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.
