PHOTOBIOLOGY OF RHODOPSIN, BACTERIORHODOPSIN & VIODOPSIN
Robert Richards Birge, Distinguished Professor
Syracuse University Office Of Sponsored Programs Syracuse, Ny 13244
Grant 5R01GM034548-14 from National Institute Of General Medical Sciences IRG: BBCA
Abstract: The nature of the chromophore binding sites and the primary photochemical events of rhodopsin, bacteriorhodopsin and the Xenopus violet cone pigment, viodopsin, will be studied by using spectroscopic and theoretical techniques. The goals are to understand the photophysical properties of the bound chromophores. The principal spectroscopic methods to be used in these studies include two-photon spectroscopy, Stark effect spectroscopy, Fourier transform infra-red spectroscopy, microwave spectroscopy, and pulsed laser photocalorimetry. The principal chemical studies to be undertaken include organic cation and chromophore analog substitutions, as well as site directed mutagenesis. The theoretical methods include semiempirical molecular orbital theory and molecular dynamics theory. The goal is to combine experiment and theory in a synergistic program which enhances both. In addition to the more global goals outlined above, Dr. Birge will seek to answer the following specific questions (1) What is the principal mechanism of wavelength modulation in the blue and violet cones? (2) Where are the cation binding sites in bacteriorhodopsin, and how do these sites mediate the properties of the bound chromophore? (3) Is there a chloride binding site in viodopsin, and what impact does this site have on the photophysical properties of the chromophore? (4) What are the molecular origins of energy storage in the primary events of these three proteins? (5) What is the principal molecular mechanism of dark noise in vertebrate and invertebrate vision? (6) What are the molecular origins of the unusual photochemical properties of the 4-keto retinal bacteriorhodopsin analog? (7) Can one improve the accuracy of the MNDO-PSDCI semi-empirical molecular orbital theory by using ab-initio effective Hamiltonian parameterizaion?
Keywords: bacteriorhodopsin, photochemistry, protein structure, rhodopsin, visual pigment, analog, biophysics, cation, chemical substitution, chromophore, molecular site, quantum chemistry, Xenopus, calorimetry, infrared spectrometry, interferometry, microwave spectrometry, molecular dynamics, photoelectron spectrometry, site directed mutagenesis
Project start date: 1988-08-01
Project end date: 2000-02-29
5R01GM034548-14 (1999): $205127
Sponsored Links Excellgen http://Excellgen.com
PHOTOPHYSICS OF VISUAL CHROMOPHORES AND RHODOPSIN
Robert Richards Birge, Distinguished Professor
Carnegie-mellon University 5000 Forbes Ave Pittsburgh, Pa 15213
Grant 5R01GM034548-03 from National Institute Of General Medical Sciences IRG: BBCA
Abstract: The long-term objectives of this research project are to assign the structure of the chromophore binding sites in rhodopsin and light-adapted bacteriorhodopsin and to define the molecular electronic details of the primary photochemical events in these two protein systems. Three projects will be carried out in order to accomplish these goals (1) Two-photon spectroscopy will be used to identify the location and photophysical properties of the low-lying "forbidden" pi pi* states in various visual chromophore analogs in solution and in the binding sites of the proteins. This information, when combined with the one-photon spectroscopic data, will provide insights into the nature and magnitude of the electrostatic and dispersive perturbations induced by the protein binding sites. (2) All-valence electron (INDO-PSDCI) molecular orbital theory will be used to help interpret the spectral data and calculate the ground and excited state potential surfaces, and excited state reaction paths, for double bond isomerization for various models of the binding sites. Semiclassical molecular dynamics theory will be used to calculate the trajectories and the quantum yields of the primary photochemical events. The effect of the protein will be included in the above calculations using classical force field procedures to determine the equilibrium geometry of the amino acid residues on the alpha helices in the vicinity of the binding site. Various models for the binding site can then be tested by comparing the calculated results with the data obtained in the experimental portions of this program as well as from the literature. (3) Energy storage in the primary events in artificial rhodopsin and bacterio-rhodopsin analogs will be measured using pulsed laser photocalorimetry. By changing the position of methyl groups along the polyene chain and determining the energy storage associated with the photoisomerization it should be possible to map out the geometry of the binding sites. These data will provide information concerning the geometry of the binding site. These studies will provide new insights into the molecular basis of vertebrate visual transduction.
