William A Cramer
Purdue University West Lafayette
Project start date: 1987-04-01
Project end date: 2013-01-31
Sponsored Links Excellgen http://Excellgen.com
STRUCTURE-FUNCTION OF PHOTOSYNTHETIC CYTOCHROME COMPLEX
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM038323-07 from National Institute Of General Medical Sciences IRG: PB
Abstract: The structure and function of the energy-transducing cytochrome b6f complex and cytochrome b-559 intrinsic to the photosystem II reaction center will be studied. The topography of the cytochrome b6 and cytochrome f polypeptide has been determined. (i) The topographical characterization of the cytochrome b6f complex will be completed by extending these studies to the Rieske iron-sulfur protein and subunit IV. The orientation in the membrane will be determined using protease sensitivity of polar epitopes for peptide-directed antibodies, and immunogold labeling of thylakoid vesicles of different sidedness. (ii) Crystallization studies will be continued on the components of the b6f complex, cytochrome f that has been crystallized, co-crystallization of cytochrome f- plastocyanin, crystallization of the Rieske iron-sulfur protein, and of the cytochrome b6f complex using a detergent-solubilized monodisperse preparation of pure dimeric complex. (iii) The function of heme bn of cytochrome b6 in the trans-membrane photosystem I cyclic phosphorylation pathway and as a branch point in the electron transport chain connecting with electron donors on the n-side of the membrane will be assayed by measuring the dependence of ATP synthesis by a single light flash on the state of reduction of heme bn, as well as the dependence of phosphorylation on the inhibitor NQNO whose presence uniquely increases the amplitude of cytochrome b6 reduction by a flash. (iv) Inhibitor-resistant mutants to the inhibitor NQNO will be isolated from the cyanobacteria, Synechococcus sp. PCC 7002 and 7942, and their loci determined by sequencing petD and petB genes. (v) Site-directed mutagenesis of the Arg-86 and Thr-187 residues would address (a) the role of a unique charged residue located near heme bp and (b) the effect of an extra residue inserted between the two His residues in helix IV of cytochrome b6 on its spectral and redox properties and interheme electron transfer rate. (vi) The role of the cytochrome b-559 psbE gene product in binding manganese or calcium essential for oxygen evolution on the lumen (p-) side of the membrane will be tested by inserting a stop codon after the trans-membrane hydrophobic domain. The requirement for the b-559 psbF gene product will be tested by inserting a stop early (G1n-3) in its gene. (vii) A role for cytochrome b-559 as an alternative donor to the PSII reaction center in photoinhibition and stress response, and (viii) the role of thylakoid cytochromes in the pathway of desaturation of the fatty acids of the highly unsaturated thylakoid membrane will be studied.
Keywords: cytochrome b, photosystem, protein structure function, adenosine triphosphate, antibody, biological signal transduction, electron transport, hydrogen transport, iron sulfur protein, membrane permeability, membrane protein, oxidation /reduction, Cyanophyta, crystallization, flash photolysis, nucleic acid sequence, site directed mutagenesis, spinach
Project start date: 1987-04-01
Project end date: 1994-11-30
5R01GM038323-07 (1994): $172695
STRUCTURE/FUNCTION OF PHOTOSYNTHETIC CYTOCHROME COMPLEX
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM038323-06 from National Institute Of General Medical Sciences IRG: PB
Abstract: The structure and function of the energy-transducing cytochrome b6f complex and cytochrome b-559 intrinsic to the photosystem II reaction center will be studied. The topography of the cytochrome b6 and cytochrome f polypeptide has been determined. (i) The topographical characterization of the cytochrome b6f complex will be completed by extending these studies to the Rieske iron-sulfur protein and subunit IV. The orientation in the membrane will be determined using protease sensitivity of polar epitopes for peptide-directed antibodies, and immunogold labeling of thylakoid vesicles of different sidedness. (ii) Crystallization studies will be continued on the components of the b6f complex, cytochrome f that has been crystallized, co-crystallization of cytochrome f- plastocyanin, crystallization of the Rieske iron-sulfur protein, and of the cytochrome b6f complex using a detergent-solubilized monodisperse preparation of pure dimeric complex. (iii) The function of heme bn of cytochrome b6 in the trans-membrane photosystem I cyclic phosphorylation pathway and as a branch point in the electron transport chain connecting with electron donors on the n-side of the membrane will be assayed by measuring the dependence of ATP synthesis by a single light flash on the state of reduction of heme bn, as well as the dependence of phosphorylation on the inhibitor NQNO whose presence uniquely increases the amplitude of cytochrome b6 reduction by a flash. (iv) Inhibitor-resistant mutants to the inhibitor NQNO will be isolated from the cyanobacteria, Synechococcus sp. PCC 7002 and 7942, and their loci determined by sequencing petD and petB genes. (v) Site-directed mutagenesis of the Arg-86 and Thr-187 residues would address (a) the role of a unique charged residue located near heme bp and (b) the effect of an extra residue inserted between the two His residues in helix IV of cytochrome b6 on its spectral and redox properties and interheme electron transfer rate. (vi) The role of the cytochrome b-559 psbE gene product in binding manganese or calcium essential for oxygen evolution on the lumen (p-) side of the membrane will be tested by inserting a stop codon after the trans-membrane hydrophobic domain. The requirement for the b-559 psbF gene product will be tested by inserting a stop early (G1n-3) in its gene. (vii) A role for cytochrome b-559 as an alternative donor to the PSII reaction center in photoinhibition and stress response, and (viii) the role of thylakoid cytochromes in the pathway of desaturation of the fatty acids of the highly unsaturated thylakoid membrane will be studied.
Keywords: cytochrome b, photosystem, protein structure function, adenosine triphosphate, antibody, biological signal transduction, electron transport, hydrogen transport, iron sulfur protein, membrane permeability, membrane protein, oxidation /reduction, Cyanophyta, crystallization, flash photolysis, nucleic acid sequence, site directed mutagenesis, spinach
Project start date: 1987-04-01
Project end date: 1994-11-30
5R01GM038323-06 (1993): $181693
5R01GM038323-05 (1992): $157399
STRUCTURE/FUNCTION OF MEMBRANE BOUND CYTOCHROMES
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM038323-11 from National Institute Of General Medical Sciences IRG: BMT
Abstract: The highly conserved cytochrome b6f complex and the reaction center cytochrome b-559 of oxygenic photosynthesis will be used to study basic aspects of the structure and function of integral membrane cytochromes. Thr proposed studies are based on the following results from previous grant support (i) the solution of the crystal structure at 2.3A resolution of the active major extrinsic domain of cytochrome f, the first structure at the atomic level of a subunit of the cytochrome bc1 or b6f complexes. Cytochrome f has three unprecedented structural features for a c-type cytochrome of (a) a predominant beta-strand motif, (b) two distinguishable domains, and (c) the N-terminal alpha-amino group of Tyr-1 as the axial sixth heme ligand the latter result provided specific information about the sequence of events in the translocation of cyt f across the membrane, i.e. that processing must precede completion of heme coordination and final assembly. (ii) The purified b6f complex was characterized as a structural and functional dimer. (iii) The interhelix forces of the b6f complex were found to be relatively weak. (iv) The orientation of the beta-subunit of that heme cross-linked cytochrome b-559 was found to be parallel to that of the alpha, in agreement with (a) the prediction that it is a heterodimer, (b) the cis-positive rule for orientation of membrane proteins, and (c) the calculated contribution of the dipole potential of the alpha and beta helices to its very positive midpoint potential. It is proposed; (I) to use the cytochrome f structure and existing cross- linking information as the basis for intelligent site-directed mutagenesis to (a) determine the position of the docking site(s) for plastocyanin, and (b) to make a set of single histidine surface mutants that will be utilized, after modification wit Ru (bpy)2 adducts, to measure the reorganization energy, gamma, and optimum pathway associated with the intraprotein electron transfer. It is hypothesized that this transfer will have an unusually small gamma because the transfer to the plastocyanin acceptor is isopotential. (c) The consequences for assembly of cyt f and the subunits of the complex will be tested of inhibition of the processing and liberation of the Tyr-1 amino group. (II,a) The homodisperse Mr 230,000 b6f dimer will be used to crystallize this integral membrane protein complex. (b) The function of dimeric b6f complex will be tested in trans-membrane signaling involving the n-side kinase, as will (c) the role of the very highly conserved n-side extrinsic loops of cyt b6 in the docking of peripheral proteins. (iii) The effect of topographical inversion of the cyt b-559 heme will be examined by applying the cis- positive rule and reversing its trans-membrane distribution of positively charged amino acids.
