James Eric Gouaux
Oregon Health And Science University
Project start date: 2012-02-01
Project end date: 2016-01-31
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to James Eric Gouaux
Structure And Function Of Neurotransmitter Transporters
James Eric Gouaux, Professor
Oregon Health And Science University 3181 Sw Sam Jackson Pk Rd Portland, Or 972393098
Grant 5R01MH070039-05 from National Institute Of Mental Health IRG: BSCT
Abstract: Transporters for neutral and anionic amino acids play keys roles in human physiology and are active in organs as diverse as the kidney and brain. In the central nervous system, glutamate mediates the majority of fast excitatory signaling, a form of neuron-neuron communication that is essential to the development and maintenance of the nervous system. A fundamental component of glutamate-mediated signaling is the removal of glutamate from the synaptic cleft, following an excitatory stimulus, by sodium-dependent, high affinity glutamate transporters in neurons and other cells, such as glial cells. At the present time, there is no atomic resolution structural information on a glutamate transporter, which greatly hampers our understanding of their architecture and mechanism of action. In this application I propose to determine the structure of a bacterial protein that has significant sequence identity to the eukaryotic glutamate transporters, using x-ray crystallography. Furthermore, I plan to determine the functional behavior of the bacterial homolog, and to test structure-based mechanisms of transporter function. In addition, by using the crystal structure(s) of the bacterial protein as a guide, I will create a homology model of selected eukaryotic transporters and, together with previously determined structure and function information, this will place structure and function relationships of the eukaryotic transporters in an atomic-resolution, three-dimensional context. Taken together, the proposed research will substantially further our understanding of both eukaryotic and prokaryotic glutamate transporters, and, because they are related to transporters of dicarboxylic acids and of neutral amino acids, our knowledge of these secondary transporters will be increased as well. Lastly, because glutamatergic signaling is pervasive in the human nervous system, the structure of the bacterial transporter, along with the homology models of the eukaryotic transporters, should facilitate the design of new molecules that may have therapeutic potential.
Keywords: bacterial protein, glutamate transporter, protein structure function, structural biology, chemical model, electrophysiology, eukaryote, prokaryote, stoichiometry, X ray crystallography, Xenopus oocyte, protein purification, radiotracer, site directed mutagenesis
Project start date: 2004-07-01
Project end date: 2009-04-30
5R01MH070039-05 (2007): $335729
5R01MH070039-04 (2006): $344955
5R01MH070039-02 (2005): $362421
1R01MH070039-01A1 (2004): $350610
MOLECULAR FUNCTION OF RECEPTORS AND TRANSPORTERS AT CHEMICAL SYNAPSES
James Eric Gouaux
Cornell University Ithaca, Office Of Sponsored Programs, Ithaca, Ny 14850-2820
Grant 5P41RR015301-07_5366 from National Center For Research Resources
Abstract: This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project involves three different and unique integral membrane proteins (1) a bacterial transport protein (´X4´); (2) a eukaryotic P2X ion channel; and (3) a eukaryotic, full-length ionotropic glutamate receptor (iGluR). For all three projects, the scientific or technical purpose is the same to measure the highest resolution diffraction data sets possible. This presents a great challenge because all these crystals diffract weakly they are crystals of integral membrane proteins and a large fraction of the crystal volume is composed of aqueous solution and disordered detergent and lipid molecules. In particular, for the X4 project, we are aiming to collect high resolution native data on the NE-CAT microfocus beam line and multiwavelength data on the ID-24 beam line. For the P2X project, since we have low resolution phases, we are measuring native data sets (with and without agonists and modulators) on the microfocus beam line. In the case of the glutamate receptor project, we are using the known structures of the extracellular domains in molecular replacement to obtain phase information, and thus the emphasis is on measuring single wavelength data on a large number of agonist, antagonist and allosteric modulator complexes. The importance of all three projects is substantial. To the best of my knowledge, the folds for the X4 and P2X proteins are currently unknown, i.e. the determined folds are likely to be unique, and thus these initial structures will provide the architectural blueprint for two large and very important classes of membrane proteins. The detailed structural analysis, in addition, will provide deep and as-of-yet unknown insights into molecular mechanism. In the case of the glutamate receptor, the structure of the intact receptor will provide profound insights into the mechanism of ion channel gating and its modulation, as well as into ion permeation and block
