Sunney I. Chan - US grants

Two membrane proteins with important and well-defined physiological functions in cellular respiration (cytochrome c oxidase from beef heart and yeast mitochondria; HCO3-/Cl- anion exchange protein from human red blood cells) are under study to relate their biological functions to their chemical structures and three-dimensional structures in the membrane. (1) Work toward elucidation of the structure of the metal sites of cytochrome c oxidase, their spatial distribution and functional role in electron transfer, dioxygen reduction and energy conservation will be continued. Particular emphasis will be devoted to the ligands of CuB, the nature of the "pulsed" enzyme, the electronic structures of the two dioxygen intermediates at the three-electron level of reduction, the sequence of intramolecular electron transfer, redox interactions among the metal sites and their role in the energy conservation process, and the site of redox-linked proton translocation in the protein. A hypothesis of redox-linked proton-translocation based on the concept of electron-gating will be tested and the details extended or refined as necessary. (2) Similarly, the structure of the anion transport sites of the Cl-/HCO3- anion exchange protein is being studied by 35Cl/37Cl nuclear magnetic resonance (NMR) spectroscopy and amino acid-specific modification experiments, followed by chemical analysis by HPLC. The goals are to develop a molecular picture describing the anion translocation event, the structure of the transport site(s), their location(s) in the primary sequence of the protein and relative to the membrane, and the minimal structure containing the intact anion transport machinery. (3) To investigate the interactions of these proteins with the lipids in the membranes in which they are embedded, purified cytochrome c oxidase as well as the HCO3-/Cl- exchange protein are being reconstituted into lipid membranes of well-defined chemical compositions. The state of aggregation of the intramembraneous particles in the reconstituted membranes is being visualized by freeze-fracture electron microscopy, and the protein distributions thus derived are correlated with the motional state of the bilayer inferred from magnetic resonance experiments to provide insights on the nature of the lipid deformations induced by the membrane proteins in their immediate surroundings. These studies on lipid-mediated protein-protein forces are designed to develop an understanding of the significance of protein-lipid interactions in biological function.

Two membrane proteins with important and well-defined physiological functions in cellular respiration (cytochrome c oxidase from beef heart and yeast mitochondria; HCO3-/Cl- anion exchange protein from human red blood cells) are under study to relate their biological functions to their chemical structures and three-dimensional structures in the membrane. (1) Work toward elucidation of the structure of the metal sites of cytochrome c oxidase, their spatial distribution and functional role in electron transfer, dioxygen reduction and energy conservation will be continued. Particular emphasis will be devoted to the ligands of CuB, the nature of the "pulsed" enzyme, the electronic structures of the two dioxygen intermediates at the three-electron level of reduction, the sequence of intramolecular electron transfer, redox interactions among the metal sites and their role in the energy conservation process, and the site of redox-linked proton translocation in the protein. A hypothesis of redox-linked proton-translocation based on the concept of electron-gating will be tested and the details extended or refined as necessary. (2) Similarly, the structure of the anion transport sites of the Cl-/HCO3- anion exchange protein is being studied by 35Cl/37Cl nuclear magnetic resonance (NMR) spectroscopy and amino acid-specific modification experiments, followed by chemical analysis by HPLC. The goals are to develop a molecular picture describing the anion translocation event, the structure of the transport site(s), their location(s) in the primary sequence of the protein and relative to the membrane, and the minimal structure containing the intact anion transport machinery. (3) To investigate the interactions of these proteins with the lipids in the membranes in which they are embedded, purified cytochrome c oxidase as well as the HCO3-/Cl- exchange protein are being reconstituted into lipid membranes of well-defined chemical compositions. The state of aggregation of the intramembraneous particles in the reconstituted membranes is being visualized by freeze-fracture electron microscopy, and the protein distributions thus derived are correlated with the motional state of the bilayer inferred from magnetic resonance experiments to provide insights on the nature of the lipid deformations induced by the membrane proteins in their immediate surroundings. These studies on lipid-mediated protein-protein forces are designed to develop an understanding of the significance of protein-lipid interactions in biological function.