Keywords: cytochrome, enzyme complex, photosynthetic reaction center, protein structure /function, adduct, chemical binding, electron transport, gene mutation, heme, histidine, molecular site, protein biosynthesis, protein kinase, Chlamydomonas, crystallization, laboratory rabbit, site directed mutagenesis
Project start date: 1987-04-01
Project end date: 1999-06-30
5R01GM038323-11 (1998): $216909
5R01GM038323-10 (1997): $208748
PROBLEMS IN MEMBRANE PROTEIN CRYSTALLOGRAPHY: HETERO-OLIGOMERIC CYTOCHROME B6F
William A Cramer
Purdue University West Lafayette, 155 S Grant Street, West Lafayette, In 47907-2114
Grant 5R01GM038323-22 from National Institute Of General Medical Sciences
Abstract: The hetero-oligomeric cytochrome b6f and bc1 complexes are in the center of the electron transfer chains in photosynthetic and respiratory energy transducing membranes. Such membranes contain the majority of the relatively few hetero-oligomeric integral membrane proteins that have been solved by X-ray crystallography to a resolution d 3.0 E. Studies on crystallization of the dimeric 220 kDa eight subunit integral b6f complex would analyze problems of proteolysis and lipid-protein interactions that are of general relevance to the crystallization of integral membrane proteins. Structure-function analysis would focus on the properties of a unique redox group, heme cn, and on the mechanism of transfer across the membrane of qui (ol) that carries the electrons and protons and is reduced by heme cn. Proposed studies (1) Crystal preparation; proteolysis. b6f complex cannot be isolated from transformable unicellular cyanobacteria because the b6f dimer is monomerized and rendered inactive and non-crystallizable upon extraction from the membrane. Successful crystallization of cyanobacterial b6f has utilized the filamentous M. laminosus, in which the extent of proteolysis is smaller. However, M. laminosus is not transformable. Therefore, the cyanobacterial source of b6f complex will be changed to the filamentous Nostoc (Anabaena) sp. PCC 7120, from which active and crystallizable complex has been obtained. Because this kind of proteolysis problem frequently hinders efforts to crystallize membrane proteins, the critical protease(s) in the unicellular cyanobacteria would be identified by mass spectroscopic and proteomic analysis. (2) Function of phospholipids. The function of intra-protein lipids has been analyzed in only a few multi-subunit membrane proteins. Our novel lipid augmentation procedure resulted in a major increase in the rate of crystallization and improvement in crystal quality. The properties of crystals of the plant thylakoid membrane b6f complex, which contains a ninth (FNR) subunit and whose crystallization depends uniquely on a different (anionic DOPG) lipid, is under study, as is the dependence of electron transfer activity and rate of crystallization on the nature of added lipids. (3) Functions of heme cn; evolution of b6f complex. The function of the unique heme cn, not found in ubiqui-containing cyt bc1 complexes, will be studied by site-directed mutagenesis in Nostoc and, through structure-function analysis, in firmicutes such as Bacillus subtilis that are phylogenetically close to cyanobacteria. His-tagged, promoter-augmented firmicute "qcr" complex will be purified and screened for crystallization and electron transfer reactions with menaqui. (4) Qui transfer though the narrow p-side portal. Electrons and protons are carried across an inter-monomer qui exchange cavity in bc1 and b6f complexes by lipophilic ubi- and plastoqui (PQ). The mechanisms by which PQ/PQH2 finds, enters, and exits a narrow 11 x 12 E p-side portal will be studied through mutagenesis of portal residues and computational analysis of the portal force field. Some of the biomedically relevant aspects of these studies are that they are directed toward an understanding of the detailed internal structure of the proteins that mediate all traffic, including nutrients and drugs, across biological membranes. Via the membrane, the set of energy-transducing proteins determines the level of energy and its regulation in the human cell
Keywords: 1, 2, 3-Propanetriol; 1, 2, 3-Trihydroxypropane; 1, 2-diacylglycerol; 2, 5-Cyclohexadiene-1, 4-dione, 2, 3-dimethyl-5-(3, 7, 11, 15, 19, 23, 27, 31, 35-nonamethyl-2, 6, 10, 14, 18, 22, 26, 30, 34-hexatriacontanonaenyl)-, (all-E)-; 2-Hydroxy-N, N, N-trimethylethanaminium; 4-hydroxyquinoline; 4H-1-Benzopyran-4-one, 2-(4, 6-dimethoxy-3, 5, 11-trimethyl-7, 9, 11-tridecatrienyl)-8-hydroxy-5, 7-dimethoxy-3-methyl-; Abbreviations; Acceleration; Active Oxygen; Adrenodoxin Reductase; Algae; Algae, Blue-Green; Aminoethanols; Anabaena; Antibody Fragments; Antiviral Agents; Antiviral Drugs; Antivirals; Appearance; Bacillus subtilis; Benzodiones; Benzoquis; Binding; Binding (Molecular Function); Binding Sites; Biological; Blue-Green Bacteria; CYP; Cardiolipins; Cells; Chlorophyll; Chloroplasts; Choline; Choline Glycerophospholipids; Choline Phosphoglycerides; Coenzyme Q; Combining Site; Complex; Computer Analysis; Computing Methodologies; Coupling; Crude Extracts; Crystallization; Crystallographies; Crystallography; Crystallography, X-Ray; Crystallography, X-Ray Diffraction; Crystallography, X-Ray/Neutron; Crystallography, Xray; Cyanobacterium; Cyanophyceae; Cyanophyta; Cytochrome b(6)-f Complex; Cytochrome b6-f; Cytochrome b6f Complex; Cytochromes; DAG; DGDG; Data; Dependence; Diacylglycerols; Diglycerides; Drugs; EPR spectroscopy; Electron Paramagnetic Resonance; Electron Spin Resonance; Electron Spin Resonance Spectroscopy; Electron Transport; Electrons; Electrophoresis; Esteroproteases; Ethanaminium, 2-hydroxy-N, N, N-trimethyl-; Ethanolamines; Evolution; Extracts, Complex; Fe element; Ferrate(2-), (7, 12-diethenyl-3, 8, 13, 17-tetramethyl-21H, 23H-porphine-2, 18-dipropanoato(4-)-N21, N22, N23, N24)-, dihydrogen, (SP-4-2)-; Ferredoxin-NADP Reductase; Ferredoxin[{..}]NADP+ oxidoreductase; Ferroprotoporphyrin; Fractionation, Electrophoretic; G Protein-Complex Receptor; G Protein-Coupled Receptor Genes; G-Protein-Coupled Receptors; GPCR; GPR; Genetics-Mutagenesis; Genome; Genomics; Glycerin; Glycerol; Goals; H(+) Pump; H+ element; Harvest; Heme; Heme Group; Heme b; Human; Human, General; Hydrogen Ions; Hydroquis; Immunoblotting; Immunoglobulin Fragments; Integral Membrane Protein; Intrinsic Membrane Protein; Iron; Iron-Sulfur Protein Reductase; Iron-Sulfur Proteins; Lecithin; Ligands; Link; Lipids; Man (Taxonomy); Man, Modern; Measures; Mediating; Medication; Membrane; Membrane Proteins; Membrane-Associated Proteins; Menaqui; Molecular Biology, Mutagenesis; Molecular Interaction; Movement; Mutagenesis; Mutagenesis, Site-Directed; NADPH-Ferredoxin Reductase; NQ-N-oxide; NQNO; Na element; Nature; Negative Beta Particle; Negatrons; Nostoc; Nutrient; Oxidation-Reduction; Oxides; Oxygen Radicals; PCR; Paramagnetic Resonance; Pathway interactions; Peptidases; Peptide Hydrolases; Pharmaceutic Preparations; Pharmaceutical Preparations; Phosphatides; Phosphatidylcholines; Phospholipids; Physiologic; Physiological; Plant Components; Plant Structures; Plants; Plants, General; Plastocyanin; Plastocyanine; Plastoqui; Plastoqui-9; Polymerase Chain Reaction; Preparation; Pro-Oxidants; Procedures; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Property; Property, LOINC Axis 2; Prosthesis; Prosthetic device; Prosthetics; Proteases; Protein Cleavage; Proteinases; Proteins; Proteolysis; Proteolytic Enzymes; Proteomics; Protoheme; Protoheme IX; Proton Pump; Protons; Quinols; Qui Compound; Qui Reductases; Quis; Reaction; Reactive Oxygen Species; Reactive Site; Redox; Regulation; Resolution; Rhinovirus; Role; S element; Screening procedure; Side; Single Crystal Diffraction; Site-Directed Mutagenesis; Site-Specific Mutagenesis; Sodium; Source; Spectroscopy, ESR; Structure; Sulfur; Surface Proteins; Survey Instrument; Surveys; System; System, LOINC Axis 4; Targeted DNA Modification; Targeted Modification; Thylakoid Membranes; Transmembrane Protein; Trees; Ubiqui; Vitamin K 2; Vitamin K Qui; Vitamin K2; X Ray Crystallographies; X-Ray Crystallography; Yeasts; ing; analog; body movement; comparative; computational analysis; computational methodology; computational methods; computer methods; cytochrome b6f; diacyl glyceride di Gal; diacylglycerol; digalactosyldiacylglycerol; diglyceride; dimer; drug/agent; electron paramagnetic resonance spectroscopy; electron transfer; experience; ferroheme; gene product; genome sequencing; his-PG; improved; in vivo; inhibitor; inhibitor/antagonist; insight; membrane structure; monomer; nonyl-4-hydroxyquinoline-N-oxide; novel; oxidation reduction reaction; p-Dihydroxybenzenes; para-Dihydroxybenzenes; pathway; photosystem; polyacrylamide; polypeptide; progesterone 11-hemisuccinate-(2-iodohistamine); protein structure; respiratory; rhomboid; screening; screenings; social role; stigmatellin; trafficking; ubiquinol
Relevance: Narrative Some of the biomedically relevant aspects of these studies are that they are directed toward an understanding of the detailed internal structure of the proteins that mediate all traffic, including nutrients and drugs, across biological membranes. Via the membrane, the set of energy-transducing proteins determines the level of energy and its regulation in the human cell
Project start date: 1987-04-01
Project end date: 2013-01-31
Budget start date: 1-FEB-2010
Budget end date: 31-JAN-2011
PFA/PA: PA-07-070
5R01GM038323-22 (2010): $330659
STRUCTURE/FUNCTION OF THE CYTOCHROME B6F COMPLEX
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM038323-16 from National Institute Of General Medical Sciences IRG: PB
Abstract: The cytochrome b6f complex has many structure-function homologies with the cytochrome bc1 complex of the mitochondrial respiratory chain. Much of this arises from the identity of cytochrome b, especially marked on the p-side of the membrane. From the high resolution structures of cytochrome f and the Rieske (ISP) protein, it is known that the peripheral domains differ significantly between bc1 and b6f. The further the domain from the membrane, the greater the difference. Thus, cytochromes f and c1 are completely different proteins and have been from their evolutionary origin, and the domain of the ISP distal to membrane folds differently in the b6f and bc1 complexes. However, the ISP domain containing the iron-sulfur cluster that must come close to the membrane has a conserved fold. Thus, detailed information on structure-function for the b6f complex complements that obtained for the bc1 and seems likely to provide differences in detailed functional mechanisms. A high resolution structure of 3-D crystals of the b6f complex from the thermophilic cy- anobacterium, M. laminosus, would provide this information. The crystals presently show ordered diffraction to 10 Angstrom units. When diffraction (less than or equal to 3 Angstrom units) appropriate for a structure analysis is obtained, the solution of the structure will be expedited by the fact that we have high resolution structures (less than 2.0 Angstrom units) for the p- side of the complex, cytochrome f and the iron-sulfur protein, 40 percent of the total mass of the complex. Issues of function to be determined by the structure include the position and function of the n-side quinone, the pathway of trans-membrane H+ transfer, and the role of intramembrane bound water. From the existing p- side structures, the local mobility of the ISP will be analyzed in vivo, in situ, and in vitro. The basis for the non-concerted reduction of high and low potential chains will be studied. Catalysis of electron transfer by conserved aromats in the Rieske protein, and in cytf where they shield the heme, will be tested my mutagenesis. The role of the water chain in the coupling of intraprotein electron and proton transfer will be examined by stopped flow kinetics in D2O, together with the properties of the bound H2O in cytf by FTIR. With a ruthenium derivative of cytf, "photo-cytf", light-induced intraprotein electron transfer rates, optimum paths of intraprotein electron transfer, and reorganization energy will be measured. Ruthenated cytf will also be used to investigate intraprotein protonation- deprotonation at specific carboxylates, associated with coupled electron and proton transfer.
