Time-Resolved and Single-Molecule Dynamics of Membrane Proteins

该项目通过将生物化学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。化学方面的创新包括：(1)合成新型有机金属光谱探针，用于监测纳米到毫秒时间尺度上的分子运动，以及在单分子水平上量化分子动力学和热力学；(2)产生新型重组膜蛋白，作为酶受体，可以研究膜表面结合和催化的结构-功能关系。材料工程创新是(1)生成均质和非均质模型脂质双层，支撑在玻璃微细血管的内表面，嵌入酶受体，将催化作用限制在膜表面；(2)流动装置提供一系列剪切条件，影响膜表面的酶催化；(3)时间分辨显微镜，用于在系综平均和单分子水平上观察和量化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开发膜结合检测系统，例如生物传感器。特别是，了解剪切在表面介导催化中的作用将有助于设计将可溶性底物引入膜结合或固定化传感器系统的设备。该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丝氨酸蛋白酶与膜蛋白受体结合的催化活性。该项目的具体目标是：(1)了解膜的脂质组成如何影响内在膜蛋白的构象动力学和自结合，并探索脂筏（膜生物学功能中有争议的范式）是否在酶与内在膜蛋白之间的动力学和相互作用中发挥作用；(2)确定蛋白质-蛋白质组装对膜相关复合物动力学和结合酶催化的影响。该项目将在蒙大拿大学完成，并在新罕布什尔大学Tom Laue的实验室进行荧光检测分析超离心。该合作项目将解决关于脂质膜上酶催化的结构动力学和脂质依赖性的未解决的基本问题。该研究通过在显微镜和分析超离心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，如激素受体、跨膜转运蛋白和抗体，以及许多领域，如生物学、动物学、环境化学和生物技术。此外，蒙大拿州大学和新罕布什尔大学这两个EPSCoR机构的同事之间的合作将有助于新科学的传播，并将促进生物物理学不同领域之间的整合。同样，PI与意大利都灵大学的Roberto Gobetto博士之间的国际合作促进了知识的共享，并促进了科学学科之间的创造性合作。此外，该项目将通过提供生物物理化学方面的培训机会，促进中学生、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物化学技术。此外，这项研究将增加三个代表性不足的群体的机会：来自地理上孤立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；和印第安人。该项目的传统和非传统学生将有机会接受与生物技术相关的专业培训，并将为经营新企业提供技术人员。此外，蒙大拿州是十个部落民族的家园，这所大学积极招收美国原住民学生。特别是，PI的实验室与Salish-Kootenai学院的学生和教师积极合作，该学院是MT . Pablo的一所部落学院，他们将参与该项目。最后，蒙大拿大学参与了全州范围内的公私部门合作，以鼓励生物技术产业的发展。该项目代表了学术研究与成熟生物技术产业之间的合作；基因泰克公司为该项目捐赠了必要的材料。

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.