Keywords: Agonist; CRISP; Carrier Proteins; Chemical Synapse; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Data Set; Dataset; Detergents; Disease; Disorder; External Domain; Extracellular Domain; Funding; Glutamate Receptor; Grant; Institution; Integral Membrane Protein; Intrinsic Membrane Protein; Investigators; Ion Channel; Ion Channel Gating; Ion Channel Gatings; Ionic Channels; Ions; Knowledge; Length; Lipids; Measures; Membrane Channels; Membrane Proteins; Membrane-Associated Proteins; Molecular; NIH; National Institutes of Health; National Institutes of Health (U.S.); P2X; P2X-receptor; Phase; Proteins; Receptor Protein; Research; Research Personnel; Research Resources; Researchers; Resolution; Resources; Solutions; Source; Structure; Surface Proteins; Transmembrane Protein; Transport Proteins; Transporter Protein; United States National Institutes of Health; aqueous; disease/disorder; gene product; insight; receptor; receptor function; structural biology
Project start date: 2009-04-01
Project end date: 2010-03-31
Budget start date: 1-APR-2009
Budget end date: 31-MAR-2010
5P41RR015301-07_5366 (2009): $38581
Training Program In Molecular Biophysics
James Eric Gouaux, Professor
Columbia University Health Sciences Columbia University Medical Center New York, Ny 100323702
Grant 5T32GM008281-17 from National Institute Of General Medical Sciences IRG: BRT
Abstract: This application seeks renewal of the highly-successful Biophysics Training Grant Program at Columbia University. Training for the students in the Program is performed in the Departments of Biochemistry and Molecular Biophysics and Microbiology on the Health Sciences campus and the Departments of Biological Sciences and Chemistry on the Morningside campus. The Program provides a rich and diverse environment in which students apply a host of biophysical approaches to the investigation of important biological problems. The Program emphasizes the application of state-of-the-art biophysical techniques to provide trenchant answers to detailed, specific questions while at the same time it endeavors to expose the trainees to a vast array of biological processes and to fertile areas of future research. Forming the underpinnings of the Training Program are exceptional laboratories housed in four departments and outstanding facilities located on the two campuses, weekly seminars in biophysics given by outstanding visiting speakers, an annual biophysics retreat where trainees present their research to all of the students and faculty in the Program, and a core, year-long biophysics course. Over the past 5 years since the last competitive renewal, the Biophysics Training Program has made important strides in several critical areas (i) the number of underrepresented minority students in the Program has been dramatically increased from one to five, (ii) several new trainers have been brought into the Program and the Program now has participation from two additional departments (Biological Sciences and Microbiology), and (iii) new procedures have been instituted for the appointment of trainees so as to assure that the most qualified students are selected for support. In the coming five year period, the Program will (i) reinforce ties between participating laboratories, departments and campuses, (ii) continue to improve the recruitment of all students, with an emphasis on underrepresented minority students, (iii) recruit additional talented faculty, and (iv) strengthen and update the pedagogical component of the program. The past historical successes and anticipated future achievements in the next funding period of the Biophysics Training Grant Program at Columbia will maintain this Program as a flagship for education and training of graduate students in New York City.
Project start date: 1988-09-30
Project end date: 2008-06-30
5T32GM008281-17 (2004): $317420
2T32GM008281-16 (2003): $311342
WATER SOLUBLE, MONOMERIC FORM OF LUKF FROM STAPHYLOCOCCUS AUREUS
James Eric Gouaux, Professor
Cornell University Ithaca Office Of Sponsored Programs Ithaca, Ny 148502820
Grant 3P41RR001646-16S10028 from National Center For Research Resources
Abstract: The research in my lab has focused on the structure determination of (i) bacterial channel-forming toxins, such as Staphylococcal leukocidin (LukF) and (ii) the ligand binding domain of ligand gated ion channels. Data from the F2 beamline has enabled us to solve the structure of the water-soluble form of LukF and the ligand binding domain of the GluR2, AMPA-selective glutamate receptor. Both structures were solved by MAD phasing from selenomethionine crystals at ~2.3 A resolution. Presently, both structures have been refined and we are currently in the process of manuscript preparation. From these structures we have furthered our understanding, at the molecular level, of the conformational changes that bacterial toxins undergo as a consequence of membrane binding and oligomerization and we have provided, for the first time, a view of the extracellular domain of a ligand gated ion channel.
