Daniel D. Oprian, Ph.D. - US grants

DESCRIPTION (provided by applicant): The long-term focus of this grant continues to be the structure and function of the visual pigment rhodopsin and its interactions with other members of the vertebrate phototransduction cascade. The studies are geared toward an understanding of the protein in terms of its mechanism of activation, its interaction with downstream proteins of the phototransduction cascade, and its function and dysfunction in health and disease. The new studies focus on two areas: the development and exploitation of a single-molecule approach to the study of interactions among signaling partners in the visual phototransduction cascade, and the characterization of novel mutants of rhodopsin in which the active site of the protein has been significantly altered from the wild-type. There are two Specific Aims: 1. to establish, characterize, and expand Total Internal Reflection Fluorescence Microscopy (TIRFM) as a tool in the single-molecule regime to study the dynamics of assembly and disassembly of key signaling complexes in the rod cell phototransduction cascade. One of the major goals of this aim is to better understand the role of constitutively active mutants in the diseases retinitis pigmentosa and congenital stationary night blindness. 2. To characterize novel rhodopsin mutants in which the highly conserved active-site Lys296 has been moved to different locations in the protein. This Aim expands upon surprising preliminary results showing that the Lys can be moved while maintaining near wild-type spectral properties and ability to activate transducin in a light-dependent manner. The focus is to better understand the evolutionary relatedness of retinylidene proteins and to probe our understanding of the molecular mechanism of photoactivation of rhodopsin.

Human color vision is trichromatic, we use three pigments (blue, green and red) to discriminate different colors. Inherited red/green color vision deficiencies are X-linked and occur with a rather high frequency (about 8%) in the male population. These inherited deficiencies are thought to arise by homologous recombination between the highly homologous red and green genes which are both located on the X chromosome. Although this is supported by much genetic evidence, the isolated pigments were not heretofore available to test this model directly. The broad aim of the research outlined in this proposal is to elucidate the structure, spectral properties, and mechanism of action of the human color vision pigments, and to test molecular models for inherited human color vision deficiencies. The experimental approach will be to use site-directed mutagenesis in combination with mammalian cell culture to produce the human pigments. The wild-type and modified pigments will be purified to homogeneity and characterized in vitro. There are two specific goals. First is to test the hypothesis that human red/green color vision deficiencies arise by unequal homologous recombination events. Second is to characterize the isolated human color vision pigments in terms of their general spectral and biochemical properties.

Human color vision is trichromatic, we use three pigments (blue, green and red) to discriminate different colors. Inherited red/green color vision deficiencies are X-linked and occur with a rather high frequency (about 8%) in the male population. These inherited deficiencies are thought to arise by homologous recombination between the highly homologous red and green genes which are both located on the X chromosome. Although this is supported by much genetic evidence, the isolated pigments were not heretofore available to test this model directly. The broad aim of the research outlined in this proposal is to elucidate the structure, spectral properties, and mechanism of action of the human color vision pigments, and to test molecular models for inherited human color vision deficiencies. The experimental approach will be to use site-directed mutagenesis in combination with mammalian cell culture to produce the human pigments. The wild-type and modified pigments will be purified to homogeneity and characterized in vitro. There are two specific goals. First is to test the hypothesis that human red/green color vision deficiencies arise by unequal homologous recombination events. Second is to characterize the isolated human color vision pigments in terms of their general spectral and biochemical properties.

One of the central problems in visual science is the mechanism of spectral tuning among the visual pigments. How is it that the absorption maxima can range from 380 nm to over 600 nm when the actual molecule that absorbs light, the 11-cis-retinal chromophore, is identical in all of the pigments? Clearly, there are interactions of the retinal chromophore with amino acid sidechains in the proteins that modify the spectral properties the chromophore. It is the purpose these studies to unravel these mechanisms for one extremely well defined experimental system: the human color vision pigments. This work will focus on three groups of amino acids in these proteins known to profoundly affect the spectral properties of the chromophore: 1) the 7 amino acids that cause the spectral difference between the red and green pigments; 2) the protonated Schiff base of the chromophore and Schiff base counterion; and 3) the chloride binding site of the red and green pigments. The approach will be to use site-directed mutagenesis, chemical modification of the proteins, and chromophore analogs in combination with in vitro characterization of the purified pigments. As a result of a recent technical breakthrough in the application of resonance Raman spectroscopy to the study of these pigments, this powerful spectroscopic tool will be used extensively to probe the structure of the retinal chromophore in all aspects of this work. The enormous potential of this application is apparent from a realization that the source of visual pigment for the Raman studies will be a library of over 150 mutants in the blue, green and red visual pigments that was constructed over the original grant period.

