Solvent Structure and Function in Macromolecules

University Of Toledo, Toledo OH

Investigators

Abstract

Project: Hydrogen bonds and proton transfer lie at the heart of biochemistry. Experimentally determined hydrogen and solvent positions and orientations are intrinsic to understanding fine-scale biochemical interactions within and between proteins. When studying the atomic structures of proteins, neutron macromolecular crystallography (NMC) is unparalleled for determination of the position of H atoms involved in enzyme catalysis and the role of the solvent surrounding the molecule. For this study, a suite of proteins will be examined at ambient and cryogenic temperatures with NMC and X-ray macromolecular crystallography to experimentally determine the protonation state and position of hydrogens within proteins, and the position and orientation of water molecules in the surrounding solvent shell, with the goal of elucidating enzymatic mechanisms and other unresolved functionalities of the proteins. Neutron diffraction studies require the use of large, high-quality single crystals. The proteins chosen (flap endonuclease 1, a-carbonic anhydrase V, haloalkane dehalogenase, D-xylose isomerase, and equine hemoglobin) have well-defined isolation, purification and crystallization protocols that have produced large crystals, but still have unresolved mechanistic questionss. Most of the proteins used in these studies will be produced from recombinant material in deuterated expression facilities provided at Oak Ridge National Laboratory (ORNL) and at Los Alamos National Laboratory (LANL), respectively. Data measurement will be performed at protein crystallography beamlines at LANL, the Institut Laue-Langevin in Grenoble, France, and, when online, the neutron sources at ORNL. The extended funding period will enable the use of the Macromolecular Neutron Diffraction beamline at the ORNL Spallation Neutron Source during its commissioning period. Broader impacts: This project will develop and promulgate the expertise for NMC among a new generation of scientists. The increasing number of neutron sources for macromolecular diffraction, especially in this country, will mean increased opportunities for both the interpretation of neutron data and an understanding of the sorts of questions that can be resolved with neutron diffraction. The PI is a member of the Instrument Development team for the Macromolecular Neutron Diffraction (MaNDi) beamline at the ORNL Spallation Neutron Source (SNS). Because of this and the planned activities of this project, graduate students and post-doctoral fellows will be introduced at an early stage of their training to the power of the SNS and of the challenges associated with NMC. Following successful refinement and analysis, deposition and publication of structures is planned. These structures will serve as the nucleus of a database of protein structures with experimentally determined hydrogen and solvent positions.