Keywords: biological product, biomedical resource, protein, radiography, structural biology, bioimaging /biomedical imaging
Project start date: 1998-09-15
Project end date: 1999-08-14
STRUCTURE/FUNCTION OF S AUREUS A HEMOLYSIN
James Eric Gouaux, Professor
Biochem & Molecular Biophysicscolumbia University Health Sciences
columbia University Medical Center
new York, Ny 100323702
Grant 5R01GM051987-04 from National Institute Of General Medical Sciences IRG: BBCB
Abstract: The long range objectives of this research are to determine molecular mechanisms for the pathogenic action of cytolytic toxins and for the folding, assembly, pore-forming properties and regulation of oligomeric transmembrane pores in general. alpha-Hemolysin (alpha-He), one of the important virulence factors from Staphylococcus aureus, provides an excellent system for elucidating the molecular mechanisms for pore-forming toxins, protein insertion into a membrane, folding and assembly of a defined multimeric aggregate, and for formation of a transmembrane pore which is regulated by divalent cations. alpha-He is secreted from S. aureus as a water-soluble monomer (MW 33 kDa) and upon encountering an appropriate membrane environment, such as that provided by an erythrocyte, the monomer binds to the membrane, oligomerizes to form a hexamer and the hexamer then inserts through the bilayer to create a water-filled transmembrane pore. This initiates the leakage of ions and small molecules and finally, cell lysis occurs. The specific aims proposed in this application are to determine the 3-D structures of the monomer and the hexamer, to map the surfaces of the monomer and the hexamer that interact with the membrane, to elucidate the conformational changes that accompany the monomer to hexamer oligomerization, to identify the amino acids that line the interior of the pore, to locate the binding sites for inhibitory divalent cations and to formulate detailed molecular mechanisms to describe the assembly, function and regulation of alpha-He. These goals will be accomplished by determining 3-D structures of alpha-He using x-ray diffraction. Specifically, the structures of the detergent- solubilized hexamer will be solved using the multiple isomorphous replacement (MIR) technique, the structure of the monomer will be determined by either MIR or by molecular replacement (MR), and the structures of important site-directed mutants, protein-ligand complexes and alternative crystal forms will be determined by MR. Over 12 different crystal forms of the detergent-solubilized hexamer have been obtained, at least one of which is suitable for a high resolution structure determination, and two crystal forms of the monomer have been found. Interpretation of the structures will be aided by molecular graphics and computational techniques. On the basis of the proposed studies, general models will be developed for the assembly and function of oligomeric membrane proteins which will be relevant to other membrane proteins such as the acetylcholine receptor, the gap junction and the complement attack complex. In addition, an understanding of the structural mechanism of action of alpha-He may reveal new approaches for the treatment of bacterial infections. Furthermore, these structural studies will provide a starting point for the rational and systematic design of immunotoxins that could be used to selectively target and kill cancer cells
Keywords: conformation, hemolysin, membrane protein, pore forming protein, protein structure /function, staphylococcal exotoxin chemical aggregate, chemical model, deoxycholate, divalent cation, erythrocyte membrane, molecular rearrangement, monomer, mutant, phosphatidylcholine, phospholipid, protein structure, sialate, structural biology X ray crystallography, computer graphics /printing, computer program /software, computer simulation, crystallization, protein purification
Project start date: 1995-01-01
Project end date: 1998-12-31
5R01GM051987-04 (1998): $139019
Sponsored Links Excellgen http://Excellgen.com
7R01GM051987-03 (1997): $153814
5R01GM051987-02 (1996): $113143
3-D CRYSTALLIZATION OF MEMBRANE PROTEINS
James Eric Gouaux, Professor
Biochem & Molecular Biophysicscolumbia University Health Sciences
columbia University Medical Center
new York, Ny 100323702
Grant 5R01GM054112-05 from National Institute Of General Medical Sciences IRG: PB
Abstract: The long range objectives of this research are to determine efficient methods for the formation of well ordered, 3-D crystals of Kdp ATPase from E. coli, cytochrome bd oxidase from E. coli, cytochrome aa3 oxidase from Rhodobacter sphaeroides, and cytochrome bo3 oxidase and complex II from E. coli. The Kdp ATPase is a member of the P-type ATPase family which includes the Na+/K+ - and Ca2+-ATPases. This enzyme provides an excellent system for determining the molecular basis for overall subunit organization, the nature of the K+ channel and the relationship between the site of phosphorylation and the ion channel, for example. The cytochrome oxidases from E. coli and Rb.sphaeroides are well studied members from the oxidase family of respiratory proteins. The selected proteins have been thoroughly characterized using biochemical, biophysical and genetic approaches and they are amenable to detailed structure/function studies. Complex II catalyzes the conversion of succinate to fumarate and carries out a key process in the tricarboxylic acid cycle and in bacterial and mitochondrial respiratory chains. The amino acid sequence of the multisubunit protein is highly conserved from bacteria to mammals and the E. coli complex is an ideal model system. The specific aims are to test new detergents for crystallization, to prepare Fv fragments which bind to tertiary epitopes on the membrane protein complexes, to carry out crystallization trials on the membrane proteins, alone and in combination with Fv domains, and to analyze the resulting crystals by x-ray diffraction. The Ferguson-Miller, Gennis, Kranz and Gouaux labs are expert in membrane protein/detergent interactions, in