们将重点关注生物医学关键 需要了解使用TEM β-内酰胺酶作为初始靶标的抗生素耐药性的起源。 同样，我们努力开发新的方法来组织和操纵生物膜，现在集中在 病毒膜融合的机制。虽然这两个地区在欧洲有着完全不同的起源， 在MIRA中合并的父R 01，它们都为新的和有影响力的 research. 我的实验室开发了光谱方法来探测蛋白质施加的电场， 获得关于电场如何在酶的活性位点促进催化的定量信息。 我们领导了振动斯塔克效应光谱的发展，作为绘制这些领域的一般方法。 使用这种方法，我们可以，第一次，量化静电的催化作用， 酶的能力。超越理想的模型酶，我们将使用这种方法来提供更深入的 了解TEM-β-内酰胺酶进化以科普人造抗生素的机制。 通过研究演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起源。拟议 文书补充将对这些项目产生重大影响。 我们发现“分裂”的GFP可以被光解离，并且我们研究了其潜在的机制。 这是一个不寻常的光遗传学应用过程。这种更深入的链光解离的观点沿着 我们的工作阐明了控制键特异性光异构化途径的因素， 研究蛋白质静电学，并将为理解GFP的电光特性提供一个框架。 我们的实验室开创了模型膜结构的发展，沿着成像和分析 探索生物膜组织和动力学的基本方面的方法。我们目前 重点是应用这些架构和新颖的单粒子分析来表征 包膜病毒（如甲型流感病毒）与靶膜融合的基本步骤。同时， 我们使用成像质谱以高横向分辨率表征脂质的组织。 最近，我们发现，原子重组可以用来确定哪些脂质和蛋白质是非常重要的。 在生物膜中紧密接近（<3 nm）。这一新方法解决了以下主要挑战： 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 The theme that unifies the research supported by this MIRA grant 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. The proposed instrument supplement will have a major impact on these projects. 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.