Time-Resolved and Single-Molecule Dynamics of Membrane Proteins

膜蛋白的时间分辨和单分子动力学

• 批准号: 0517644
• 负责人: J.B. Alexander Ross
• 金额: --
• 依托单位: University of Montana
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0517644&HistoricalAwards=false
• 关键词: Time; Resolved; Single; Molecule; Dynamics

This project addresses fundamental questions in molecular recognition and catalysis on membrane surfaces by integrating biochemical structure-function studies with innovative chemical and materials engineering approaches and time-resolved excited-state spectroscopy. The chemical innovations are (1) synthesis of novel organometallic spectroscopic probes designed to monitor molecular motions on the nano- to millisecond timescale, as well as to quantitate molecular kinetics and thermodynamics at the single-molecule level, and (2) generation of novel recombinant membrane proteins that act as enzyme receptors, which allow study of structure-function relationships in binding and catalysis at membrane surfaces. The materials engineering innovation is the combination of (1) generation of homogeneous and non-homogeneous model lipid bilayers, supported on the inside surface of glass micro-capillaries, with embedded enzyme receptors that confine catalysis to the membrane surface, (2) a flow apparatus to provide a range of shear conditions that affect enzyme catalysis at the membrane surface, and (3) a time-resolved microscope designed to observe and quantify the physical events that regulate catalysis at the ensemble average and single-molecule levels. The knowledge gained from these studies is expected to have applications to the development of membrane-bound detections systems used, for example, in biosensors. In particular, understanding the role of shear in surface-mediated catalysis will aid the design of devices that introduce soluble substrates to membrane-bound or immobilized sensor systems. This project will explore, at a molecular level, how lipid composition and protein-protein assembly on membrane surfaces regulate the catalytic activity of serine proteases that bind to membrane-protein receptors. The specific objectives of this project are: (1) to learn how the lipid composition of the membrane affects the conformational dynamics and self-association of the intrinsic membrane protein, and to explore whether lipid rafts, a controversial paradigm in the biological function of membranes, play a role in the dynamics and interactions between the enzyme and the intrinsic membrane protein; and (2) to determine the impact of protein-protein assembly on the dynamics of this membrane-associated complex and catalysis by the bound enzyme. The project will be accomplished at The University of Montana, and with fluorescence-detected analytical ultracentrifugation in the lab of Tom Laue at The University of New Hampshire. This collaborative project will address unresolved, fundamental questions about the structural dynamics and lipid-dependence of enzyme catalysis occurring on lipid membranes. The research addresses these questions through the development and application of new fluorescence spectroscopic approaches in microscopy and analytical ultracentrifugation. This work will have broad application to scientific inquiries involving membrane-localized catalysis, i.e., hormone receptors, transmembrane transporters, and antibodies, and across many fields, i.e., biology, zoology, environmental chemistry, and biotechnology. Also, the collaboration between colleagues at The University of Montana and The University of New Hampshire, both EPSCoR institutions, will contribute to the dissemination of new science and will promote integration between different areas of biophysics. Similarly, the international collaboration between the PI and Dr. Roberto Gobetto, University of Turin, Italy, promotes sharing of knowledge and fosters creative cooperation between scientific disciplines. In addition, the project will foster the scientific education of secondary school, undergraduate and graduate students by providing training opportunities in biophysical chemistry. Students will learn state-of-the-art biophysical spectroscopic approaches and data analysis of sophisticated physical models. They will also learn the requisite molecular biology and biochemical techniques. In addition, this research will enhance opportunities for three underrepresented groups: Students from a geographically isolated and economically disadvantaged state; non-traditional adult students seeking new career paths; and Native Americans. Traditional and non-traditional students in this project will have the opportunity to train for biotechnology related professions encouraged by the initiatives and will provide skilled personnel to run new businesses. Also, the State of Montana is home to ten tribal nations, and the University actively recruits Native American students. In particular, the laboratory of the PI has an active collaboration with students and faculty from the Salish-Kootenai College, a tribal college in Pablo, MT, who will participate in this project. Finally, The University of Montana is engaged in a statewide public-private sector partnership to encourage growth in the biotechnology industry. This project represents collaboration between academic research and established biotechnology industry; Genentech, Inc. is donating essential materials to the project.