The California Institute of Technology will develop and deliver through a collaborative effort by scientists, teacher- educators, and master teachers, a one-semester course for the elementary teacher education program. Future elementary teachers will construct their own science knowledge and work cooperatively in a "hands on" science classroom environment. These prospective teachers will do challenging investigations with sophisticated tools while modeling good science teaching. Simultaneously, the course will provide a solid foundation in fundamental principles of biology, chemistry, physics and earth science. The course will be integrated into the elementary science teacher program.

High field NMR spectroscopy has emerged as one of the key tools for biophysical investigation of molecular conformations. With the rapid evolution of techniques for discovering, synthesizing and designing molecules of biomedical importance, there is an increasing need to characterize their structural details in developing a rational basis for elucidating the relationship between structure and function. In this proposal a number of investigators are requesting funds to upgrade facilities for applying NMR to such problems. This will enable them to expand their research horizons in this area. A broad range of projects are described from synthesis of natural products to studies of proteins, DNA, and their intermolecular interactions.

Two membrane proteins with important and well-defined physiological functions in cellular respiration (cytochrome c oxidase from beef heart and yeast mitochondria; HCO3-/Cl- anion exchange protein from human red blood cells) are under study to relate their biological functions to their chemical structures and three-dimensional structures in the membrane. (1) Work toward elucidation of the structure of the metal sites of cytochrome c oxidase, their spatial distribution and functional role in electron transfer, dioxygen reduction and energy conservation will be continued. Particular emphasis will be devoted to the ligands of CuB, the nature of the "pulsed" enzyme, the electronic structures of the two dioxygen intermediates at the three-electron level of reduction, the sequence of intramolecular electron transfer, redox interactions among the metal sites and their role in the energy conservation process, and the site of redox-linked proton translocation in the protein. A hypothesis of redox-linked proton-translocation based on the concept of electron-gating will be tested and the details extended or refined as necessary. (2) Similarly, the structure of the anion transport sites of the Cl-/HCO3- anion exchange protein is being studied by 35Cl/37Cl nuclear magnetic resonance (NMR) spectroscopy and amino acid-specific modification experiments, followed by chemical analysis by HPLC. The goals are to develop a molecular picture describing the anion translocation event, the structure of the transport site(s), their location(s) in the primary sequence of the protein and relative to the membrane, and the minimal structure containing the intact anion transport machinery. (3) To investigate the interactions of these proteins with the lipids in the membranes in which they are embedded, purified cytochrome c oxidase as well as the HCO3-/Cl- exchange protein are being reconstituted into lipid membranes of well-defined chemical compositions. The state of aggregation of the intramembraneous particles in the reconstituted membranes is being visualized by freeze-fracture electron microscopy, and the protein distributions thus derived are correlated with the motional state of the bilayer inferred from magnetic resonance experiments to provide insights on the nature of the lipid deformations induced by the membrane proteins in their immediate surroundings. These studies on lipid-mediated protein-protein forces are designed to develop an understanding of the significance of protein-lipid interactions in biological function.

9877039

Abstract

This project involves the acquisition of a 600 MHz NMR spectrometer for the Instrumentation facilities of the Division of Chemistry and Chemical Engineering of California Institute of Technology. The major purpose of this high field instrument will be for the determination of the structures of complex biological molecules of biosynthetic, semisynthetic and synthetic origin. The research activities of several groups in the Division will benefit greatly from the new spectrometer and in many cases, entirely new projects will be made possible by the new instrument. The 600 MHz spectrometer has the resolution and sensitivity needed to determine structures of complexes in the 5 to 30 kD range. Research for which the instrument is required includes the study of metal-DNA complexes; protein- and peptide-DNA complexes; the structure of DNA intercalators; studies of the hyperthermostability of rubredoxin from P. furiosus; structure and dynamics of novel proteins; artificial proteins; ion channel proteins; and electron transfer reactions in wild-type and mutant netalloproteins. The availability of this instrument will have a major impact on both the research and training activities of the Division.