membrane protein overexpression and purification, in monoclonal antibody production, screening and Fv construction and expression, and in crystallization of membrane proteins and in x-ray crystallography, respectively. These goals will be accomplished by determining the thermal stability and biological function of the membrane proteins in the presence of new detergents (Ferguson-Miller), by isolating and purifying Fv fragments which bind to tertiary epitopes using standard immunological and molecular biological techniques (Kranz & Gennis) and by subjecting the proteins to sparse and systemic crystallization matrices designed for membrane proteins (Gouaux). On the basis of the proposed studies, general methods will be investigated and developed for 3-D crystallization of membrane proteins; for the proteins under study, crystals which are suitable for a high resolution structure determination may be obtained. Extending the methods elucidated from this research to other systems will provide a structural understanding of such human conditions as cystic fibrosis, retinitis pigmentosa and multidrug resistance
Keywords: crystallization, membrane protein, method development, protein structure /function adenosinetriphosphatase, cytochrome oxidase, enzyme complex, structural biology, succinate dehydrogenase, thermostability X ray crystallography, detergent, monoclonal antibody, protein purification
Project start date: 1996-04-01
Project end date: 2000-03-31
5R01GM054112-05 (1999): $234685
5R01GM054112-03 (1997): $217251
5R01GM054112-04 (1998): $225798
1R01GM054112-01 (1996): $209408
STRUCTURE AND FUNCTION OF NEUROTRANSMITTER TRANSPORTERS
James Eric Gouaux, Senior Scientist
Oregon Health And Science University, 3181 Sw Sam Jackson Pk Rd, Portland, Or 97239-3098
Grant 5R37MH070039-08 from National Institute Of Mental Health
Abstract: The function of the human nervous system is dependent upon billions of nerve cells. A primary mechanism by which this vast number of cells communicates involves chemical synapses - specialized junctions where a small molecule neurotransmitter released by one cell binds to and activates receptors on an adjacent cell. In order for this cycle of neurotransmission to rapidly and faithfully repeat, the neurotransmitter must be cleared or removed from synapses. There are a large number of therapeutic drugs and a wide array of illicit compounds that modulate transporter function, including antidepressants, cocaine and amphetamines. At most chemical synapses, the removal of transmitter is accomplished by integral membrane proteins called transporters. In many cases, such as with glutamate, GABA, glycine and the biogenic amine transporters, the transporter proteins harness ion gradients established by ATP-dependent pumps to thermodynamically drive or pump transmitter into adjacent cells; these proteins are commonly referred to as ion-coupled symporters. In other cases, such as with the glutamate/cystine exchanger, the transporter protein obligatorily exchanges one substrate (glutamate) for another (cystine); these transporters are generally referred to as antiporters. Because both symporters and antiporters are highly hydrophobic integral membrane proteins, studies of their atomic structures by x-ray diffraction methods have proven difficult. I propose to carry out high resolution crystallographic studies of bacterial orthologs of neurotransmitter symporters and antiporters and, in combination with complimentary functional studies, develop molecular mechanisms for the function of these crucial transporter proteins. In addition, I propose to commence structural studies of eukaryotic neurotransmitter transporters facilitated by new technology developed in my laboratory. By accomplishing the proposed studies, we will not only learn how these proteins function, but we will also have the fundamental information for the development of new compounds to treat a wide range of neurological diseases and disorders. Integral membrane transport proteins remove chemical messengers or neurotransmitters from special junctions between nerve cells called synapses and their dysfunction is associated with numerous neurological diseases and disorders. The transport proteins are the targets of both therapeutic agents, such as antidepressants, and illicit substances, such as cocaine. The aims of the work proposed in this application are to determine the molecular structure and function of these important transporter proteins
Keywords: 4-Aminobutanoic Acid; 4-Aminobutyric Acid; 8-Azabicyclo(3.2.1)octane-2-carboxylic acid, 3-(benzoyloxy)-8-methyl-, methyl ester, (1R-(exo, exo))-; Abscission; Acids; Aminalon; Aminalone; Amino Acids; Aminoacetic Acid; Amphetamines; Anions; Antibodies; Antidepressant Agent; Antidepressant Drugs; Antidepressants; Antidepressive Agents; Antigenic Determinants; Architecture; Aspartate; Bacteria; Binding; Binding (Molecular Function); Binding Determinants; Binding Sites; Biogenic Amines; Butanoic acid, 4-amino-; Carrier Proteins; Cations; Cell Communication and Signaling; Cell Count; Cell Number; Cell Signaling; Cells; Chemical Synapse; Chemicals; Chromatography, Molecular Sieve; Co-Transporters; Cocaine; Combining Site; Complex; Cystine; Cytoplasm; Detection; Development; Disease; Disorder; Drugs; Dysfunction; Engineering / Architecture; Environment; Epitopes; Eukaryota; Eukaryote; Eukaryotic Cell; Excision; Extirpation; Family; Fluorescence; Functional disorder; GABA; Glutamates; Glycine; Human; Human, General; Integral Membrane Protein; Intracellular Communication and Signaling; Intrinsic Membrane Protein; Investigation; Ion Cotransport; Ions; Knowledge; L-Aspartate; L-Cystine; L-Glutamate; Laboratories; Learning; Life; Macromolecular Structure; Man (Taxonomy); Man, Modern; Medication; Membrane; Membrane Transport Proteins; Membrane Transporters; Methods; Molecular; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Sieve Chromatography; Molecular Stereochemistry; Molecular Structure; Movement; NRVS-SYS; Na element; Na+ element; Nerve Cells; Nerve Impulse