Biophysical Studies of Macromolecules and Molecular Assemblies

大分子和分子组装体的生物物理研究

• 批准号: 10440897
• 负责人: STEVEN G. BOXER
• 金额: $ 12.54万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/10440897
• 关键词: Active Sites; Address; Antibiotic Resistance; Antibiotics; Architecture; Area; Biological; Biological Assay; Biophysics; Catalysis; Communicable Diseases; Development; Dissociation; Electrostatics; Enzymes; Evolution; Freedom; Genetic Recombination; Grant; Health; Human; Image; Influenza A virus; Lactamase; Lateral; Lipids; Maps; Mass Spectrum Analysis; Membrane; Membrane Fusion; Methods; Optics; Parents; Pathway interactions; Process; Property; Proteins; Research; Research Support; Resolution; Spectrum Analysis; System; Time; Viral; Work; analytical method; biological systems; biophysical analysis; complex biological systems; design; electric field; enzyme model; imaging modality; instrument; macromolecule; man; membrane model; model development; molecular assembly/self assembly; novel; novel strategies; optogenetics; particle; resistance mechanism; structural biology; vibration; virus envelope

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.

项目摘要/摘要 统一此MIRA赠款支持的研究的主题是开发和应用 可以影响复杂生物系统的定量分析的新物理方法。自由 MIRA支持提供和扩大我们提供的研究已导致 强调我们部分关于传染病的工作。具体而言，我们将重点放在生物医学上的关键上 需要使用TEM-内乳酶作为初始靶标了解抗生素抗性的起源。 同样，我们为组织和操纵生物膜的新颖方法的努力现在关注 病毒膜融合的机制。尽管这两个领域在 在Mira合并的家长R01，他们都为新的和有影响力的领域提供了丰富的领域 研究。 我的实验室开发人员的光谱方法用于探测我们用来探测蛋白质的电场 获取有关电场如何在酶的活性部位催化的定量信息。 我们领导了振动性鲜明效应光谱的发展，作为绘制这些磁场的一般方法。 使用这种方法，我们可以首次量化对催化的静电贡献 酶的熟练度。超越理想模型酶，我们将使用这种方法来提供更深的 理解温度 - 内酰胺酶演变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，我们希望开发一般设计 这些酶的原理，发现抗生素抗性的物理起源。提议 仪器补充剂将对这些项目产生重大影响。 我们发现可以将“拆分” GFP与照片解散，我们研究了 这种用于光遗传应用的不寻常过程。这种更深刻的链片段散开的看法以及 我们的工作阐明控制键特异性的光相异构化途径的因素与我们的 在蛋白质静电方面进行工作，并将提供一个理解GFP的电形特性的框架。 我们的实验室开发了模型膜体系结构的开发，以及成像和分析 探测生物膜组织和动态基本方面的方法。我们的目前 焦点是这些架构和新颖的单粒子测定的应用来表征 基本步骤，通过将诸如影响力的病毒（例如靶向膜）构成的基本步骤。并联， 我们表征了使用成像质谱法进行高侧分辨率的脂质的组织。 最近，我们表明原子重组可用于确定哪些脂质和蛋白质非常 生物膜中的紧密接近（<3nm）。这种新方法解决了 膜生物物理学和结构生物学，其中本地组织是新兴功能的关键。