Keywords: EYE, VISUAL PIGMENTS, RHODOPSIN, MOLECULAR AND CELLULAR BIOPHYSICS STUDY SECTION, PIGMENTS, BACTERIAL, BACTERIORHODOPSIN-RETINAL, photochemistry, CHEMICAL BONDS, BINDING AND BINDING SITES, CHEMICAL STRUCTURE, ISOMERS, STEREOISOMERS (GENERAL), CHEMICAL STRUCTURE, STEREOCHEMISTRY, CONFORMATIONS, CHEMISTRY, QUANTUM (GENERAL), ELECTRONIC SPECTRA, MOLECULAR ORBITALS, MODELS, PHANTOM MODELS, MOLECULAR ENERGY LEVELS, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, PHOTOELECTRON, TEMPERATURE, HEAT, CALORIMETRY
Project start date: 1985-04-01
Project end date: 1988-03-31
5R01GM034548-08 (1992): $166429
Photobiology Of Rhodopsin And Bacteriorhodopsin
Robert Richards Birge, Distinguished Professor
University Of Connecticut Storrs 438 Whitney Road Extension, Unit 1133 Storrs-mansfield, Ct 06269
Grant 5R01GM034548-19 from National Institute Of General Medical Sciences IRG: BBCA
Abstract: The long-term objectives of this research program are to understand the molecular details of protein photochemical mediation and wavelength regulation in bacteriorhodopsin and the visual pigments of rods and cones. Our emphasis for the present grant period is to study the blue, violet and uv cones because relatively little is known about these pigments. The key spectroscopic tools that will be used include one-photon and two-photon spectroscopy, Fourier-transform infrared spectroscopy, Raman spectroscopy, CD spectroscopy and pulsed laser photocalorimetry. The principal biochemical studies to be undertaken include site-directed mutagenesis and chromophore analog substitutions as well as random mutagenesis followed by screening for wavelength and photochemical properties. The theoretical studies will rely heavily on the use of MNDOPSDCI molecular orbital theory to probe the photophysical properties of the protein-bound chromophores. An important and new goal of this grant period is to add the prediction of circular dichroism spectra to the MNDOPSDCI procedures, with the immediate goal of using experimental CD spectra to analyze the protein binding sites of rhodopsin and the cone pigments. We will also use ab-initio and semiempirical molecularorbital theory to examine the ground state properties of the chromophore binding sites while using molecular mechanics to describe the remaining portions of the protein. Our goal is to combine experiment and theory in a synergistic program that enhances both. In addition to the more global goals outlined above, we will seek to answer the following specific questions (1) What are the principal mechanisms of wavelength regulation in the cone pigments? (2) What is responsible for the significant difference in the absorption spectra of bacteriorhodopsin versus sensory rhodopsin II? (3) What are the mechanisms through which the chloride binding sites in the cone pigments influence the spectroscopic properties of the chromophores in the long wavelength cone pigments? (4) Where are the calcium binding sites in bacteriorhodopsin, and how do these sites influence the photophysical properties of the bound chromophore? (5) What specific proteinchromophore interactions are responsible for selecting 6-s-cis versus 6-s-trans ring conformations of the bound chromophore, and why does rhodopsin select the 6-s-cis while bacteriorhodopsin and sensory rhodopsin select the 6-s-trans conformation?
