BBSRC-NSF/BIO: Real-time Nanosensing of Membrane Protein Assembly in a TXTL Synthetic Cell System

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The bottom-up construction of synthetic cells that emulate specific biological functions has the potential to address certain societal problems related to human health and the environment. A serious limitation in the current state-of-the-art production of synthetic cells is the lack of basic knowledge of how particular membrane-associated functions can be engineered into the cells. Indeed, integrating membrane functions into synthetic cells requires a quantitative understanding of basic aspects of membrane-protein interactions and dynamics. The goal of this collaborative project is to advance our fundamental understanding of membrane protein functions in genetically programmed synthetic cells using a cutting-edge sensor technology. This project, which is a collaboration between the University of Minnesota-Twin Cities (US) and the University of Exeter (UK), also aims to provide early career scientists with opportunities to do interdisciplinary research and to introduce cutting-edge biotechnology into the classroom of undergraduate students. This interdisciplinary project will provide novel quantitative information on various dynamic features of membrane proteins and their self-assembly by applying highly-sensitive whispering gallery mode single-molecule sensors to a set of three biological membrane functions recapitulated in a synthetic cell system: membrane channels, cytoskeleton and two-component signal transduction systems. The single molecule experiments will be paralleled by a quartz crystal microbalance with dissipation approach to provide complementary information on large scales and collective effects of membrane proteins at the lipid bilayer. The research effort is divided into three objectives, to be carried out during the three-year project. First, the synthetic cell, consisting of a liposome loaded with a cell-free expression reaction to express the membrane proteins, will be set up on the single molecule sensor to monitor the insertion of native membrane protein channels and to characterize their activity. Second, we will use the whispering gallery mode sensors and the microbalance to monitor previously inaccessible adsorption kinetics of cytoskeletal proteins that mediate cell shape and division at the membrane of synthetic cell, as a function of lipid membrane properties. Third, the single molecule experiments will be performed by several independent sensing channels to characterize in real time biomolecular structural changes during signaling of a two-component system. Nanoseconds to hour’s detection timescales of sensor channels will provide information to analyze the hierarchy of protein motions in two component systems signaling, with respect to physical and chemical stimuli. The microbalance will provide information on the relationship between the membrane biophysical and biochemical properties of the membrane and the interaction between the proteins of the two-component system. This project will support the interdisciplinary training of undergraduates and post-doctoral researchers and aspects of the project will be integrated into curriculum of an undergraduate student course.This collaborative US/UK project is supported by the US National Science Foundation and the UK Biotechnology and Biological Sciences Research Council.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.