Proton Loading Clusters and Complex Proton Pathways in Proton Pumping Proteins

Cuny City College, New York NY

Investigators

Abstract

Living organisms store metabolic energy by having different concentrations of hydrogen ions (H+), on each side of the membranes of mitochondria or bacterial cells. The proteins to be studied in this project are embedded in these membranes, taking H+ from the side at lower concentration and ‘pumping’ them to the side at higher concentration. H+ from the high concentration reservoir is used to fuel essential biological processes such as the production of ATP, which supplies chemical energy for many cellular reactions. The positions of the atoms in several H+ pumping proteins are known. This project uses computer simulations to investigate: (1) How the protein interior can switch between having a high or low affinity for H+ so it can be first bound and then released; (2) The pathway the H+ take through the protein interior; and (3) How proteins from different organisms conserve their function, even as the proteins change through evolution. The project will also develop novel computational tools and these tools, source code, manuals and video instructions will be made freely, publicly available. The research projects will be carried out by undergraduates, masters and PhD students, leading to a broad-based, interdisciplinary training for the next generation of scientists. The findings of this research will be integrated into a project-based class for students majoring in all STEM fields, from engineering to biology, to introduce students on how the structure of a protein determines its function. Complex I is the first protein, and cytochrome c oxidase (CCo) the last, in the aerobic electron transfer chain that uses energy-releasing redox reactions to drive H+ ions (protons) from the lower (N-side) to the higher (P-side) concentration side of the membrane. These proteins use very different structures to accomplish their task but both contain three proton transfer elements: (1) Water filled channels, anchored by acidic, basic and polar amino acid side chains, through which protons move; protons pass via hydrogen bonded connections from one water or side chain to the next; (2) Proton Loading Sites (PLS) along the path, which are transient proton binding sites; These site change between having a high proton affinity to load protons to a low affinity one to release them; (3) Gates that allow the path to be open to the N-side when the PLS loads and are open to the P-side when it unloads. Water moving out of a path is one way to close a gate. Proton pumps are driven by their reaction cycle to move between proton transfer states with PLS loaded or unloaded and gates open or closed. This research project will investigate these conformational changes. The multi-conformation continuum electrostatics (MCCE) method, developed in the Gunner lab, will be integrated with molecular dynamics and network analysis. MCCE calculates the distribution of protonation microstates and the side chain and water hydrogen bonds in Monte Carlo calculations. A protonation microstate defines the protonation state of every residue. A PLS is identified as residues that change protonation state and network analysis finds proton transfer paths in the jumble of hydrogen bonds. Molecular dynamics simulations with different residue protonation and cofactor redox states change the protein conformations. Sequence and structure comparisons of Complex I and Cytochrome c oxidase from different organisms have shown that the residues that make up the PTS and their location can change. The project will elucidate these elements in multiple CcO and Complex I that have the same function but low sequence identity. Better understanding of the range and flexibility of requirements for proton transfer can provide methods to control the many biochemical reactions that gain or lose protons at buried active sites. This research is funded by the Molecular Biophysics program in the Division of Molecular and Cellular Biosciences in the Directorate of Biological Sciences. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.