Keywords: bacteriorhodopsin, photobiology, protein structure function, rhodopsin, visual pigment, Schiff base, analog, binding site, chemical substitution, chromophore, cone cell, molecular site, photochemistry, protein binding, protonation, rod cell, ultraviolet radiation, Raman spectrometry, circular dichroism, infrared spectrometry, interferometry, photoelectron spectrometry, site directed mutagenesis
Project start date: 1988-08-01
Project end date: 2007-08-31
5R01GM034548-19 (2005): $202244
5R01GM034548-18 (2004): $212888
5R01GM034548-17 (2003): $212888
PHOTOPHYSICS OF RHODOPSIN AND BACTERIORHODOPSIN
Robert Richards Birge, Distinguished Professor
Syracuse University Office Of Sponsored Programs Syracuse, Ny 13244
Grant 5R01GM034548-11 from National Institute Of General Medical Sciences IRG: BBCA
Abstract: Rhodopsin, the protein responsible for converting light into an optic nerve impulse, and bacteriorhodopsin, the light transducing protein of the purple membrane of Halobacterium halobium, have significantly different biological roles. Nevertheless, natural selection has converged on very similar designs for both proteins, and these similarities have prompted comparative experimental and theoretical studies. The lack of high resolution X-ray structural data has precluded precise assignment of the tertiary structure of either protein, and hence our knowledge of the binding sites and the primary events is based on indirect analyses of chemical and spectroscopic data. Despite a number of important advances during the past decade much remains to be understood and a number of interesting experimental paradoxes remain unexplained. The nature of the chromophore binding sites and the nature of the primary photochemical events of rhodopsin and bacteriorhodopsin will be studied by using spectroscopic and theoretical techniques. The goals are to understand the photophysical properties of the bound chromophores and how the proteins mediate the photochemical properties of the bound chromophores. The principal experimental methods to be used in these studies include two-photon spectroscopy, Stark effect spectroscopy, Fourier transform near infra-red spectroscopy, microwave spectroscopy, pulsed laser photocalorimetry, and time-resolved photovoltaic spectroscopy. The theoretical methods include semiempirical molecular orbital theory and molecular dynamics theory. In addition, we will seek to answer the following specific questions (1) To what extent does water participate in mediating the photochemical properties of the bound chromophores in both proteins? (2) What is the origin of the fast photoelectric signal that is generated during the primary photochemical events in rhodopsin and bacteriorhodopsin? (3) Can we observe and use protein-chromophore charge transfer bands to help analyze the nature of the protein chromophore interactions within the protein binding site? (4) What is the origin of the strong microwave absorptivity of bacteriorhodopsin and the origin of the M - bR microwave difference spectrum? (5) What is the nature of energy storage in the primary events of rhodopsin and bacteriorhodopsin? (6) Are there charged amino acids near the ring of the chromophore in bacteriorhodopsin? Furthermore, we will seek to develop a new method of collecting two-photon spectra of photochemically labile biological molecules based on Fourier transform optical techniques.
Keywords: bacteriorhodopsin, biophysics, photochemistry, protein structure function, rhodopsin, analog, biomedical equipment development, chemical binding, chromophore, computer program /software, computer simulation, conformation, mathematical model, quantum chemistry, retinaldehyde, visual phototransduction, animal tissue, calorimetry, infrared spectrometry, interferometry, laser, microwave spectrometry, molecular dynamics, photoelectron spectrometry, photon absorptiometry
Project start date: 1988-08-01
Project end date: 1997-07-31
5R01GM034548-11 (1995): $177347
Sponsored Links Excellgen http://Excellgen.com
5R01GM034548-10 (1994): $180602
Robert Richards Birge
University Of Connecticut Storrs
Project start date: 1988-08-01
Project end date: 2013-02-28
PHOTOPHYSICS OF RHODOPSIN AND BACTERIORHODOPSIN
Robert Richards Birge, Distinguished Professor
Syracuse University Office Of Sponsored Programs Syracuse, Ny 13244
Grant 5R01GM034548-12 from National Institute Of General Medical Sciences IRG: BBCA
Project start date: 1988-08-01
Project end date: 1998-02-28
5R01GM034548-12 (1996): $180682
Grants awarded to Robert Richards Birge
PHOTOBIOLOGY OF RHODOPSIN, BACTERIORHODOPSIN & VIODOPSIN
Robert Richards Birge, Distinguished Professor
Chemistryuniversity Of Connecticut Storrs