The long term goal of these studies is to unravel the molecular mechanism of action of the visual pigment rhodopsin. This work will lead to the design of specific, active-site directed, and/or mechanism-based reagents aimed at treatment of diseases such as retinitis pigmentosa and congenital stationary night blindness. There are two specific aims in this proposal: (i) to identify and elucidate the role of essential amino acid residues in rhodopsin, and (ii) to use this information to design active-site directed reagents that target mutant rhodopsins. The experimental approach will be to use site-directed mutagenesis combined with extensive in vitro functional analysis and chemical modification of the altered rhodopsins. Mutagenesis will be used to identify essential amino acids in the activation and inactivation of rhodopsin and opsin. Crosslinking studies will be used to determine how close together are essential residues in the active site. Transducin activity assays will be performed to determine how closely the activated mutants resemble photoactivated rhodopsin. Finally, the increased understanding of mechanism in the wild-type and mutant rhodopsins will be used to design active-site directed reagents based on retinal analogs that specifically target mutant rhodopsins found in patients with retinitis pigmentosa and congenital stationary night blindness.

DESCRIPTION: The enzyme Myristoyl CoA-transferase (NMT) transfers myristate to an N terminal glycine of a target protein or peptide. Myristoylation may serve to anchor peptides or proteins in a hydrophobic region. The mechanism of action of NMT will be investigated. An important question is whether myristate forms a covalent intermediate with the enzyme. To answer this question [3H] myristoyl CoA will be added to NMT in the absence of peptide acceptor, or in the presence of a peptide analog which is not myristoylated. The enzyme will be degraded with trypsin and chymotrypsin. The resulting peptides will be separated and examined for radio-activity. If radiolabeled material is associated with one of the peptides, formation of a covalent enzyme-substrate complex probably occurred. Aza peptides are peptides in which the S1 amino acid is replaced by NH-NR-CONH. These peptides inhibit serine and thiol proteases. Inhibition is due to acylation of the active site -OH or -SH. The resulting acyl-enzyme hydrolyses slowly. The structure of the inactive enzyme will be determined by NMR and chemical approaches. Understanding of the mechanism will be important in improving these inhibitors. Aza-peptide inhibitors are attractive for pharmacological applications, since they are not subject to proteolytic digestion and are resistant to chemical degradation. They also are highly selective. CO formation occurs in organisms ranging from fungi to mammals. CO has biological effects similar to NO. It has been called a neuro transmitter. Little is known about the enzymatic mechanism of CO formation. The investigator and his colleagues have purified an enzyme from K. Pneumoniae which catalyzes the formation of CO. To ascertain the enzyme mechanism, non enzymatic model systems will be investigated. For instance: CH3-CO-CH2-CO-CH3 is non-enzymatically converted to CO and other products in the presence of H2O2. ESR experiments will be done to detect free radicals. It is anticipated that an understanding of the mechanism will enable the applicant to design an inhibitor which can be used to inhibit CO production in intact bacterial cells.

The Bioorganic Training Grant, which is now submitted for renewal, has played an important role in science education at Brandeis. It gave rise to a group of graduate students with novel interests and novel capabilities. It was the goal of this program to train investigators who can work at the interphase of biology, biochemistry, and chemistry. We have been successful. It is apparent, from listening to rotation reports, that chemists can use, and understand techniques of molecular biology, and biologists can synthesize compounds. This combination of skills is in demand by the pharmaceutical industry, and should facilitate future health related research. The program started with zero students and now contains 18 students. Trainees are appointed on the basis of their strong academic record and evidence of their research ability. In the first year of graduate training they begin to participate in a program, which consists of a core curriculum of seven courses. Students take these courses in their first two years of the program. The core curriculum consists of two semesters of Advanced Biochemistry (Biochemistry 101A & 101B), one semester of Advanced Organic Synthesis (Chemistry 134), one semester of Molecular Biology (Biology 105), one semester of Mechanistic Organic Chemistry (selected from Chemistry 131, 133, or Biochemistry 202), one semester of an advance course in spectroscopy or crystallographic structure determination (selected from Chemistry 132, 229, or 235), and finally one elective, selected from offerings in the Chemistry, Biochemistry, Biology or Neuroscience programs. First year students also undertake six, six-week rotations in different laboratories in the program. The students must complete half of their rotations on problems which focus on chemical synthesis. This can be accomplished by rotating in laboratories of faculty from the Chemistry Department or, where appropriate, in laboratories from the Biochemistry Department. A written report and oral presentation of the results are given at the end of each rotation. The purpose of the rotations is two-fold. First, is to give students exposure to many different faculty so as to facilitate their choice of mentor and to give them a broad experience with different approaches to scientific problems. Second, the rotations are a means of ensuring hands-on synthesis experience for all of our students early in the scientific careers. In addition students participate in seminars and journal clubs throughout their stay.

DESCRIPTION: The focus of this project will be to elucidate the mechanism of spectral tuning in the subgroup of short-wave visual pigments. First, the protonation state of the Schiff base nitrogen in the 11-cis retinal chromophore of the ultraviolet, human blue and bovine blue pigments will be determined by 15N-solid state magic angle spinning (MAS) NMR spectroscopy. Second the amino acid residues responsible for spectral tuning within the short-wave group will be identified. Finally the mechanism of spectral tuning in the short-wave subgroup will be compared to that of other subgroups of visual pigments. In particular, attention will be focused on the zebrafish blue pigment because it is a member of the middle-wavelength subgroup of pigments (based on its amino acid sequence) but has a maximum centered in the short wavelength range (419nm). Results from this work will shed light on the mechanisms of spectral tuning among the visual pigments to illuminate the mechanistic basis for the evolutionary development of human color vision.