Keywords: cytochrome, enzyme complex, protein structure function, cytochrome b, electron transport, iron sulfur protein, membrane protein, oxidation reduction reaction, photochemistry, protonation, X ray crystallography, crystallization, laboratory rabbit, photosynthetic bacteria, ruthenium, stop flow technique
Project start date: 1987-04-01
Project end date: 2003-06-30
5R01GM038323-16 (2003): $147700
5R01GM038323-15 (2002): $252164
5R01GM038323-14 (2001): $245108
5R01GM038323-13 (2000): $240244
Sponsored Links Excellgen http://Excellgen.com
STRUCTURE And FUNCTION OF THE PHOTOSYNTHETIC CYTOCHROME B6
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM038323-03 from National Institute Of General Medical Sciences IRG: PB
Abstract: The cytochrome b6-f and b-c1 complex of chloroplasts, mitochondria, and photosynthetic bacteria is an almost universal trans-membrane electron transport and H+ translocating membrane protein complex in energy-transducing membranes. The amino acid sequence and hydropathy functions of the heme binding domain (first 200 residues) of cytochrome b6 is particularly highly conserved. The topography of cytochrome b6 in the membrane will be studied by (i) determining conditions under which the isolated complex is a monomer or dimer; (ii) purifying the cytochrome from the rest of the complex; (iii) incorporating the cytochrome into liposomes and testing the exposure of predicted surface-exposed peptides using (a) antibodies against these peptides, (b) proteolysis by trypsin at a predicted labile site, (c) azido-labaled photoreactive phospholipids, and (d) DCCD as a probe of predicted acidic residues in helix IV. If DCCD is found to react with one of these acidic residues, and it is conserved in cyanobacteria, it would be changed by directed mutagenesis. Crystallization wells are set up of pure active b6-f prepared by a method allowing 5-10 times more yield of complex from the starting material. Studies of the function of cytochrome b6 is thylakoid membranes, reconstituted liposomes, and membranes of cyanobacteria would determine (i) whether heme bp or bn is reduced by a single flash in the presence of NQNO, (ii) whether b6 is reduced by quinol added to liposomes containing b6-f, and whether the Em values of the two hemes can be distinguished in the reconstituted system. (iii) The dependence of b6 flash reduction of the DeltaMuH+ would be determined. Experiments (i) - (iii) should tell whether a Q cycle can be operating. (iv) The questions of whether the msec DeltaPsi that occurs under reducing conditions actually arises from H+ pumping will be tested using an internal pH indicator. (v) The molecular basis of the heterogeneity of the hemes will be tested by deleting Thr-184. (vi) The sites of resistance to inhibitors in the cyanobacterial cyt b6 would be determined by DNA sequencing of selected mutants. These experiments should provide details about the structure of this protein in the membrane, and also determine whether it has a trans-membrane function in non-cyclic or cyclic electron transport.
Keywords: HEMOPROTEINS, CYTOCHROME B, PHOTOCHEMISTRY, PHOTOSYNTHESIS, protein structure, BIOLOGICAL TRANSPORT, MEMBRANE MODELS, LIPOSOMES, BIOLOGICAL TRANSPORT, MEMBRANE PERMEABILITY AND TRANSPORT, CHEMICAL TRANSFER, ELECTRON TRANSPORT, GENETICS, MUTATION, IMMUNOLOGY, ANTIBODIES, NUCLEIC ACIDS STRUCTURE, NUCLEOSIDES (TIDES) SEQUENCE, OXIDATION-REDUCTION, PHOSPHOLIPIDS, PROTEINS, TRANSPORT PROTEINS, ION PUMPS, HYDROGEN (PROTON) PUMPS, QUINONES, biological signal transduction, PHYSICAL SEPARATION, CHROMATOGRAPHY, HIGH PERFORMANCE LIQUID CHROMATOGRAPHY, PHYSICAL SEPARATION, ELECTROPHORESIS, GEL, PLANTS, CROPS AND FOODS, VEGETABLES, SPINACH, PLANTS, THALLOPHYTES, ALGAE, CYANOPHYTA, RADIATION EFFECTS, FLASH PHOTOLYSIS
Project start date: 1987-04-01
Project end date: 1990-11-30
Structural Analysis Of Integral Membrane Proteins
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM038323-20 from National Institute Of General Medical Sciences IRG: PB
Abstract: Structure-function studies on the hetero-oligomeric cytochrome b6 f complex have led to successful crystallization and initial characterization of well-diffracting crystals of the complex from the thermophilic cyanobacterium, Mastigocladus laminosus, whose structure analysis is underway. They have also led to a new "lipid enrichment" methodology for the crystallization of such integral membrane proteins, considering the protein in detergent micelles as a protein-detergent-lipid complex. The presence and the masses of eight subunits of the complex from M. laminosus and nine subunits from the higher plant complex, including the newly discovered FNR component, have been characterized by mass spectroscopy. The MS analysis removed uncertainty as to exactly how many small polypeptides are present in the complex. A unique aspect of the role of lipid in the function and structural stabilization of the complex arose through the discovery of one equivalent of lipid-like beta-carotene and chlorophyll alpha in the complex. The lipid parameters of the "enrichment methodology will be examined. It is proposed to test the applicability of the new lipid enrichment methodology to the b6f complex in a transformable cyanobacterium and four other unrelated integral membrane protein systems. The potential application of this methodology is important for studies on integral membrane proteins, as there are at present only 23 solved independent IMP structures,and 56 in total. This creates a limiting step in proteomic analysis since 20-30 % of gene products are membrane proteins. The function of the Rieske iron-sulfur protein (ISP), which has been proposed to undergo a unique large amplitude rotation-translation in its electron transfer function, was analyzed through (i) the viscosity dependence of the electron transfer function, (ii) stopped-flow kinetics of diffusion limited delectron transfer in solution, and (iii) mutagenesis analysis of the linker region, the "hinge of the proposed rotation. The ISP motion is constrained in vivo. The motion does not depend sensitively on the structure of the linker region, but the linker has a minimum length of 7 residues. It is proposed to directly measure the kinetics of motion of the ISP soluble domain triggered by electron transfer.
Keywords: cytochrome, enzyme complex, membrane protein, protein structure function, cytochrome b, electron transport, iron sulfur protein, oxidation reduction reaction, photochemistry, protonation, X ray crystallography, crystallization, fluorescence resonance energy transfer, laboratory rabbit, photosynthetic bacteria, ruthenium, stop flow technique
Project start date: 1987-04-01
Project end date: 2007-08-02
5R01GM038323-20 (2006): $312394
5R01GM038323-19 (2005): $316909
5R01GM038323-18 (2004): $315189
STRUCTURE/FUNCTION OF MEMBRANE BOUND CYTOCHROMES
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM038323-09 from National Institute Of General Medical Sciences IRG: BMT
Project start date: 1987-04-01
Project end date: 1998-11-30
5R01GM038323-09 (1996): $213891
Grants awarded to William A Cramer
IMPROVING RATE/QUALITY LIMITATIONS IN MEMBRANE PROTEIN STRUCTURE DETERMINATION
William A Cramer
Purdue University West Lafayette, 155 S Grant Street, West Lafayette, In 47907-2114
Grant 5P50GM088499-02 from National Institute Of General Medical Sciences
Abstract: This proposal is directed to solving the problem of the slow rate of generation of high resolution structures of integral membrane proteins that can be useful as drug targets and in medicine. The group combines established expertise in membrane protein structural biology, which has led to 16 structures deposited in the Protein Data Bank, as well as cutting-edge advances in methods development. Experience has shown that the paucity of high resolution membrane protein structure is a consequence of several recognizable bottlenecks in the structure determination pipeline, including (i) co-expression of the subunits of hetero-subunit prokaryotic proteins; (ii) expression of eukaryotic membrane proteins; (iii) determination of stability and functionality; (iv) recognition of (a) the role of lipid in the structure and in the rate of crystallization and (b) proteolytic degradation that can preclude purification of active protein. A suite of new expression approaches will be developed targeting (i) and (ii), with complementary analysis approaches in (ii). Problems (i-iv) lead to uncertainties in the formation of well-diffracting crystals, which can take weeks or months to generate. Early detection of crystal growth would greatly decrease the time required for crystal screening and allow access to a greater phase-space of crystallization conditions. We describe an innovative methodology, SONICC (second order nonlinear optical imaging of chiral crystals), for sensitive and selective detection of incipient protein crystals as small as 100 nm, prepared in screening platforms with volumes as low as 0.5 picoliter. Its ability to distinguish membrane protein crystals of the maltose transporter and cytochrome b6f complex has been confirmed. This approach will greatly speed generation of diffraction-quality 3-dimensional and 2-dimensional crystals, generated both in surfo and in meso. A range of integral membrane protein targets has been selected, with an emphasis on ABC and hetero-oligomeric proteins; (i) ABC maltose and ribose transporters, ABCBl, ABCG2, and Sur-Kir6.2; (ii) non-ABC twin-arginine translocase; Kdp-ATPase; NADH dehydrogenase. Integral membrane proteins control all traffic between cell compartments, assembly of the membranes themselves, the supply of nutrients to the cell, its energization, and communication of the cell with the extracellular environment. The atomic structures of membrane proteins is crucial for an understanding of the molecular basis of human diseases and the development of drugs to combat them or ameliorate their consequences
Project start date: 2009-09-30
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: RFA-RM-08-019
5P50GM088499-02 (2010): $392688
1P50GM088499-01 (2009): $656767
STRUCTURE/FUNCTION OF THE COLICIN E1 CHANNEL
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM018457-23 from National Institute Of General Medical Sciences IRG: BBCB
Abstract: The structure of the colicin El channel domain is studied by (i) spectroscopic (FTIR and CD) determination of the secondary structure of the channel peptide incorporated into artificial membranes; (ii) site-directed mutagenesis of the prominent hydrophobic sequence near the COOH-terminus to determine the limits of the membrane-spanning domain, the residues that interact with the polar channel interior, and the regions of local hydrophobicity. Mutagenesis will also be used to test two other highly amphipathic nineteen residue segments that are proposed to be membrane spanning alpha=helices, and to generate Cys-containing mutants that can be labeled with heavy atoms for the phase analysis of well-diffracting crystals ([2.5 Alpha resolution_ of tryptic and thermolytic channel peptides. Initial experiments on preparation of heavy atom derivatives will use the one cysteine in the channel peptides, Cys-505, that has been previously modified without loss of function. (iii) The minimum fully active peptide will be determined, as will the monomeric or multimeric state of the functional channel. Studies on channel formation and the associated translational dynamics would focus on (iv) tryptophan residues in site-directed mutants containing a single tryptophan; (v) the using directed mutagenesis to test residues implicated in binding to the membrane surface, using directed mutagenesis to test residues implicated from the structure of the soluble colicin Alpha peptide; (vi) the dynamics and the time course of voltage-dependent insertion into the membrane bilayer, as well as pH-dependent reversible insertion and extrusion. The segments of the channel peptide that insert into the bilayer in the presence of the segments and individual amino acids by lipophilic and phospholipid photoaffinity probes. Initial studies have identified one such segment. (vii) The colicin E3-btuB and El colicin and receptor binding domains defined, the role of receptor examined in the unfolding of the imported colicin, and the mechanism of receptor into liposomes. The structure of the colicin channel and the mechanism and dynamics of its import bear on the problems of protein import and secretion, translocation of toxin molecules, eukaryotic voltage-dependent and ligand-gated channels, and receptor function.