438 Whitney Road Extension, Unit 1133
storrs-mansfield, Ct 06269
Grant 7R01GM034548-15 from National Institute Of General Medical Sciences IRG: BBCA
Abstract: The nature of the chromophore binding sites and the primary photochemical events of rhodopsin, bacteriorhodopsin and the Xenopus violet cone pigment, viodopsin, will be studied by using spectroscopic and theoretical techniques. The goals are to understand the photophysical properties of the bound chromophores. The principal spectroscopic methods to be used in these studies include two-photon spectroscopy, Stark effect spectroscopy, Fourier transform infra-red spectroscopy, microwave spectroscopy, and pulsed laser photocalorimetry. The principal chemical studies to be undertaken include organic cation and chromophore analog substitutions, as well as site directed mutagenesis. The theoretical methods include semiempirical molecular orbital theory and molecular dynamics theory. The goal is to combine experiment and theory in a synergistic program which enhances both. In addition to the more global goals outlined above, Dr. Birge will seek to answer the following specific questions (1) What is the principal mechanism of wavelength modulation in the blue and violet cones? (2) Where are the cation binding sites in bacteriorhodopsin, and how do these sites mediate the properties of the bound chromophore? (3) Is there a chloride binding site in viodopsin, and what impact does this site have on the photophysical properties of the chromophore? (4) What are the molecular origins of energy storage in the primary events of these three proteins? (5) What is the principal molecular mechanism of dark noise in vertebrate and invertebrate vision? (6) What are the molecular origins of the unusual photochemical properties of the 4-keto retinal bacteriorhodopsin analog? (7) Can one improve the accuracy of the MNDO-PSDCI semi-empirical molecular orbital theory by using ab-initio effective Hamiltonian parameterizaion?
Keywords: bacteriorhodopsin, photochemistry, protein structure, rhodopsin, visual pigment analog, biophysics, cation, chemical substitution, chromophore, molecular site, quantum chemistry Xenopus, calorimetry, infrared spectrometry, interferometry, microwave spectrometry, molecular dynamics, photoelectron spectrometry, site directed mutagenesis
Project start date: 1988-08-01
Project end date: 2002-08-31
7R01GM034548-15 (2000): $195499
2R01GM034548-13A1 (1998): $177575
PHOTOPHYSICS OF RHODOPSIN AND BACTERIORHODOPSIN
Robert Richards Birge, Distinguished Professor
Syracuse University At Syracuse
syracuse, Ny 13210
Grant 2R01GM034548-09 from National Institute Of General Medical Sciences IRG: BBCA
Abstract: Rhodopsin, the protein responsible for converting light into an optic nerve impulse, and bacteriorhodopsin, the light transducing protein of the purple membrane of Halobacterium halobium, have significantly different biological roles. Nevertheless, natural selection has converged on very similar designs for both proteins, and these similarities have prompted comparative experimental and theoretical studies. The lack of high resolution X-ray structural data has precluded precise assignment of the tertiary structure of either protein, and hence our knowledge of the binding sites and the primary events is based on indirect analyses of chemical and spectroscopic data. Despite a number of important advances during the past decade much remains to be understood and a number of interesting experimental paradoxes remain unexplained. The nature of the chromophore binding sites and the nature of the primary photochemical events of rhodopsin and bacteriorhodopsin will be studied by using spectroscopic and theoretical techniques. The goals are to understand the photophysical properties of the bound chromophores and how the proteins mediate the photochemical properties of the bound chromophores. The principal experimental methods to be used in these studies include two-photon spectroscopy, Stark effect spectroscopy, Fourier transform near infra-red spectroscopy, microwave spectroscopy, pulsed laser photocalorimetry, and time-resolved photovoltaic spectroscopy. The theoretical methods include semiempirical molecular orbital theory and molecular dynamics theory. In addition, we will seek to answer the following specific questions (1) To what extent does water participate in mediating the photochemical properties of the bound chromophores in both proteins? (2) What is the origin of the fast photoelectric signal that is generated during the primary photochemical events in rhodopsin and bacteriorhodopsin? (3) Can we observe and use protein-chromophore charge transfer bands to help analyze the nature of the protein chromophore interactions within the protein binding site? (4) What is the origin of the strong microwave absorptivity of bacteriorhodopsin and the origin of the M - bR microwave difference spectrum? (5) What is the nature of energy storage in the primary events of rhodopsin and bacteriorhodopsin? (6) Are there charged amino acids near the ring of the chromophore in bacteriorhodopsin? Furthermore, we will seek to develop a new method of collecting two-photon spectra of photochemically labile biological molecules based on Fourier transform optical techniques