DESCRIPTION (provided by applicant): The focus of this proposal is on the structure and function of the visual pigment rhodopsin. There are two Specific Aims: 1. The 3-dimensional crystallization and x-ray structure determination of recombinant wild-type and mutant rhodopsins. While the x-ray crystal structure of bovine rhodopsin was solved several years ago, the protein used in that study was isolated from native sources. No one has as yet crystallized rhodopsin from a recombinant source. This is an important goal because once achieved it will allow a high resolution structural analysis of rhodopsin mutants. Structural analysis of rhodopsin mutants is a crucial next step for a fundamental understanding of the functioning of this protein. The mutants of greatest interest in this work will be those found in the retinal diseases retinitis pigmentosa and congenital night blindness, specifically those that constitutively activate the protein. 2. To use transgenic Xenopus laevis to explore the molecular mechanisms of retinal disease arising from mutations in the genes for rhodopsin and its downstream signaling component transducin. The main focus of this Aim will be on the disease, retinitis pigmentosa.

DESCRIPTION (provided by applicant): The long-term goal of this work is to elucidate molecular mechanisms of vertebrate photoresponse signal transduction. In the current application we propose experiments to characterize the interaction of recoverin with rhodopsin kinase. This proposal is based upon preliminary experiments from the last grant period in which a previously unknown conformation of recoverin was formed when Ca2+-activated recoverin was bound to either the full RGS domain of rhodopsin kinase or an N-terminal peptide fragment of the kinase. Importantly, this new conformation appears to be present as a minor species (RvCaB) in an equilibrium mixture (RvCaA/RvCaB) formed upon the binding of Ca2+ to recoverin even in the absence of the kinase. Our approach will be to use a combination of biophysical techniques (primarily NMR spectroscopy) and biochemical analyses with purified components under in vitro conditions to unravel details of both the structure and mechanism of action for the interaction of these two proteins. There are three Specific Aims in this proposal: 1. To complete structural characterization of RvCaB;2. To probe through mutagenesis studies which amino acid residues in recovern contribue to stabilization of RvCaB;and 3. To determine in atomic detail the structure of the recoverin/rhodopsin kinase complex with initial focus on the RGS domain of the kinase. PUBLIC HEALTH RELEVANCE: This study will have an impact in three areas: 1. Understanding fundamental mechanisms of signaling molecules involved in protein-protein interactions;2. Understanding of aberrant recoverin expression for cancer-associated retinopathy and cell proliferation;and 3. Understanding general mechanisms of neuronal calcium sensor proteins.

Project Summary This Training Program provides graduate students with advanced education in the principles and practice of macromolecular chemistry, mechanism, and structure. All aspects of the program - formal course curriculum, laboratory rotations, informal specialized area-interest seminars, and intensive research in laboratories operating on the edge of discovery - are aimed at the question: how do biological macromolecules work? How do proteins, membranes, nucleic acids, and high-order complexes of these huge molecules use physical-chemical and structural principles to act in the enormous variety of contexts that underlie biological function? The Training Program provides support for selected students in the Graduate Program in Biochemistry and Biophysics at Brandeis University. This is a flexible PhD program designed for two broad types of students: those with strong quantitative backgrounds but who may have weaker prior training experience in biological chemistry, and those with more traditional training in biochemistry and cell biology. Our intention is to bring these two groups of students to the same end-point and to prepare them for careers in basic research. Currently, 34 students are enrolled in this Ph.D. program; the Training Program includes 25 participating faculty, from four departments, working in the following areas: macromolecular structure determination by x-ray crystallography and NMR, mechanistic enzymology, bioinorganic chemistry emphasizing epr and Mössbauer spectroscopy, membrane transport and ion channel mechanisms, single- molecule analysis, virology, chemical biology, computational biophysics, and protein evolution.

? DESCRIPTION (provided by applicant): This Training Program provides graduate students with advanced education in the principles and practice of macromolecular chemistry, mechanism, and structure. All aspects of the program - formal course curriculum, laboratory rotations, informal specialized area-interest seminars, and intensive research in laboratories operating on the edge of discovery - are aimed at the question: how do biological macromolecules work? How do proteins, membranes, nucleic acids, and high-order complexes of these huge molecules use physical-chemical and structural principles to act in the enormous variety of contexts that underlie biological function? The Training Program provides support for selected students in the Graduate Program in Biochemistry and Biophysics at Brandeis University. This is a flexible PhD program designed for two broad types of students: those with strong quantitative backgrounds but who may have weaker prior training experience in biological chemistry, and those with more traditional training in biochemistry and cell biology. Our intention is to bring these two groups of students to the same end-point and to prepare them for careers in basic research. Currently, 3 students (which will rise to 35 students in September '14) are enrolled in this Ph.D. program; the Training Program includes 26 participating faculty (in four departments) working in the following areas: macromolecular structure determination by x-ray crystallography and NMR, mechanistic enzymology, membrane transport and ion channel mechanisms, single-molecule analysis, high- resolution mass spectroscopy and proteomics, computational biophysics, protein evolution.