Intellectual Merit: The defining step in evolution of life was the encapsulation of bio-molecules within a lipid membrane. However, to grow, heal, divide, transport, and secrete, cellular membranes need to undergo fission and fusion. Fundamental life processes such as neurotransmission, or the secretion of hormones and enzymes, require membrane fusion. Therefore, understanding the molecular mechanism of membrane fusion will not only enable an understanding of the above mentioned processes, but help in the design and development of biosensors, drugs to block infections, and the development of treatment technologies for exposure to toxic industrial chemicals. Furthermore, from this understanding of membrane fusion, efficient drug delivery systems could be developed for specialized and targeted delivery, and sustained release.
Although distinct membrane proteins, such as tv-SNARE (soluble NSF attachment protein receptor, where NSF = N-ethyl-maleimide-sensitive fusion protein), have been identified to facilitate and confirm specificity to membrane fusion in cells, the chemistry of the process at the atomic level is still unclear. Therefore the overall objective of this work is to understand membrane fusion at the atomic level. Specifically, the PIs seek to identify the mechanism through which Ca2+ triggers the tv-SNARE mediated fusion reaction, and the role of the membrane fusion protein synaptotagmin-I on the regulation of membrane fusion.
In this hypothesis driven work, molecular dynamics simulations are used to determine quantitatively how the release of Ca2+ between vesicles alters the interactions between apposed lipid vesicles through the calculation of solvation force, the potential of mean force, interfacial tension and elastic modulus, as a function of [Ca2+], bilayer composition, and temperature. Molecular dynamics simulations are used to investigate the formation of water pores in the presence of a Ca2+ concentration gradient, and the effect of pore formation on the rate of membrane fusion. Molecular dynamics simulations are used to provide insight into the role of synaptotagmin-I on the regulation of membrane fusion. In all cases, complimentary experiments are performed using atomic force microscopy (AFM), optical tweezers, light scattering, zeta potential measurements and x-ray scattering.
Novelty of the proposed research: Experiments have been used extensively to study the Ca2+ and membrane fusion protein interactions with lipid vesicles; however, probing specific interactions in dynamic, heterogeneous fluid systems at the atomic level is extremely difficult. As a result, the molecular mechanism of membrane fusion in vivo is still the subject of considerable debate. Molecular simulation provides an alternative means to study complex biological systems at the atomic-level, however, recent simulations of membrane fusion have largely ignored the core machinery responsible for triggering the event: Ca2+ and various membrane proteins, such as tv-SNARE and synaptotagmin-I. The proposed research therefore addresses two key limitations in the field. Simulations are used to study interactions between Ca2+, lipids and membrane fusion proteins, such as synaptotagmin-I and tv-SNARE, with atomic scale resolution not possible with experiments.
Broader Impacts: The proposed work is transformative because the fundamental atomic level understanding of the fusion event that we seek will enable the development of a wide range of medical advances, which include smart membrane-based bio-sensors, drug delivery devices and treatment technologies for exposure to toxic industrial chemicals. Improved understanding of membrane fusion, a fundamental life process, will enable advances in a wide array of fields from disease detection to treatment.
As part of the proposed work, the PIs will develop courses on molecular simulation and modeling of chemical and biological systems using the OpenCourseWare model. Lecture notes and problem sets will be posted on our group website, while videos of lectures for the entire course will be posted on YouTube. Given the increasing use of simulation in research, the OpenCourseWare model provides an opportunity to reach thousands of students in Chemical Engineering, Physics, Chemistry and the Biomedical Sciences, thereby providing a broad impact. The undergraduate research experience will be used to recruit and mentor students from under-represented groups.