Transmission; Nerve Transmission; Nerve Transmitter Substances; Nerve Unit; Nervous System; Nervous System Diseases; Nervous system structure; Neural Cell; Neurocyte; Neurologic Body System; Neurologic Disorders; Neurologic Organ System; Neurological Disorders; Neuronal Transmission; Neurons; Neurotransmitters; Nutrient; Ortholog; Orthologous Gene; Pharmaceutic Preparations; Pharmaceutical Preparations; Physiopathology; Polyamine Compound; Polyamines; Process; Proteins; Pump; Reactive Site; Receptor Protein; Removal; Research; Resolution; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Molecule; Site; Size Exclusion Chromatography; Sodium; Structure; Surgical Removal; Symport; Synapses; Synaptic; Testing; Therapeutic; Therapeutic Agents; Transmembrane Protein; Transport Proteins; Transporter Protein; Vestibular Apparatus; Vestibule; Work; aminoacid; antiport; antiporter; base; biological signal transduction; body movement; cellular development; conformation; conformational state; disease/disorder; drug/agent; eukaryotida; experiment; experimental research; experimental study; extracellular; frontier; gamma-Aminobutyric Acid; gene product; inhibitor; inhibitor/antagonist; member; membrane structure; mutant; nervous system disorder; neurological disease; neuronal; neurotransmission; neurotransmitter release; new technology; pathophysiology; protein function; public health relevance; receptor; research study; resection; small molecule; sodium ion; symporter; symporter (molecular); uptake
Relevance: Integral membrane transport proteins remove chemical messengers or neurotransmitters from special junctions between nerve cells called synapses and their dysfunction is associated with numerous neurological diseases and disorders. The transport proteins are the targets of both therapeutic agents, such as antidepressants, and illicit substances, such as cocaine. The aims of the work proposed in this application are to determine the molecular structure and function of these important transporter proteins
Project start date: 2003-12-01
Project end date: 2014-04-30
Budget start date: 1-MAY-2010
Budget end date: 30-APR-2011
PFA/PA: PA-07-070
5R37MH070039-08 (2010): $378265
2R37MH070039-07 (2009): $378265
CRYSTALLOGRAPHIC REFINEMENT OF A HEMOLYSIN, HEPTAMERIC TRANSMEMBRANE PORE
James Eric Gouaux, Professor
Mellon Pitts Corporation (mpc Corp) Pittsburgh, Pa 152133890
Grant 5P41RR006009-100136 from National Center For Research Resources
Abstract: Sets of six essential and three to four stimulating genes for DNA replication have been identified in 2 Baculoviruses AcMNPV and OpMNPV. In addition, both viruses have multiple origins of DNA replication that fall into groups of relative short origins with a Palindromic structure and larger origins that are more complex and not well-defined. Despite this wealth of information, limite information about the function of the encoded replication faction is available Therefore, these sets of proteins will be subjected to analyses for possible motifs, database searches with different parameters (PAM s), multiple sequence alignments, etc. Furthermore, searches for homology with DNA replication proteins of other viruses and searches with short conserved patterns will be carried out with the entire AcMNPV genome (134kb). Analyses of origin structures, search for commonalities among different origins of replication.
Keywords: biological product, biomedical resource, computer, genetics, skin
Project start date: 1999-08-01
Project end date: 2000-07-31
Sponsored Links Excellgen http://Excellgen.com
3D Structure And Function Of Ligand-Gated Ion Channels
James Eric Gouaux, Professor
Oregon Health And Science University 3181 Sw Sam Jackson Pk Rd Portland, Or 972393098
Grant 5R01NS038631-10 from National Institute Of Neurological Disorders And Stroke IRG: MDCN
Abstract: Ionotropic glutamate receptors mediate the majority of excitatory signal transmission in the mammalian nervous system and are essential to its normal development and function. There are three distinct and well defined receptor subtypes that are the AMPA, kainate and NMDA receptors. Ionotropic glutamate receptors work by coupling the binding of glutamate to gating, or opening, of a transmembrane ion channel. Because ionotropic glutamate receptors are linchpins of the human nervous system, they have been the target of a large number of pharmacological investigations and, therefore, a vast number of agonists, antagonists and allosteric effectors are known. Because in many cases these molecules elicit specific functional responses, they serve as excellent probes of the relationships between receptor structure and function. A substantial portion of the research proposed in this application is aimed at determining atomic resolution structures of the extracellular domains of AMPA and NMDA receptors, and understanding how agonists, antagonists and allosteric modulators exert their functional effects on the receptor. In addition, we have recently discovered bacterial homologs of the eukaryotic receptors and these molecules are promising species for over expression, crystallization and structure determination. Thus, a second element of the research proposed in this application is concentrated on determining the high resolution, three-dimensional structure of a bacterial receptor, in order to understand how ligand-binding is coupled to channel gating. A final element of the research proposed in this application is related to testing structure-based mechanistic hypotheses using site-directed mutagenesis and electrophysiology. By carrying out two electrode voltage clamp experiments in my laboratory, we will be able to evaluate the validity of specific mechanistic hypotheses. Taken together, the overall aims of the proposed research are to provide new and deep insights into the relationships between structure and function in ionotropic glutamate receptors. The resulting atomic structures and the mechanistic schemes will likely prove useful in the design of novel molecules that may have significant therapeutic value.