Keywords: bacteriorhodopsin, biophysics, photochemistry, protein structure function, rhodopsin analog, biomedical equipment development, chemical binding, chromophore, computer program /software, computer simulation, conformation, mathematical model, quantum chemistry, retinaldehyde, visual phototransduction animal tissue, calorimetry, infrared spectrometry, interferometry, laser, microwave spectrometry, molecular dynamics, photoelectron spectrometry, photon absorptiometry
Project start date: 1988-08-01
Project end date: 1997-07-31
2R01GM034548-09 (1993): $173682
3R01GM034548-12S1 (1997): $50828
Photobiology Of Rhodopsin And The Cone Pigments
Robert Richards Birge, Distinguished Professor
Chemistryuniversity Of Connecticut Storrs
Grant 2R01GM034548-20A1 from National Institute Of General Medical Sciences IRG: BDPE
Abstract: Cone cells are responsible for photopic vision, the visual process under normal light conditions. The cone receptors must operate over a wide range of light intensities and cover the full range of the visible spectrum. The ability to function under these diverse conditions is due primarily to the highly optimized GPCR light-transducing proteins informally called cone pigments. These proteins have absorption maxima that range from 350 to 660 nm, and upon the absorption of light, undergo an efficient photobleaching sequence to produce an activated protein. Subsequent binding of transducin to the activated protein results in a nerve impulse and vision. A key observation made during the previous NIH funded study was that cone pigments undergo a counterion switch during photoactivation. A key aim of this study is to explore whether a counterion switch mechanism is also active in the red and blue cone pigments, and if so, to characterize the molecular details. To achieve this goal, we will use vibrational and electronic spectroscopy at temperatures from 10K to ambient to trap and characterize the photobleaching intermediates. Site directed mutagenesis will be used to identify the key residues responsible for wavelength selection and the nature of the counterion switch. It is clear from homology studies that many of the red cones differ from the green, blue and UV cones in nature and implementation of the counterion switch. Indeed, it is possible that the red cones lack this mechanistic feature entirely. An additional aim of this study is to systematically identify the mechanisms of wavelength selection in the UV, blue, green and red cones. Although our research identified key features of wavelength selection in the blue and UV cones, much remains to be understood. Our inclusion of the red cones in this study is new, and our enthusiasm for this topic rests in part on our belief that the red cones are fundamentally different. We have preliminary evidence, presented in our preliminary studies discussion, that the deep red cones use at least one new mechanism for wavelength selection involving manipulation of the chromophore ring conformation. The combination of unique wavelength selection and a significantly different (or absent) counterion switching mechanism make the red cones an important target. Our studies will include the use of molecular orbital theory to probe structure-function relationships in the cone pigments, and to calculate the spectroscopic properties of the bound chromophores. We will refactor our MNDO-PSDCI code and improve the interface to make these procedures more useful to the scientific community. As before, we will provide these procedures to interested researchers without charge. There is a growing need to understand the photobleaching and recovery mechanisms associated with light exposure in cone photoreceptors. Because these cells are essential for human photopic vision, it is important to understand the structure and function relationships in the associated light transducing pigments. The project goals of this research may help understand macular disease, which involves loss of cone cells, cone dystrophy, and other eye diseases, which involve damage or diminished function of retinal photoreceptors
Project start date: 1988-08-01
Project end date: 2013-02-28