Keywords: colicine, liposome, membrane channel, protein structure function, chemical binding, lipid bilayer membrane, membrane permeability, membrane potential, membrane structure, mutation, phospholipid, receptor, receptor binding, vitamin B12, voltage gated channel, Escherichia coli, X ray crystallography, affinity labeling, circular dichroism, fluorescence spectrometry, hydropathy, infrared spectrometry, interferometry, laboratory rabbit, plasmid, site directed mutagenesis, solution
Project start date: 1974-09-01
Project end date: 1995-03-31
5R01GM018457-23 (1994): $182930
STRUCTURE And FUNCTION OF THE COLICIN E1 CHANNEL
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM018457-22 from National Institute Of General Medical Sciences IRG: BBCB
Abstract: The structure of the colicin El channel domain is studied by (i) spectroscopic (FTIR and CD) determination of the secondary structure of the channel peptide incorporated into artificial membranes; (ii) site-directed mutagenesis of the prominent hydrophobic sequence near the COOH-terminus to determine the limits of the membrane-spanning domain, the residues that interact with the polar channel interior, and the regions of local hydrophobicity. Mutagenesis will also be used to test two other highly amphipathic nineteen residue segments that are proposed to be membrane spanning alpha=helices, and to generate Cys-containing mutants that can be labeled with heavy atoms for the phase analysis of well-diffracting crystals ([2.5 Alpha resolution_ of tryptic and thermolytic channel peptides. Initial experiments on preparation of heavy atom derivatives will use the one cysteine in the channel peptides, Cys-505, that has been previously modified without loss of function. (iii) The minimum fully active peptide will be determined, as will the monomeric or multimeric state of the functional channel. Studies on channel formation and the associated translational dynamics would focus on (iv) tryptophan residues in site-directed mutants containing a single tryptophan; (v) the using directed mutagenesis to test residues implicated in binding to the membrane surface, using directed mutagenesis to test residues implicated from the structure of the soluble colicin Alpha peptide; (vi) the dynamics and the time course of voltage-dependent insertion into the membrane bilayer, as well as pH-dependent reversible insertion and extrusion. The segments of the channel peptide that insert into the bilayer in the presence of the segments and individual amino acids by lipophilic and phospholipid photoaffinity probes. Initial studies have identified one such segment. (vii) The colicin E3-btuB and El colicin and receptor binding domains defined, the role of receptor examined in the unfolding of the imported colicin, and the mechanism of receptor into liposomes. The structure of the colicin channel and the mechanism and dynamics of its import bear on the problems of protein import and secretion, translocation of toxin molecules, eukaryotic voltage-dependent and ligand-gated channels, and receptor function.
Keywords: colicine, liposome, membrane channel, protein structure function, chemical binding, lipid bilayer membrane, membrane permeability, membrane potential, membrane structure, mutation, phospholipid, receptor, receptor binding, vitamin B12, voltage gated channel, Escherichia coli, X ray crystallography, affinity labeling, circular dichroism, fluorescence spectrometry, hydropathy, infrared spectrometry, interferometry, laboratory rabbit, plasmid, site directed mutagenesis, solution
Project start date: 1974-09-01
Project end date: 1995-03-31
5R01GM018457-22 (1993): $177304
5R01GM018457-21 (1992): $171003
OPTICAL BIOSENSOR TO STUDY MACROMOLECULE INTERACTIONS
William A Cramer, Henry Koffler Professor
Biological Sciencespurdue University West Lafayette
302 Wood St
west Lafayette, In 479072108
Grant 1S10RR013662-01 from National Center For Research Resources IRG: ZRG2
Abstract: The properties and capabilities are described of a biosensor instrument that will be used to define interactions between macromolecules. Its acquisition would establish one of an array of instruments to be used for this purpose. The proposal is submitted by seven investigators involved in studies on protein-protein, ligand-protein, peptide-protein, or protein-membrane interactions, who have expertise in high resolution protein structure studies, and in areas of biochemistry, biophysics, and molecular genetics. The biosensor instrumentation would complement and extend the information obtained from the high resolution structure studies by providing information on protein function and mechanism. A wide range of problems will be addressed. These include (i) the nature of the membrane binding affinity of "membrane-active" proteins; (ii) the interaction between colicins and their cellular receptor; (iii) the binding affinity of antibodies to human rhinovirus; (iv) receptor interactions of enterotoxins; (v) altered binding of NAD+ by Asian alcohol dehydrogenase; (vi) binding of mitochondrial leader peptides to their receptors; (vii) interaction of G protein receptors with caveolin; (viii) interactions of erythrocyte band III peptide with glycolytic enzymes, and of lyn tyrosine kinase and B-cell antigen receptor; (ix) SH2-mediated interactions between p72 syk and the src-family kinase lck; (x) profilin-actin interactions and their modulation by ligands including proline-rich proteins and phosphoinositide lipids
Keywords: biomedical equipment, biomedical equipment purchase, intermolecular interaction, surface plasmon resonance
Project start date: 1999-03-15
Project end date: 2000-03-14
1S10RR013662-01 (1999): $238000
CYTOCHROME REDOX PROPERTIES IN A MEMBRANE ENVIRONMENT
William A Cramer, Henry Koffler Professor
Biological Sciencespurdue University West Lafayette
302 Wood St
west Lafayette, In 479072108
Grant 1R03TW000063-01A1 from Fogarty International Center IRG: SRC
Abstract: The mechanism of intramembrane electron transfer and structure-function relationships in the energy-transducing cytochrome beta complexes will be studied. The long-term objectives of this study are a deeper understanding of physico-chemical mechanisms governing charge transfer reactions in bioenergetics and development of an improved approach to prediction of structures of membrane proteins and their complexes structure predicted on the basis of experimental biochemical, spectroscopic, and kinetic data, and molecular modeling will be tested by theoretical evaluation of thermodynamic and kinetic parameters. Studies already carried out provide an explanation for the anomalous positive midpoint oxidation-reduction potential (Em) of the thylakoid cytochrome beta-559 in terms of (i) the Bornian solvation energy of the heme, (ii) the positive dipole potential near the N-termini of the two coordinating trans-membrane alpha-helices, in the context of a three phase model of the membrane. The novel third phase is a proteinaceous surface layer of intermediate dielectric constant that creates a hydrophobic niche for the heme and protects it from water. Similar considerations imply that the measured Em values of the two hemes of cyt beta of the cytochrome beta6 and betac1 complexes can only be explained if the dielectric environment of the hemes is asymmetric, more polar on the electrochemically positive side. It is proposed to (i) examine the correlation between the integrity of the surface layer and the high Em state of the heme; (ii) calculate the Em of cytochromes of known structure, and the kinetics of interheme transfer between the two hemes of the thylakoid cytochrome beta6; (iii) examine the molecular and electrostatic basis for the asymmetric dielectric environment of the two hemes of cyt beta6; (iv) rigorously determine the reference (aqueous) potential of protoheme; (v) determine the factors that control the rate of in situ interheme electron transfer of cyt beta6; and (vi) undertake a spectroscopic study of reorganization energy in electron transport proteins
Project start date: 1993-09-30
Project end date: 1996-03-31
1R03TW000063-01A1 (1993): $21473
THE MECHANISM OF ACTION OF COLICIN E1
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM018457-18 from National Institute Of General Medical Sciences IRG: BBCB
Abstract: The long-term goal of this study is to understand the molecular mechanisms by which a particular toxin-like protein binds to and penetrates into a cell. The problem concerns mechanisms of receptor function, protein translocation through one membrane, movement through an intermembrane compartment, binding to a second membrane, insertion into the membrane, channel formation, and voltage control of channel function. The toxin-like protein under discussion here is colicin E1. Solution to all of the above problems are of general relevance to the mechanism of action of toxins such as diphtheria and tetanus, and insertion of viruses into membranes. This similarity appears particularly graphic when the interaction of colicin E1 and diphtheria toxin with artificial planar membranes is compared. The two molecules show a very similar dependence for channel activity on pH, membrane potential, and lipid charge. Colicin El is a particularly good molecule in which to study all of these problems because it is naturally cloned, easily reclonable, and its sequence and that of its usual plasmid are known. Four aspects of the mechanism of action of colicin E1 will be analyzed (1) Function of the colicin E1 receptor Purification and characterization of the E1 receptor, binding of different colicin domains and peptide fragments to the receptor to localize the receptor binding site on the colicin polypeptide, incorporation of the receptor into liposomes, translocation of colicin through incorporated receptor. (2) Interaction of the C-terminal channel forming channel domain with membranes pH dependence of binding, pH dependence of conformation in the presence and absence of membranes, role of a particular residue, glu 468, in the pH dependence, tested by chemical labeling and directed mutagenesis. The role of other residues in binding and control of voltage dependence would also be tested by directed mutagenesis. (3) Structure of colicin channel Cleavage of exposed loops of channel forming polypeptide in liposomes, determination of Alpha-helical secondary structure content of colicin channel in liposomes, electron diffraction analysis of two dimensional membrane crystals of C-terminal fragment. (4) Mechanism of inhibition of channel formation by immunity protein Use of artificial liposome system to study interaction of imm protein with colicin, question of whether imm protein acts from cytoplasm or inner membrane to block channel formation.
