Proteins are large biological molecules that are an essential part of all living organisms, in charge of all biochemical processes and of all cellular housekeeping tasks. Proteins are the most complex molecules known. This project will use pressure to examine the molecular determinants of stability and other fundamental properties of proteins. One goal is to understand why the structure of proteins, and in particular, the presence of empty cavities inside the protein, renders them sensitive to pressure. These cavities are involved in many important biochemical processes, such as enzymatic catalysis (enzymes are proteins that facilitate chemical reactions), thus it is important to understand properties such as their volume, whether they are empty or whether water is found inside them, how they change over the course of functional processes, and how they affect the essential communication between different parts of the protein. Graduate, undergraduate, and high school students will be trained in computational and experimental biophysics. Dr. GarcÃa-Moreno is actively engaged in the development of a modern science curriculum focused on the training of biology students in more physical, computational and quantitative approaches. He also has been very active and will continue to be engaged in his role as mentor and advisor to students from many groups that are underrepresented in science.
Proteins can be unfolded with pressure. This implies that the net volume of the unfolded state is smaller than the volume of the folded state. The origins of these volume differences are not obvious. This project is focused on fundamental and systematic studies of cavities and pressure unfolding of proteins, with emphasis on the roles of cavities as determinants of pressure unfolding and on the properties of the cavities. One goal is to examine the relationship between the size, character, state of hydration and location of cavities, and a protein's sensitivity to pressure, its thermodynamic stability, and the magnitude of volume change measured upon unfolding. A second goal is to study the effects of cavities on fluctuations and correlated motions, on dynamics in the hydrophobic interior, on structural cooperativity, and on internal hydration. The experiments that will be performed for this project will further fundamental understanding of structural and thermodynamic consequences of cavities in proteins, especially under conditions of high hydrostatic pressure. The approach that will be pursued involves equilibrium thermodynamic experiments to measure volume changes with pressure unfolding monitored by Trp fluorescence and high pressure NMR, measurement of thermodynamic stability with chemical denaturation, x-ray crystallography to determine crystal structures of proteins to describe the location and volume of internal cavities, and MD simulations to examine the state of hydration of cavities and structural consequences of the application of pressure. High pressure NMR spectroscopy will also be used to examine the response of proteins to pressure with atomic resolution, by characterizing the pressure dependence of fast backbone and side chain dynamics.
This project is jointly funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Physics of Living Systems Program in the Division of Physics in the Directorate of Mathematical and Physical Sciences.
