PROJECT SUMMARY Our long-term goal is to understand how microbes are able to withstand remarkable extremes of temperature, pressure, and chemical composition (P-T-X), by determining how the macromolecular structures comprising the microbes are preserved. Our focus is on effects of high pressure, which are much less understood than temperature effects. Since high-pressure methods are increasingly being used for sterilization and food preservation, understanding the limits of pressure is important for human health and welfare. A disturbing finding is that some mesophilic microbes appear to able to withstand ~10 kbar pressures, while “piezophiles”, microbes that thrive at high pressures, have been found at maximum pressures of only ~1.1 kbar. Thus, an overall aim is to help define the limiting pressures that microbes can endure and recover from upon return to normal conditions, which is relevant to determining whether pathogenic microbes can survive high pressure treatments, by determining what pressures will permanently disrupt structures of proteins in the intracellular environment. Our over-arching question for the proposed work is how mesophiles can rapidly develop 10 kbar resistance given that piezophiles have been found only at 1.1 kbar. Our hypotheses are that enzymes from piezophiles are actually more stable at high pressure than those from mesophiles and that piezolytes (osmolytes up-regulated by microbes in response to pressure) may further protect enzymes from high pressure. In particular, piezolytes could lead to rapidly acquired pressure resistance for the entire organism. Our specific aims are: Aim 1. To test existing force fields for a range of P and T and to develop new force fields if needed. Aim 2. To determine the effects of the flexibility-stability balance on enzymes from mesophiles and piezophiles at ultra-high P. Aim 3. To determine the effects of piezolytes on enzymes at ultra-high P.