Keywords: ANTIBIOTICS, COLICINES, BIOLOGICAL TRANSPORT, MEMBRANE MODELS, LIPOSOMES, PROTEINS, TRANSPORT PROTEINS, ION CHANNELS, BIOLOGICAL TRANSPORT, MEMBRANE PERMEABILITY AND TRANSPORT, ELECTROPOTENTIALS, MEMBRANE POTENTIALS, GENETICS, GENES, GENE EXPRESSION, GENETICS, GENES, STRUCTURAL GENES, GENETICS, MUTATION, MEMBRANE, MEMBRANE (BIOLOGICAL) STRUCTURE, PROTEINS, TRANSPORT PROTEINS, RECEPTORS, immunity, BACTERIA, ENTEROBACTERIACEAE, ESCHERICHIA COLI, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, FLUORESCENCE, CHEMISTRY, ANALYTICAL METHODS, X-RAY STRUCTURE ANALYSIS, GENETICS, EXTRACHROMOSOMAL INHERITANCE, PLASMIDS
Project start date: 1974-09-01
Project end date: 1990-03-31
Sponsored Links Excellgen http://Excellgen.com
STRUCTURE/FUNCTION OF MEMBRANE BOUND CYTOCHROMES
William A Cramer, Henry Koffler Professor
Biological Sciencespurdue University West Lafayette
302 Wood St
west Lafayette, In 479072108
Grant 3R01GM038323-11S1 from National Institute Of General Medical Sciences IRG: BMT
Abstract: The highly conserved cytochrome b6f complex and the reaction center cytochrome b-559 of oxygenic photosynthesis will be used to study basic aspects of the structure and function of integral membrane cytochromes. Thr proposed studies are based on the following results from previous grant support (i) the solution of the crystal structure at 2.3A resolution of the active major extrinsic domain of cytochrome f, the first structure at the atomic level of a subunit of the cytochrome bc1 or b6f complexes. Cytochrome f has three unprecedented structural features for a c-type cytochrome of (a) a predominant beta-strand motif, (b) two distinguishable domains, and (c) the N-terminal alpha-amino group of Tyr-1 as the axial sixth heme ligand the latter result provided specific information about the sequence of events in the translocation of cyt f across the membrane, i.e. that processing must precede completion of heme coordination and final assembly. (ii) The purified b6f complex was characterized as a structural and functional dimer. (iii) The interhelix forces of the b6f complex were found to be relatively weak. (iv) The orientation of the beta-subunit of that heme cross-linked cytochrome b-559 was found to be parallel to that of the alpha, in agreement with (a) the prediction that it is a heterodimer, (b) the cis-positive rule for orientation of membrane proteins, and (c) the calculated contribution of the dipole potential of the alpha and beta helices to its very positive midpoint potential. It is proposed; (I) to use the cytochrome f structure and existing cross- linking information as the basis for ´intelligent´ site-directed mutagenesis to (a) determine the position of the docking site(s) for plastocyanin, and (b) to make a set of single histidine surface mutants that will be utilized, after modification wit Ru (bpy)2 adducts, to measure the reorganization energy, gamma, and optimum pathway associated with the intraprotein electron transfer. It is hypothesized that this transfer will have an unusually small gamma because the transfer to the plastocyanin acceptor is isopotential. (c) The consequences for assembly of cyt f and the subunits of the complex will be tested of inhibition of the processing and liberation of the Tyr-1 amino group. (II,a) The homodisperse Mr 230,000 b6f dimer will be used to crystallize this integral membrane protein complex. (b) The function of dimeric b6f complex will be tested in trans-membrane signaling involving the n-side kinase, as will (c) the role of the very highly conserved n-side extrinsic loops of cyt b6 in the docking of peripheral proteins. (iii) The effect of topographical inversion of the cyt b-559 heme will be examined by applying the cis- positive rule and reversing its trans-membrane distribution of positively charged amino acids
Keywords: cytochrome, enzyme complex, photosynthetic reaction center, protein structure /function adduct, chemical binding, electron transport, gene mutation, heme, histidine, molecular site, protein biosynthesis, protein kinase Chlamydomonas, crystallization, laboratory rabbit, site directed mutagenesis
Project start date: 1987-04-01
Project end date: 1999-06-30
3R01GM038323-11S1 (1999): $72696
Problems In Membrane Protein Crystallography: Hetero-Oligomeric Cytochrome B6f
William A Cramer, Henry Koffler Professor
Biological Sciencespurdue University West Lafayette
Grant 2R01GM038323-21A2 from National Institute Of General Medical Sciences IRG: BBM
Abstract: The hetero-oligomeric cytochrome b6f and bc1 complexes are in the center of the electron transfer chains in photosynthetic and respiratory energy transducing membranes. Such membranes contain the majority of the relatively few hetero-oligomeric integral membrane proteins that have been solved by X-ray crystallography to a resolution d 3.0 E. Studies on crystallization of the dimeric 220 kDa eight subunit integral b6f complex would analyze problems of proteolysis and lipid-protein interactions that are of general relevance to the crystallization of integral membrane proteins. Structure-function analysis would focus on the properties of a unique redox group, heme cn, and on the mechanism of transfer across the membrane of qui (ol) that carries the electrons and protons and is reduced by heme cn. Proposed studies (1) Crystal preparation; proteolysis. b6f complex cannot be isolated from transformable unicellular cyanobacteria because the b6f dimer is monomerized and rendered inactive and non-crystallizable upon extraction from the membrane. Successful crystallization of cyanobacterial b6f has utilized the filamentous M. laminosus, in which the extent of proteolysis is smaller. However, M. laminosus is not transformable. Therefore, the cyanobacterial source of b6f complex will be changed to the filamentous Nostoc (Anabaena) sp. PCC 7120, from which active and crystallizable complex has been obtained. Because this kind of proteolysis problem frequently hinders efforts to crystallize membrane proteins, the critical protease(s) in the unicellular cyanobacteria would be identified by mass spectroscopic and proteomic analysis. (2) Function of phospholipids. The function of intra-protein lipids has been analyzed in only a few multi-subunit membrane proteins. Our novel lipid augmentation procedure resulted in a major increase in the rate of crystallization and improvement in crystal quality. The properties of crystals of the plant thylakoid membrane b6f complex, which contains a ninth (FNR) subunit and whose crystallization depends uniquely on a different (anionic DOPG) lipid, is under study, as is the dependence of electron transfer activity and rate of crystallization on the nature of added lipids. (3) Functions of heme cn; evolution of b6f complex. The function of the unique heme cn, not found in ubiqui-containing cyt bc1 complexes, will be studied by site-directed mutagenesis in Nostoc and, through structure-function analysis, in firmicutes such as Bacillus subtilis that are phylogenetically close to cyanobacteria. His-tagged, promoter-augmented firmicute "qcr" complex will be purified and screened for crystallization and electron transfer reactions with menaqui. (4) Qui transfer though the narrow p-side portal. Electrons and protons are carried across an inter-monomer qui exchange cavity in bc1 and b6f complexes by lipophilic ubi- and plastoqui (PQ). The mechanisms by which PQ/PQH2 finds, enters, and exits a narrow 11 x 12 E p-side portal will be studied through mutagenesis of portal residues and computational analysis of the portal force field. Some of the biomedically relevant aspects of these studies are that they are directed toward an understanding of the detailed internal structure of the proteins that mediate all traffic, including nutrients and drugs, across biological membranes. Via the membrane, the set of energy-transducing proteins determines the level of energy and its regulation in the human cell
Project start date: 1987-04-01
Project end date: 2013-01-31
Structure-Function Of Hetero-oligomeric Integral Membrane Proteins
William A Cramer, Henry Koffler Professor
Biological Sciencespurdue University West Lafayette
302 Wood St
west Lafayette, In 479072108
Grant 2R56GM038323-21 from National Institute Of General Medical Sciences IRG: BBM
Abstract: The cytochrome b6f complex is one of a small number (presently, 17) of hetero-oligomeric integral membrane proteins whose crystal structure has been solved to a resolution better than 3.0 A. The structure is a dimer that encloses a large (10,000 A3) lipophilic inter-monomer ´qui exchange cavity that exchanges qui/quinol with the lipid bilayer, and connects the qui reduction site on the electrochemically negative n-side of one monomer with the oxidation site at the [2Fe-2S] cluster on the positive p-side. The qui pathway between n- and p-sides involves passage through anllAx!2A portal. We have solved four b6f structures from the thermophilic cyanobacterium, M. laminosus, (i) a native structure with resolution recently improved to 2.95 A in the presence of Cd2+ cations, and three structures of complexes with qui- analogue inhibitors, p-side TDS and DBMIB, and n-side NQNO. These structures have provided markers for the route of qui passage across the complex and diffusion within it. Further understanding of the structure and dynamics of qui transfer is relevant to understand transfer of hydrophobic drugs and metabolites across membrane proteins, and will be pursued through higher resolution structures, site-directed mutagenesis and molecular/steered dynamics. The complex contains 8 subunits 4 of these form a core of ´large´ subunits that bind 8 prosthetic groups. Each b6f monomer contains 4 hemes, one [2Fe-2S] cluster, one plastoqui, and 3 unique prosthetic groups (i) a novel heme en with one covalent linkage to a Cys residue of cytochrome b, and no amino acid side chains as axial ligands; (ii) one chlorophyll a, for which two H2O have been resolved as its 5th ligand; (iii) one beta-carotene to which the Chi, can transfer excited triplet state energy in spite of their 14A separation. These prosthetic groups unique to b6f raise new questions about redox function in be complexes. We will focus on the unique heme en, whose functions are linked to the ability of the b6f complex to carry out ferredoxin-dependent cyclic electron transport and of the photosynthetic membrane to evolve O2. A unique coupling between heme en and the nearby (4 A) heme bn was established, which implies an ability of hemes bn - en to serve as a 2 electron donor, thus providing a new mechanism to protect against formation of superoxide and reactive oxygen species (ROS). Studies on a second hetero- oligomeric integral membrane protein, Ndh-1 from the cyanobacterial membrane that also reduces qui and generates ROS, have masses of 12 of the 14-15 subunits, and in which the goal is to obtain a crystal structure
Project start date: 1987-04-01
Project end date: 2009-07-31
2R56GM038323-21 (2007): $335937
STRUCTURE/FUNCTION OF THE CYTOCHROME B6F COMPLEX
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 2R01GM038323-12A1 from National Institute Of General Medical Sciences IRG: PB
Abstract: The cytochrome b6f complex has many structure-function homologies with the cytochrome bc1 complex of the mitochondrial respiratory chain. Much of this arises from the identity of cytochrome b, especially marked on the p-side of the membrane. From the high resolution structures of cytochrome f and the Rieske (ISP) protein, it is known that the peripheral domains differ significantly between bc1 and b6f. The further the domain from the membrane, the greater the difference. Thus, cytochromes f and c1 are completely different proteins and have been from their evolutionary origin, and the domain of the ISP distal to membrane folds differently in the b6f and bc1 complexes. However, the ISP domain containing the iron-sulfur cluster that must come close to the membrane has a conserved fold. Thus, detailed information on structure-function for the b6f complex complements that obtained for the bc1 and seems likely to provide differences in detailed functional mechanisms. A high resolution structure of 3-D crystals of the b6f complex from the thermophilic cy- anobacterium, M. laminosus, would provide this information. The crystals presently show ordered diffraction to 10 Angstrom units. When diffraction (less than or equal to 3 Angstrom units) appropriate for a structure analysis is obtained, the solution of the structure will be expedited by the fact that we have high resolution structures (less than 2.0 Angstrom units) for the p- side of the complex, cytochrome f and the iron-sulfur protein, 40 percent of the total mass of the complex. Issues of function to be determined by the structure include the position and function of the n-side quinone, the pathway of trans-membrane H+ transfer, and the role of intramembrane bound water. From the existing p- side structures, the local mobility of the ISP will be analyzed in vivo, in situ, and in vitro. The basis for the non-concerted reduction of high and low potential chains will be studied. Catalysis of electron transfer by conserved aromats in the Rieske protein, and in cytf where they shield the heme, will be tested my mutagenesis. The role of the water chain in the coupling of intraprotein electron and proton transfer will be examined by stopped flow kinetics in D2O, together with the properties of the bound H2O in cytf by FTIR. With a ruthenium derivative of cytf, "photo-cytf", light-induced intraprotein electron transfer rates, optimum paths of intraprotein electron transfer, and reorganization energy will be measured. Ruthenated cytf will also be used to investigate intraprotein protonation- deprotonation at specific carboxylates, associated with coupled electron and proton transfer.