Keywords: glutamate receptor, ligand, membrane channel, protein structure function, structural biology, AMPA receptor, NMDA receptor, conformation, receptor binding, X ray crystallography, Xenopus oocyte, affinity chromatography, analytical ultracentrifugation, cell line, immunoaffinity chromatography, ion exchange chromatography, site directed mutagenesis, voltage /patch clamp, western blotting
Project start date: 1999-03-19
Project end date: 2009-02-28
5R01NS038631-10 (2007): $335250
5R01NS038631-09 (2006): $343784
5R01NS038631-07 (2005): $370873
2R01NS038631-06 (2004): $369963
3D STRUCTURE/FUNCTION OF LIGAND GATED ION CHANNELS
James Eric Gouaux, Professor
Columbia University Health Sciences Columbia University Medical Center New York, Ny 100323702
Grant 5R01NS038631-05 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: Ionotropic glutamate receptors (iGluRs) are the primary mediators of excitatory neuronal events. iGluRs form transmembrane, cation permeable channels that open in response to agonist binding. The three different iGluR subtypes are the AMPA, NMDA and kainate receptors, named according to their ligand sensitivity. iGluRs are linchpins of the nervous system and participate in learning and memory. Consistent with the central roles of iGluRs in the nervous system, disruption of receptor function has been implicated in brain disease and trauma ranging from epilepsy to stroke. Hyperactivation of iGluRs may lead neuronal death in degenerative conditions such as Huntington s disease, Alzheimer s disease, Parkinson s disease and amyotrophic lateral sclerosis. Therefore, an understanding of the structure of iGluRs may lead to the development of valuable therapeutic agents. The proposed research involves the elucidation of molecular mechanisms for the function of iGluRs based on high resolution, three-dimensional structures determined by x-ray crystallography and on selected biochemical and biophysical techniques. Mechanisms to describe (i) ligand specificity and affinity, (ii) channel selectivity, (iii) ligand-gated channel opening and closing and (v) channel desensitization will be developed. These hypotheses will be probed by site-directed mutagenesis, measurements of biological function, and evaluation of biophysical behavior and three-dimensional structure. Achievement of the long term objectives is founded on determining the structures of (i) the ligand binding domains, (ii) constructs containing the extracellular domains and (iii) species incorporating the extracellular and channel-forming domains of the AMPA, NMDA and kainate receptor subtypes. These structures will be determined in the presence of a wide range of agonists, antagonists and allosteric effectors. Methods for the folding of water-soluble and membrane proteins and strategies for the crystallization of membrane proteins will be developed. Since there is currently no structural information on an iGluR or a domain of an iGluR, the proposed studies will significantly increase our understanding of these essential ligand gated ion channels.
Keywords: X ray crystallography, conformation, glutamate receptor, ligand, membrane channel, protein structure function, biophysics, inclusion body, protein folding, receptor binding, structural biology, Escherichia coli, affinity chromatography, analytical ultracentrifugation, bioimaging /biomedical imaging, calorimetry, cell line, circular magnetic dichroism, computer data analysis, cryoscience, crystallization, immunoaffinity chromatography, ion exchange chromatography, mutant, site directed mutagenesis
Project start date: 1999-03-19
Project end date: 2004-02-29
5R01NS038631-05 (2003): $490732
5R01NS038631-03 (2001): $461488
5R01NS038631-02 (2000): $3
1R01NS038631-01 (1999): $3
James Eric Gouaux, Professor
Biochem & Molecular Biophysicscolumbia University Health Sciences
columbia University Medical Center
new York, Ny 100323702
Grant 1S10RR012848-01 from National Center For Research Resources IRG: ZRG3
Abstract: Eight investigators from four departments at Columbia University are applying for funding to purchase an XL-I analytical ultracentrifuge to enhance research capabilities in structural and molecular biology. All of the investigators study interactions of biological molecules. The analytical ultracentrifuge will be used to (i) determine sample homogeneity (degree of polydispersity) in the presence and absence of denaturants, (ii) measure sedimentation and diffusion coefficients, (iii) determine molecular masses, (iv) study oligomerization of macromolecules and measure dissociation constants, stoichiometries and thermodynamic parameters and (v) estimate molecular shape and changes in conformation upon ligand binding. Other applications, currently worked-out and those not yet developed, will be applied as necessary. A wide range of molecules will be investigated including ligand-gated ion channels, self-assembling transmembrane toxins, mesophilic and thermophilic ribonucleases, extracellular