Pressure-Effects and Adaptation Mechanisms of Ambient and Deep-Sea Bacterial Enzymes

Abstract

Submersible expeditions into the deepest parts of Earth's oceans have led to discoveries of organisms thriving in the extremes, raising questions on the limits of life and the functioning of their central biological macromolecules. Questions arise as to how life would be able to survive under such “harsh” conditions, and what adaptations may be present in these organisms’ proteins. Our major focus is on understanding biophysical effects of extreme pressure, which is a relatively underexplored condition for protein solutions. Investigating the effects of high hydrostatic pressure (HHP) on the biophysical properties of proteins, in conjunction with structural adaptations utilized by known piezophilic organisms to maintain proper function, provide better insight into how organisms are affected by and survive at such depths. The results gathered from these data can impact our use of pressure for sterilization of food products, our understanding of piezophilic adaptations, and enhance our understanding of the origins of life. Graphics processing unit (GPU)-accelerated OpenMM molecular dynamics simulations were used to explore pressure-effects on and pressure-adaptations within a central metabolic enzyme, dihydrofolate reductase (DHFR). In-silico comparisons of hydrogen bonding, collective motion, among other phenomena, were made between bacterial DHFRs of the mesophile (ambient pressure) Escherichia coli, the deep-sea piezophiles Moritella profunda and Moritella yayanosii, as well as mutants containing specific residues identified to be important in pressure-sensitivity. Furthermore, high-pressure small-angle neutron and X-ray scattering, fluorescence and other high-pressure experimental techniques conducted up to 3.5 kbar further probed the biophysical effects of HHP on E. coli and M. profunda DHFRs. The combined results from in-silico and in-situ techniques provide enhanced insight into the interplay between the sequence, structure, and hydration of proteins underpinning life inhabiting extreme pressures.