Keywords: cytochrome, enzyme complex, protein structure /function, cytochrome b, electron transport, iron sulfur protein, membrane protein, oxidation /reduction, photochemistry, protonation, X ray crystallography, crystallization, laboratory rabbit, photosynthetic bacteria, ruthenium, stop flow technique
Project start date: 1987-04-01
Project end date: 2003-06-30
2R01GM038323-12A1 (1999): $100739
BIOPHYS STUD OF PROTEINS, NUCLEIC ACIDS, AND VIRUSES
William A Cramer, Henry Koffler Professor
Biological Sciencespurdue University West Lafayette
302 Wood St
west Lafayette, In 479072108
Grant 5T32GM008296-13 from National Institute Of General Medical Sciences IRG: BRT
Abstract: The biophysical training grant at Purdue University has continued its strong emphasis on structural biology, while increasing the representation of faculty mentors from other disciplines which involve and affect structural biology. The mentoring faculty has increased from 19 to 22 in this period, with all members from six different departments either fully or partly involved in some aspect of structural biology. Two new departments are represented, and the number of mentors from biology, biochemistry, and physics as increased. The basic core research equipment, X-ray diffractometers, NMR spectrometers, and electron microscopes, has expanded and improved greatly. Six areas of overlapping research interests (structure-function of soluble proteins, membrane proteins and toxins, nucleic acid structure-function and protein-nucleic acid interactions, virus structure-function, computational approaches and metalloproteins) can be identified with a minimum faculty cluster size of at least four faculty members. These interactions had led to fifty-three publications involving at least two faculty members over the two grant periods in which thirteen biophysics trainees have also participated. During this time, past and current biophysics have contributed to 132 publications and s of presentations and professional meetings. A high fraction of past trainees are currently working actively in research at universities or in industry. During the last grant period, there has been important and significant representation among the trainees from under-represented minorities and women. A discussion group on important issues of ethics and science, involving both students and faculty, has been enthusiastically received. The graduate course curriculum has improved and maintained a high degree of emphasis on quantitation. New educational aspects of the program have been an annual-student oriented biophysics symposium, already presented three times, and pre-seminar "biophysical discussions" given by different mentors on central concepts to be presented in upcoming seminars. The weekly structural biology seminars and bi-weekly structural biology journal club have continued to be regular research and training events
Project start date: 1989-07-01
Project end date: 2004-06-30
5T32GM008296-13 (2001): $179592
5T32GM008296-12 (2000): $170261
2T32GM008296-11 (1999): $162210
2001 Gordon Research Conference On Bioenergetics
William A Cramer, Henry Koffler Professor
Gordon Research Conferences
west Kingston, Ri 02892
Grant 1R13GM063876-01 from National Institute Of General Medical Sciences IRG: PB
Abstract: The Bioenergetics Gordon Conference is convened biannually and was last held in June, 1999. The larger (ca. 500 person) European Bioenergetics Congress, completed last week, is held in the alternate years. Traditionally, the Bioenergetics GRC has emphasized discussions on mechanisms of electron transport and oxidative phosphorylation, along with the discussions of the biomedical implications for mitochondrial myopathies and aging. However, as the majority of the first high resolution structures of integral membrane proteins were obtained from energy-transducing membranes, this Conference has become a major platform for discussions of integral membrane protein biochemistry. Structure-based discussion of mechanism involving integral membrane proteins has now become a basic theme of the Bioenergetics GRC, and contributions in this cutting-edge area are a major criteria in the planning and organization of the sessions. Thus, the determination in the last year of the high resolution (1.55 A) structure of bacteriorhodopsin, the highest resolution membrane protein structure, along with high resolution structures of halorhodopsin and rhodopsin, created a wonderful opportunity for a comparative presentation and discussion of the structures and evolution of the rhodopsin family. The present program will also have major sessions on Active Transport and Biogenesis. The special features of informality and spontaneity of Gordon Conferences allow the possibility of in-depth discussions of fundamental questions. To further stimulate such discussions, to the extent that it is feasible, this Conference is being organized around conceptual themes rather than focused on individual proteins or experimental systems. This is a bit of an experiment, but I believe it will be interesting. The major use of the funds requested in this application is to support the attendance at this conference of "young" people, graduate students and postdoctorals. Forty-six students and postdocs, out of a total of 145 in attendance, came to the last Conference. The research discoveries, relevance of spectoscopic methods, and quantitative analysis of integral membrane proteins make it essential to educate, and inform the young researchers interested in this field, who will form its next generation. The Bioenergetics GRC is a wonderfully inspiring venue for these purposes. Of course, the ideas and studies contributed by the younger folks are also inspirational to the rest of us
Keywords: bioenergetics, meeting /conference /symposium active transport, membrane biogenesis, membrane protein, postdoctoral investigator, postgraduate education, protein structure function, structural biology travel
Project start date: 2001-06-15
Project end date: 2002-06-14
1R13GM063876-01 (2001): $7000
Sponsored Links Excellgen http://Excellgen.com
STRUCTURE/FUNCTION OF COLICIN E1/RECEPTOR/IMM PROTEIN
William A Cramer, Henry Koffler Professor
Biological Sciencespurdue University West Lafayette
302 Wood St
west Lafayette, In 479072108
Grant 2R01GM018457-28 from National Institute Of General Medical Sciences IRG: BBCB
Abstract: The 522 residue colicin E1 voltage-gated ion channel is used to study basic aspects of the major structure transition of a toxin-like molecule from the water-soluble to the integral membrane-bound state, the properties associated with the unique intramembrane interaction of the colicin channel with its immunity protein, and its translocation across the cell envelope from outer membrane across the periplasmic space to the cytoplasmic membrane. The channel is highly charged with a pronounced C- terminal hydrophobic domain, and its interaction with membranes is representative of the many facets of membrane interactions of proteins contrasted with those of small peptides. Using newly developed DNA constructs and purification procedures, the studies utilize a 190 residue C-terminal colicin channel polypeptide (P190) over-produced under its own promoter, purified 113 residue hydrophobic immunity protein, and purified 368 residue soluble TolA protein from the cell intermembrane translocation system. The studies will combine a combination of structural, biochemical, biophysical, and molecular genetic approaches. The in vitro transition from H2O to the membrane involves structure intermediates, including a partly unfolded state in solution, and an unfolded state on the surface of the membrane. (I) Structure information will be obtained from (i) an X-ray structure determination of the P190 channel; (ii) NMR structure studies including solid-state analysis of the trans-membrane helix, and secondary structure and folding of the immunity protein in a membrane-mimetic organic solvent mixture; (iii) the identity of residues in contact with the channel aqueous lumen measured by single cysteine substitution and chemical modification by channel-blockers at consecutive residues around helix turns; (iv) the identity of specific lysine residues, Lys 362,395,397,402,403, inferred from structural models to be involved in the docking, will be tested by mutagenesis and quantitative analysis of binding parameters; (v) Cys-Cys crosslinking of specific helices will be used to determine the unfolding steps that must accompany binding to the membrane surface; (vi) the significance of an unusual SDS- resistant dimer of the immunity protein will be tested with imm mutants with impaired activity; (vii) imm 2nd-site revertants to existing imm bypass mutants will define sites of interaction; (viii) the TolA-colicin interaction that confers in vivo translocation-competence will be sought; (ix) 2 dimensional crystallization of the immunity protein and structure solution by EM image analysis will be performed. (II) Quantitative determination of equilibrium and kinetic binding parameters will also be carried out as a function of acidic lipid content, along with the solid- state NMR analysis, to test the inference based on data obtained in the last grant period that the insertion of the hydrophobic domain into the membrane, and the resulting channel activity, requires an optimum surface electrostatic interaction. A weak interaction precludes effective binding. One that is too strong prevents insertion into the bilayer
Keywords: colicine, immunity, membrane channel, protein structure /function, vitamin B12, vitamin receptor chemical kinetics, fluidity, hydropathy, ionic bond, ionic strength, membrane activity, membrane protein, protein folding, protein protein interaction, protein reconstitution, receptor binding, voltage gated channel circular dichroism, crystallization, fluorescence resonance energy transfer, laboratory rabbit, nuclear magnetic resonance spectroscopy, site directed mutagenesis, stop flow technique
Project start date: 1974-09-01
Project end date: 2003-03-31
2R01GM018457-28 (1999): $299118
5R01GM018457-31 (2002): $298792
5R01GM018457-30 (2001): $295375
5R01GM018457-29 (2000): $287046
MEMBRANE INTERACTIONS OF COLICIN EI IMMUNITY PROTEIN
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM018457-27 from National Institute Of General Medical Sciences IRG: BBCB
Abstract: The 522 residue colicin E1 voltage-gated ion channel is used to study basic aspects of the major structure transition of a toxin-like molecule from the water-soluble to the integral membrane-bound state, the properties associated with the unique intramembrane interaction of the colicin channel with its immunity protein, and its translocation across the cell envelope from outer membrane across the periplasmic space to the cytoplasmic membrane. The channel is highly charged with a pronounced C- terminal hydrophobic domain, and its interaction with membranes is representative of the many facets of membrane interactions of proteins contrasted with those of small peptides. Using newly developed DNA constructs and purification procedures, the studies utilize a 190 residue C-terminal colicin channel polypeptide (P190) over-produced under its own promoter, purified 113 residue hydrophobic immunity protein, and purified 368 residue soluble TolA protein from the cell intermembrane translocation system. The studies will combine a combination of structural, biochemical, biophysical, and molecular genetic approaches. The in vitro transition from H2O to the membrane involves structure intermediates, including a partly unfolded state in solution, and an unfolded state on the surface of the membrane. (I) Structure information will be obtained from (i) an X-ray structure determination of the P190 channel; (ii) NMR structure studies including solid-state analysis of the trans-membrane helix, and secondary structure and folding of the immunity protein in a membrane-mimetic organic solvent mixture; (iii) the identity of residues in contact with the channel aqueous lumen measured by single cysteine substitution and chemical modification by channel-blockers at consecutive residues around helix turns; (iv) the identity of specific lysine residues, Lys 362,395,397,402,403, inferred from structural models to be involved in the docking, will be tested by mutagenesis and quantitative analysis of binding parameters; (v) Cys-Cys crosslinking of specific helices will be used to determine the unfolding steps that must accompany binding to the membrane surface; (vi) the significance of an unusual SDS- resistant dimer of the immunity protein will be tested with imm mutants with impaired activity; (vii) imm 2nd-site revertants to existing imm bypass mutants will define sites of interaction; (viii) the TolA-colicin interaction that confers in vivo translocation-competence will be sought; (ix) 2 dimensional crystallization of the immunity protein and structure solution by EM image analysis will be performed. (II) Quantitative determination of equilibrium and kinetic binding parameters will also be carried out as a function of acidic lipid content, along with the solid- state NMR analysis, to test the inference based on data obtained in the last grant period that the insertion of the hydrophobic domain into the membrane, and the resulting channel activity, requires an optimum surface electrostatic interaction. A weak interaction precludes effective binding. One that is too strong prevents insertion into the bilayer.