该项目通过将生化结构-功能研究与创新的化学和材料工程方法以及时间分辨激发态光谱相结合，解决了膜表面分子识别和催化的基本问题。化学创新是(1)合成了新型有机金属光谱探针，旨在监测分子在纳秒到毫秒级的运动，以及在单分子水平上定量分子动力学和热力学，以及(2)产生作为酶受体的新型重组膜蛋白，这使得研究膜表面结合和催化的结构-功能关系成为可能。材料工程的创新是结合了(1)一代均一和非均一的模型脂质双层，支撑在玻璃微毛细管的内表面，与嵌入的将催化限制在膜表面的酶受体相结合，(2)流动装置，提供影响膜表面酶催化的一系列剪切条件，以及(3)时间分辨显微镜，旨在观察和量化在整体平均和单分子水平上调节催化的物理事件。从这些研究中获得的知识有望应用于膜结合检测系统的开发，例如用于生物传感器。特别是，了解剪切在表面介导的催化中的作用将有助于将可溶性底物引入膜结合或固定化传感器系统的装置的设计。这个项目将在分子水平上探索膜表面的脂质组成和蛋白质-蛋白质组装如何调节与膜-蛋白质受体结合的丝氨酸蛋白酶的催化活性。本项目的具体目标是：(1)了解膜脂组成如何影响固有膜蛋白的构象动力学和自结合，并探索脂筏(膜生物学功能中一个有争议的范式)是否在酶和固有膜蛋白之间的动力学和相互作用中发挥作用；以及(2)确定蛋白质-蛋白质组装对膜相关复合体的动力学和结合酶催化的影响。该项目将在蒙大拿大学完成，并在新汉普郡大学汤姆·劳伊的实验室通过荧光检测的分析超速离心法完成。这个合作项目将解决尚未解决的基本问题，即发生在脂膜上的酶催化的结构动力学和脂类依赖性问题。这项研究通过在显微镜和分析超速离心法中开发和应用新的荧光光谱方法来解决这些问题。这项工作将广泛应用于涉及膜定位催化的科学研究，即激素受体、跨膜转运蛋白和抗体，并跨越许多领域，即生物学、动物学、环境化学和生物技术。此外，蒙大拿大学和新汉普郡大学这两个EPSCoR机构的同事之间的合作将有助于传播新的科学，并将促进生物物理学不同领域之间的整合。同样，国际和平研究所和意大利都灵大学罗伯托·戈贝托博士之间的国际合作促进了知识共享，促进了科学学科之间的创造性合作。此外，该项目还将通过提供生物物理化学的培训机会，促进中学、本科生和研究生的科学教育。学生将学习最先进的生物物理光谱方法和复杂物理模型的数据分析。他们还将学习必要的分子生物学和生化技术。此外，这项研究将增加三个代表不足的群体的机会：来自地理上与世隔绝和经济上处于不利地位的州的学生；寻求新职业道路的非传统成人学生；以及美洲原住民。该项目中的传统和非传统学生将有机会为受倡议鼓励的生物技术相关专业进行培训，并将提供经营新企业的技术人员。此外，蒙大拿州有十个部落民族，该大学积极招收美洲原住民学生。特别是，国际和平研究所的实验室与萨利什-库特奈学院的学生和教职员工进行了积极的合作，萨利什-库特奈学院是密歇根州巴勃罗的一所部落学院，他们将参与这一项目。最后，蒙大拿大学参与了全州范围的公共-私营部门伙伴关系，以鼓励生物技术行业的增长。这个项目代表了学术研究和成熟的生物技术行业之间的合作；基因技术公司正在向该项目捐赠必要的材料。