Project Summary In order to perform some of their most important functions, cells must be able to generate, sense, and respond to mechanical forces. Many “mechanosensing” proteins have been discovered that are believed to change their behavior in a predictable and repeatable way when under mechanical tension. Yet, in most of these cases, we don’t know the molecular basis of how this force shifts the conformations adopted by the protein, or how this then leads to a concomitant change function. The molecular basis of mechanosensing can in principle be predicted using molecular simulation techniques, however this approach has either not been employed or not been successful because of the small magnitude of forces involved and the large size and complexity of the mechanosensors. In this work, we will develop a set of new simulation methodologies to properly sample protein conformations and protein-ligand biding lifetimes at a range of small forces. We will employ these techniques to study mechanosensing in three different contexts where we believe three distinct mechanisms for changing behavior in response to force are employed. Overall, the work in these studies will lead to a much greater understanding of the molecular paradigms used by cells to regulate their behavior in response to mechanical stimuli, and expand our simulation toolbox to be able to properly sample and assess their response to physiologically small forces.