Keywords: colicine, intermolecular interaction, membrane, protein structure /function, voltage gated channel, chloroform, cysteine, disulfide bond, glycophorin, methanol, phosphatidylserine, phosphoglyceride, plasmid, protein folding, tryptophan, water, X ray crystallography, crystallization, laboratory rabbit, nuclear magnetic resonance spectroscopy, site directed mutagenesis
Project start date: 1974-09-01
Project end date: 1999-03-31
5R01GM018457-27 (1998): $220028
5R01GM018457-26 (1997): $212246
Voltage-Gated Insertion Of Colicin Into Planar Bilayers
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R03TW001235-06 from Fogarty International Center IRG: ICP
Abstract: Studies on membrane import and channel formation of the pore-forming colicins concern the nature of (i) the large soluble --> membrane-bound structural transition undergone by colicins, toxins, and other membrane-active proteins, (ii) the surface-bound state that potentates helix insertion, (iii) structure changes associated with voltage-gated channel formation, and (iv) the pathway of protein insertion into the membrane, and (v) the mechanism by which the MW = 65,000 colicins are translocated across the E. coli outer membrane. The structure of the colicin E1 channel domain, solved at atomic resolution, allows structure-based mutagenesis strategies to test models for structural transitions upon membrane-binding and channel formation. Single-Trp and -Cys mutants were used in fluorescence quenching and fluorescence resonance energy transfer to define the colicin channel bound in the membrane interfacial layer as an extended, flexible, two-dimensional helical net. Planar lipid bilayer experiments have been carried out in collaboration with the lab of Y. N. Antonenko (Moscow, Russia) to observe the kinetics of colicin channel formation and related properties. There have been 4 collaborative projects (1) Colicin channel activity was photoinactivated in the presence of sensitizing dyes, and this effect depended on the presence of Trp495 in helix 9 of channel domain. Colicin photoinactivation will serve as an important model for study of photodamage of membrane proteins and photodynamic therapy, widely used in cancer treatment. (2) Colicin import and channel formation was found to be very sensitive to membrane anionic lipid content and to be "tuned" at a surface potential of -60 +/- 5 mV. (3) The channel activities of purified outer membrane receptors proteins were found to be occluded by exogenous colicin. (4) Preliminary experiments indicate that colicin El membrane-binding and channel formation is affected by the lipid interfacial dipole potential. It is proposed to simultaneously measure channel current and fluorescence with horizontal planar bilayers and to analyze the kinetics and pathway of voltage-gated colicin insertion into, and channel formation in, the membrane.
Keywords: colicine, lipid bilayer membrane, membrane protein, protein structure function, voltage gated channel, Commonwealth of Independent States, biophysics, conformation, cooperative study, intermolecular interaction, mutant, pore forming protein, transposon /insertion element, fluorescence microscopy, voltage /patch clamp
Project start date: 2000-02-01
Project end date: 2007-07-31
5R03TW001235-06 (2005): $38500
5R03TW001235-05 (2004): $35700
2R03TW001235-04 (2003): $35750
Structural Analysis Of Integral Membrane Proteins
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 2R01GM038323-17 from National Institute Of General Medical Sciences IRG: PB
Abstract: Structure-function studies on the hetero-oligomeric cytochrome b6 f complex have led to successful crystallization and initial characterization of well-diffracting crystals of the complex from the thermophilic cyanobacterium, Mastigocladus laminosus, whose structure analysis is underway. They have also led to a new "lipid enrichment" methodology for the crystallization of such integral membrane proteins, considering the protein in detergent micelles as a protein-detergent-lipid complex. The presence and the masses of eight subunits of the complex from M. laminosus and nine subunits from the higher plant complex, including the newly discovered FNR component, have been characterized by mass spectroscopy. The MS analysis removed uncertainty as to exactly how many small polypeptides are present in the complex. A unique aspect of the role of lipid in the function and structural stabilization of the complex arose through the discovery of one equivalent of lipid-like beta-carotene and chlorophyll alpha in the complex. The lipid parameters of the "enrichment methodology will be examined. It is proposed to test the applicability of the new lipid enrichment methodology to the b6f complex in a transformable cyanobacterium and four other unrelated integral membrane protein systems. The potential application of this methodology is important for studies on integral membrane proteins, as there are at present only 23 solved independent IMP structures,and 56 in total. This creates a limiting step in proteomic analysis since 20-30 % of gene products are membrane proteins. The function of the Rieske iron-sulfur protein (ISP), which has been proposed to undergo a unique large amplitude rotation-translation in its electron transfer function, was analyzed through (i) the viscosity dependence of the electron transfer function, (ii) stopped-flow kinetics of diffusion limited delectron transfer in solution, and (iii) mutagenesis analysis of the linker region, the "hinge of the proposed rotation. The ISP motion is constrained in vivo. The motion does not depend sensitively on the structure of the linker region, but the linker has a minimum length of 7 residues. It is proposed to directly measure the kinetics of motion of the ISP soluble domain triggered by electron transfer.
Keywords: cytochrome, enzyme complex, membrane protein, protein structure function, cytochrome b, electron transport, iron sulfur protein, oxidation reduction reaction, photochemistry, protonation, X ray crystallography, crystallization, fluorescence resonance energy transfer, laboratory rabbit, photosynthetic bacteria, ruthenium, stop flow technique
Project start date: 1987-04-01
Project end date: 2007-06-30
2R01GM038323-17 (2003): $314771
Sponsored Links Excellgen http://Excellgen.com
SENSITIZED PHOTOINACTIVATION OF COLICIN E1 CHANNELS
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R03TW001235-03 from Fogarty International Center IRG: ICP
Abstract: Some major problems of general interest from studies on the pore-forming colicins are the nature of (i) the large soluble yields membrane-bound structural transition undergone by colicins, toxins, and other membrane-active proteins; (ii) the surface-bound state that potentiates helix insertion; (iii) structure changes associated with voltage-gated channel formation. The structure of the colicin E1 channel domain, solved at atomic resolution, allows structure-based mutagenesis strategies to test models for structural transitions upon membrane-binding and channel formation. Single-Trp and -Cys mutants were used in fluorescence quenching and fluorescence resonance energy transfer to define the colicin channel bound in the membrane interfacial layer as an extended, flexible, two-dimensional helical net. To initiate studies on the structure transition from closed- to open-channel state, planar lipid bilayer experiments have been carried out in collaboration with the lab of Y. N Antonenko (Moscow, Russia) to observe the kinetics of colicin channel formation. Colicin channel activity was photoinactivated in the presence of sensitizing dyes, and this effect depended on the presence of Trp495 in helix 9 of channel domain. Cross-linking of the polypeptide molecule (dimerization) was detected in parallel experiments with channel domain bound to liposomes. Based on studies of the Antonenko lab on the gramicidin cation-selective channel, it is proposed to investigate the mechanism of sensitized photoinactivation of the colicin E1 channel, and associated structure changes. The Trp-dependence of photoinactivation allows the use of single-Trp mutants to infer the helices and specific side chain involvement in channel formation. New approaches that will be developed in the course of this study will extend the application of Trp- and Cys-scanning mutagenesis. Preliminary experiments show also that colicin E1 membrane-binding and channel formation is affected by the lipid interfacial dipole potential. Both single-and multi- channel measurements will be used to investigate the mechanism photoinactivation and the role of the membrane dipole potential. Colicin photoinactivation will serve as an important model for study of photodamage of membrane proteins and photodynamic therapy, widely used in cancer treatment.