matrix proteins (integrin receptors and fibronectin type III domains), cell surface molecules of the immune system and their complexes with HIV proteins, giant invertebrate hemoglobins, Taxol-tubulin complexes, ATP binding cassette transporters and so called two-component signal transduction systems. A detailed structure has been defined for the administration, operation and maintenance of the XL-I facility and the fostering of an educated group of XL-I users. This involves (i) the creation of an XL-I Advisory committee, (ii) a method for allocation of instrument time for major and outside users, (iii) a strategy for continuing education in the field of analytical ultracentrifugation to keep users up-to-date, (iv) a plan for user training, (v) a basis for management of the XL-I facility over the short and long term, and (v)a vision of how we should apply current and as-yet-to-be developed programs for data analysis
Keywords: analytical ultracentrifugation, biomedical equipment purchase, nonclinical biomedical equipment structural biology
Project start date: 1998-03-01
Project end date: 1999-02-28
1S10RR012848-01 (1998): $271496
Sponsored Links Excellgen http://Excellgen.com
3D STRUCTURE AND FUNCTION OF LIGAND-GATED ION CHANNELS
James Eric Gouaux
Oregon Health And Science University, 3181 Sw Sam Jackson Pk Rd, Portland, Or 97239-3098
Grant 3R37NS038631-13S1 from National Institute Of Neurological Disorders And Stroke
Abstract: Chemical synapses are connections between neurons in the human central nervous system that are fundamental to its development and normal function. At these points of nerve cell - nerve cell interaction, a chemical transmitter released from one cell diffuses across a small space, called the synapse, to receptors located on the opposing cell, triggering the opening of ligand-gated ion channels and the activation of other cell surface receptors. At excitatory synapses, the opening or activation of ligand-gated ion channels causes the influx of primarily sodium and calcium ions, depolarizing the cell and injecting a potent calcium signal. The activation and modulation of ligand- gated ion channels is thus a crucial component of signal transmission in the central nervous system. In this grant application, I propose to study the atomic structure of ionotropic glutamate receptors and acid sensing ion channels, two ubiquitous and important classes of ligand-gated ion channel. In the proposed experiments on essential fragments of the receptors, as well as on the intact receptors, I will define the precise atomic structure of these ligand-gated ion channels and will determine mechanisms of ion channel activation, inhibition and modulation. The proposed studies will reveal basic principles of ligand-gated ion channel function and they will also provide crucial molecular blueprints to guide the development of new pharmacological agents to treat debilitating diseases of the human nervous system. Glutamate receptors and acid sensing ion channels are ligand-gated ion channel proteins that are essential to the normal development and function of the human nervous system and are integral to such fundamental processes as learning and memory. In this proposal we aim to determine their molecular structures and define principles for their activity, and thereby provide a foundation for development of new therapeutic agents to treat diseases of the human nervous system
Relevance: Glutamate receptors and acid sensing ion channels are ligand-gated ion channel proteins that are essential to the normal development and function of the human nervous system and are integral to such fundamental processes as learning and memory. In this proposal we aim to determine their molecular structures and define principles for their activity, and thereby provide a foundation for development of new therapeutic agents to treat diseases of the human nervous system
Project start date: 1999-03-19
Project end date: 2013-02-28
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
PFA/PA: PA-07-253
3R37NS038631-13S1 (2010): $147200
STRUCTURE AND FUNCTION OF S AUREUS ALPHA HEMOLYSIN
James Eric Gouaux, Professor
Biochem & Molecular Biophysicscolumbia University Health Sciences
columbia University Medical Center
new York, Ny 100323702
Grant 5R01GM051987-07 from National Institute Of General Medical Sciences IRG: BBCB
Abstract: Adapted from ) The long-term goal is to understand mechanisms for the pathogenic action of channel-forming bacterial toxins. Work focuses on the Staphylococcal toxins a-hemolysin, g-hemolysin and leukocidin. These toxins assemble from soluble monomers to inserted transmembrane oligomers that kill several cell types, including human erythrocytes and leukocytes. The crystal structure of fully assembled a-hemolysin has been determined to a resolution of 1.9 A in the current funding period. Crystal structures of intermediates on the assembly pathway will now be determined to elucidate the mechanisms of assembly in molecular detail. Specific structures to be determined include water-soluble and membrane-bound forms of toxin protomers, oligomers complexed with phospholipids, individual components and assembled forms of g-hemolysin and leukocidin toxins, and site-directed mutants of a-hemolysin. The kinetics of assembly under conditions similar to those under which the toxins were crystallized will be determined for the purpose of structure/function correlation
