19-BBSRC-NSF/BIO

The goal of this collaborative project is to advance our fundamental understanding of membrane protein functions in live cells using a cutting-edge sensor technology developed in Vollmer's lab (Exeter University, UK) applied to genetically programmed synthetic cells developed in the Noireaux' lab (University of Minnesota, USA). The bottom-up construction of synthetic cells that emulate specific biological functions holds strong promises as potential solutions to societal problems related to human health and the environment. While engineering functional minimal cells could be achieved in the near future, fundamental approaches to synthetic cells are also needed to expand our engineering capabilities. A serious limitation in the current state-of-the-art is the lack of basic knowledge of how synthetic cells with active membrane functions can be robustly developed and characterized. Integrating membrane functions into synthetic cells requires a quantitative understanding of basic aspects of membrane-protein interactions and dynamics. This analysis is optimal when it is performed with non-invasive techniques directly in a minimal cell setting where membrane proteins are dynamically synthesized and functionally inserted into the lipid bilayer, as proposed in this project. By placing a state-of-the art single molecule visualisation technique right next to a dynamically active synthetic cell, this collaboration will investigate how nature assembles molecular nanomachines made of proteins. These nanomachines take on important function in living systems such as catalysis, cell signalling, and they give the cell its shape and structure.

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.