Keywords: colicine, lipid bilayer membrane, membrane channel, membrane protein, photoactivation, photosensitizing agent, protein structure function, cysteine, free radical oxygen, hydropathy, membrane model, tryptophan, voltage gated channel, SDS polyacrylamide gel electrophoresis, site directed mutagenesis
Project start date: 2000-02-01
Project end date: 2003-01-31
5R03TW001235-03 (2002): $35750
5R03TW001235-02 (2001): $40000
1R03TW001235-01 (2000): $35850
THE MECHANISM OF ACTION OF COLICIN
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM018457-13 from National Institute Of General Medical Sciences IRG: BBCB
Abstract: It has been found that active transport function can be restored by trypsin treatment to cells inhibited by colicin E1 if the cells are incubated at 15 degrees C. This experiment implies that the colicin molecule is exposed at the outside of the outer membrane at the time it inhibits active transport at the inner membrane. It is proposed to examine the length of the colicin molecule exposed at the outside of the outer membrane at the time of reversal of inhibitory effects by trypsin in order to define this stage in the process of penetration of the cell envelope by trypsin. Colicin El has been incorporated into membrane vesicle made of the single phospholipid dimpyristoyl phosphatidyl choline. Colicin E1 shows appreciable conductance to salts at temperatures well below the phase transition of this lipid, thus providing substantial support for a channel function of this colicin. Further studies will be made of the ion and solute selectivity of the colicin channel in this chemically well defined membrane system, as well as of the colicin conformational changes that accompany its insertion into the membrane. The orientation of colicin in this vesicle will be studied in order to define the membrane active segement. The latter studies will be complemented by using a small 20 k dalton tryptic fragment of colicin E1 which is active in the synthetic lipid vesicles.
Keywords: ANTIBIOTICS, COLICINES, BIOPHYSICAL CHEMISTRY STUDY SECTION, DRUGS, PHARMACOLOGY, BIOLOGICAL TRANSPORT, TRANSPORT PROTEINS (SEE ALSO SPECIFICS), ENZYME INHIBITORS, OXIDOREDUCTASE INHIBITORS, FATTY ACIDS, HEMOPROTEINS, CYTOCHROME B, MEMBRANE, MEMBRANE (BIOLOGICAL) STRUCTURE, PHYSICAL PROPERTIES, VISCOSITY-VISCOELASTICITY, PROTEINS-PEPTIDES STRUCTURE, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, ATP, potassium, BACTERIA, ENTEROBACTERIACEAE, ESCHERICHIA COLI, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, FLUORESCENCE, CHEMISTRY, ANALYTICAL METHODS, X-RAY STRUCTURE ANALYSIS, GENETICS, MUTATION, MUTANTS, OPTICS, MICROSCOPY, ELECTRON
Project start date: 1974-09-01
Project end date: 1984-08-31
NMR STRUCTURAL STUDIES OF COLICIN E1: CYTOTOXIN
William A Cramer
Institution:
Grant 5P41RR009793-030003 from National Center For Research Resources
Abstract: Colicins are highly effective cytotoxins. A single bound molecule can kill a cell. Several different approaches have been applied to understanding the mechanism of action of the 190 residue colicin E1 in terms of protein structure. The variety of biophysical studies that have been performed on the membrane associated form of colicin E1 have led to widely differing structural models, some of which contradict each other. The presence of trans-membrane helices in the functional form of colicin E1 is a crucial point, since it has strong implications for the structural organization and mechanism of action of the protein. The solid-state NMR experimental approaches being developed at the Resource are able to address this issue particularly well. Uniformly 15N labeled colicin E1 was prepared at Purdue University by expression of the plasmid encoded gene in bacteria. The unoriented spectrum of uniformly 15N labeled colicin E1 in bilayers is of a 15N amide chemical shift anisotropy powder pattern with some relatively narrow resonance intensity superimposed at the isotropic chemical shift frequency. This is exactly the result expected for a typical membrane protein where most of the residues are rigidly held in helices and some of the residues, most likely those at the N- and C- termini and in loops connecting the helices, are mobile. More interesting results emerge from the experiments on samples of the protein in bilayers oriented between glass plates. Since the secondary structure of the protein is known to be primarily alpha helix, these resonance bands indicate that the protein had both trans-membrane and in-plane helices. These results indicate that colicin E1 is a typical membrane protein that has the potential to form channels with its trans-membrane helices.
THE MECHANISM OF ACTION OF COLICIN
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM018457-08 from National Institute Of General Medical Sciences IRG: BBCA
Abstract: Colicin El has recently been shown by ourselves to deenergize the cell membrane with respect to active transport in a time very similar to that necessary for adsorption of the protein to the cell receptors, and significantly faster than previously thought for the depolarizing effect of colicin El. The mechanism for this rapid deenergization is being sought. A preparation of colicin E2 has been developed with much higher specific activity than any others in the literature. Effort is being directed at present toward the stablization of this high activity preparation.
Keywords: ANTIBIOTICS, COLICINES, BIOPHYSICS AND BIOPHYSICAL CHEMISTRY STUDY SECTION, DRUGS, PHARMACOLOGY, BIOLOGICAL TRANSPORT, TRANSPORT PROTEINS (SEE ALSO SPECIFICS), ENZYME INHIBITORS, OXIDOREDUCTASE INHIBITORS, FATTY ACIDS, HEMOPROTEINS, CYTOCHROME B, MEMBRANE, MEMBRANE (BIOLOGICAL) STRUCTURE, PHYSICAL PROPERTIES, VISCOSITY-VISCOELASTICITY, PROTEINS-PEPTIDES STRUCTURE, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, ATP, potassium, BACTERIA, ENTEROBACTERIACEAE, ESCHERICHIA COLI, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, FLUORESCENCE, CHEMISTRY, ANALYTICAL METHODS, X-RAY STRUCTURE ANALYSIS, GENETICS, MUTATION, MUTANTS, OPTICS, MICROSCOPY, ELECTRON
Project start date: 1974-09-01
Project end date: 1979-08-31
Receptor-mediated Colicin Import
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM018457-35 from National Institute Of General Medical Sciences IRG: BBCB
Abstract: An understanding of protein translocation and import across receptors and membranes is fundamental in biology and medicine. Receptor-mediated import of the toxin-like colicins is a paradigm for protein import to mitochondria and the import of viral proteins. Colicins E1 and E3 have different modes of bactericidal action, but parasitize the same (vitamin B12, BtuB) integral outer membrane receptor, and are used to study the mechanism of import in the E. coil inner and outer membrane, respectively. (I) OM import (1) A 2.75 A structure of the complex of the expressed 135 residue (R135) 100 A long coiled-coil receptor binding domain of colicin E3, and the BtuB receptor in detergent, is the first high resolution structure of an intact integral protein receptor and its protein ligand. This structure forms the basis for studies on mechanisms of protein translocation through the outer membrane. (2) SPR and CD show that R135 binds tightly, and its receptor-distal ends unwind upon binding to BtuB. A smaller R60 peptide rewinds upon binding. The basis for these unusual properties of the binding site will be probed by mutagenesis of both R135 and BtuB, particularly the loop regions of BtuB. An R135 Arg399Ala mutant has defined a residue involved in electrostatic binding. Disulfide cross-linking of the distal ends of R135 will test the coupling between distal unwinding and binding affinity. (3) The 2.75 A structure and the absence of large ion channels in BtuB imply that E3 does not pass through it. Occlusion of the large OmpF and TolC ion channels by E3 and E1, respectively, indicate a role for these Omp in the colicin translocon. (4) The bending and mechanical properties of the R135 coiled-coil, which might be involved in delivery of colicin domains to OmpF, will be tested by atomic force microscopy. (5) The role of two OM receptors in colicin translocation will be tested with OmpF and TolC, (i) using planar bilayers to measure changes in their channel conductance and receptor function, and the ability of individual colicin domains and TolA, TolB proteins to occlude the channels; and (ii) in co-crystallization experiments. (II) IM import We have proposed that two prerequisites for import of soluble membrane-active proteins are segmental mobility on the membrane surface and helix extension. (1) This hypothesis will be further tested using (a) the colicin channel protein, P178, membranes of defined surface potential, and assays by FRET measurements of distance changes, and (b) solid-state NMR. (2) The pathway of membrane insertion of the E1 channel domain, relative to the time course of other unfolding events, will be examined by time-resolved FRET using a large set of single Trp mutants.
Keywords: colicine, membrane channel, protein folding, protein reconstitution, protein structure function, protein transport, vitamin receptor, chemical kinetics, hydropathy, intracellular transport, ionic bond, membrane activity, membrane protein, protein protein interaction, receptor binding, vitamin B12, voltage gated channel, atomic force microscopy, calorimetry, circular dichroism, crystallization, fluorescence resonance energy transfer, laboratory rabbit, nuclear magnetic resonance spectroscopy, site directed mutagenesis
Project start date: 1974-09-01
Project end date: 2008-11-30
5R01GM018457-35 (2007): $320362
Sponsored Links Excellgen http://Excellgen.com
5R01GM018457-34 (2006): $326318
5R01GM018457-33 (2005): $330901
2R01GM018457-32A1 (2004): $331861
BIOPHYSICAL STUDIES OF PROTEINS, NUCLEIC ACIDS, VIRUSES
William A Cramer, Henry Koffler Professor
Biological Sciencespurdue University West Lafayette
302 Wood St
west Lafayette, In 479072108
Grant 5T32GM008296-10 from National Institute Of General Medical Sciences IRG: SRC
Project start date: 1989-07-01
Project end date: 1999-06-30
5T32GM008296-10 (1998): $144106
5T32GM008296-09 (1997): $161229
STRUCTURAL STUDIES OF MEMBRANE BOUND CLOSED STATE OF COLICIN E1 CHANNEL DOMAIN
William A Cramer
University Of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104
Grant 5P41RR009793-040006 from National Center For Research Resources
Keywords: bacteria, biological product, biomedical resource, drug adverse effect, nuclear magnetic resonance spectroscopy, protein
MEMBRANE INTERACTIONS OF COLICIN EI IMMUNITY PROTEIN
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5R01GM018457-25 from National Institute Of General Medical Sciences IRG: BBCB
Project start date: 1974-09-01
Project end date: 1999-03-31
5R01GM018457-25 (1996): $199968
BIOPHYSICAL STUDIES OF PROTEINS, NUCLEIC ACIDS, VIRUSES
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette 302 Wood St West Lafayette, In 479072108
Grant 5T32GM008296-08 from National Institute Of General Medical Sciences IRG: SRC
Project start date: 1989-07-01
Project end date: 1999-06-30
5T32GM008296-08 (1996): $149979
2T32GM008296-06 (1994): $128394
THE MECHANISM OF ACTION OF COLICIN
William A Cramer, Henry Koffler Professor
Purdue University West Lafayette, In 47907
Grant 5R01GM018457-05 from National Institute Of General Medical Sciences IRG: BBCA
Project start date: 1974-09-01
Project end date: 1979-08-31
Sponsored Links Excellgen http://Excellgen.com