Keywords: Staphylococcus aureus, hemolysin, membrane protein, molecular assembly /self assembly, pore forming protein, protein structure function, staphylococcal exotoxin biophysics, conformation, lipid bilayer membrane, micelle, phospholipid, structural biology X ray crystallography, crystallization, site directed mutagenesis
Project start date: 1995-01-01
Project end date: 2003-06-30
5R01GM051987-07 (2001): $159707
5R01GM051987-06 (2000): $228179
2R01GM051987-05 (1999): $252864
James Eric Gouaux
Oregon Health And Science University
Project start date: 1999-03-19
Project end date: 2013-02-28
STRUCTURAL GENOMICS AND MEMBRANE PROTEINS
James Eric Gouaux
New York Structural Biology Center, 89 Convent Ave, New York, Ny 10027
Keywords: Membrane Proteins; Membrane-Associated Proteins; Surface Proteins; structural genomics
Project start date: 2010-09-30
Project end date: 2015-06-30
Budget start date: 30-SEP-2010
Budget end date: 30-JUN-2011
PFA/PA: RFA-GM-10-006
1U54GM095315-01_5810 (2010): $135743
James Eric Gouaux, Senior Scientist
Columbia University, New York, Ny 10027
Keywords: Membrane Proteins; Membrane-Associated Proteins; Surface Proteins; ing; structural genomics
Budget start date: 1-JUL-2009
Budget end date: 30-JUN-2010
PFA/PA: RFA-GM-05-002
5U54GM075026-05_0002 (2009): $198808
Sponsored Links Excellgen http://Excellgen.com
3D Structure And Function Of Ligand-Gated Ion Channels
James Eric Gouaux
Biochem & Molecular Biophysicscolumbia University Health Sciences
3R01NS038631-07S1 (2005): $43444
James Eric Gouaux, Professor
Columbia University New York, Ny 10027
Grant 1U54GM075026-010002 from National Institute Of General Medical Sciences IRG: ZGM1
Keywords: functional /structural genomics, genetic registry /resource /referral center, membrane protein, protein structure, proteomics, cooperative study, X ray crystallography
Project start date: 2005-07-01
Project end date: 2010-06-30
3D Structure And Function Of Ligand-Gated Ion Channels
James Eric Gouaux, Professor
Columbia University Health Sciences Columbia University Medical Center New York, Ny 100323702
Grant 3R01NS038631-06S1 from National Institute Of Neurological Disorders And Stroke IRG: MDCN
Project start date: 1999-03-19
Project end date: 2009-02-28
3R01NS038631-06S1 (2004): $32724
TRAINING PROGRAM IN MOLECULAR BIOPHYSICS
James Eric Gouaux, Professor
Columbia University Health Sciences Columbia University Medical Center New York, Ny 100323702
Grant 5T32GM008281-15 from National Institute Of General Medical Sciences IRG: ZGM1
Project start date: 1988-09-30
Project end date: 2003-06-30
5T32GM008281-15 (2002): $240302
CRYSTALLOGRAPHIC REFINEMENT OF A HEMOLYSIN, HEPTAMERIC TRANSMEMBRANE PORE
James Eric Gouaux, Professor
Mellon Pitts Corporation (mpc Corp) Pittsburgh, Pa 152133890
Grant 5P41RR006009-130136 from National Center For Research Resources
Keywords: biological product, biomedical resource, computer, genetics, skin
Project start date: 2002-08-01
Project end date: 2003-07-31
TRAINING PROGRAM IN MOLECULAR BIOPHYSICS
James Eric Gouaux, Professor
Biochem & Molecular Biophysicscolumbia University Health Sciences
columbia University Medical Center
new York, Ny 100323702
Grant 5T32GM008281-14 from National Institute Of General Medical Sciences IRG: ZGM1
Project start date: 1988-09-30
Project end date: 2003-06-30
5T32GM008281-14 (2001): $254450
CRYSTALLOGRAPHIC REFINEMENT OF A HEMOLYSIN, HEPTAMERIC TRANSMEMBRANE PORE
James Eric Gouaux, Professor
Mellon Pitts Corporation (mpc Corp) Pittsburgh, Pa 152133890
Grant 5P41RR006009-120136 from National Center For Research Resources
Keywords: biological product, biomedical resource, computer, genetics, skin
Project start date: 2001-08-01
Project end date: 2002-07-31
3D CRYSTALLIZATION OF MEMBRANE PROTEINS
James Eric Gouaux, Professor
Biochem & Molecular Biophysicscolumbia University Health Sciences
columbia University Medical Center
new York, Ny 100323702
Grant 3R01GM054112-04S1 from National Institute Of General Medical Sciences IRG: PB
Project start date: 1996-04-01
Project end date: 2000-03-31
3R01GM054112-04S1 (1998): $42886
CRYSTALLOGRAPHIC REFINEMENT AT HIGH RESOLUTION OF HEPTAMERIC TRANSMEMBRANE PORE
James Eric Gouaux, Professor
Mellon Pitts Corporation (mpc Corp) Pittsburgh, Pa 152133890
Grant 5P41RR006009-080074 from National Center For Research Resources
Keywords: bacteria, biomaterial, biomedical resource, biotechnology, communicable disease
Project start date: 1997-09-01
Project end date: 1998-08-31
Sponsored Links Excellgen http://Excellgen.com