CENTER FOR PROTEIN STRUCTURE AND FUNCTION
Francis S Millett, Distinguished Professor
University Of Arkansas At Fayetteville, 120 Ozark Hall, Fayetteville, Ar 72701
Grant 5P20RR015569-10 from National Center For Research Resources
Project start date: 2000-09-30
Project end date: 2011-02-27
Budget start date: 1-MAR-2009
Budget end date: 27-FEB-2011
PFA/PA: RFA-RR-04-008
5P20RR015569-10 (2009): $1965166
Sponsored Links Excellgen http://Excellgen.com
CENTER FOR PROTEIN STRUCTURE AND FUNCTION
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 5P20RR015569-05 from National Center For Research Resources IRG: ZRR1
Abstract: This is a proposal to establish a Center for Protein Structure and Function at the University of Arkansas Protein structure and function is a central biomedical research area within structural biology, and is also crucial to the emerging field of structure-based drug discovery and design. The Center will build upon the unique research expertise of our faculty in the areas of X-ray crystallography, solution and solid-state NMR, mass spectrometry, computational chemistry, rapid laser-initiated kinetic analysis, peptide and drug design, and chemical synthesis. While building on current strengths, the establishment of the Center will substantially augment our capabilities in each of these areas, and will allow us to attract new colleagues to joint our efforts. The unique combination of scientific expertise and state-of-the-art instrumentation in the Center will foster new opportunities for collaboration, and will position our faculty to develop innovative approaches to biomedical research problems. The center will support five multi-disciplinary, collaborative research projects involving 8 junior faculty and 5 new faculty who will receive direct NIH COBRE grant support, and 9 senior faculty who will provide expertise in a broad range of techniques needed to study protein structure and function. The Center will provide the junior investigators with the support and mentoring necessary to develop nationally competitive biomedical research careers, and attract funding through the normal NIH grant mechanism. All five research projects will involve a multi-disciplinary, collaborative approach to obtaining a better understanding of the structure and function of biomedically important proteins. Project 1 will focus on protein folding and orientation within membranes, and peptide transport via ABC-type permeases. Applications will include a better understanding of Clostridium infections and development of anti- Clostridium drugs. Project 2 will involve the development of new families of specific protein inhibitors as candidates for new drugs. Current targets include nucleic acid analogues that can bind to the NS3 helicase of the hepatitis C virus. Project 3 will explore new approaches to determining the structures of signal recognizing particles, which facilitate protein targeting. Project 4 will focus on new experimental and theoretical approaches to understanding the principals governing protein folding. Project 5 will utilize powerful new rapid kinetics understanding the principals governing protein folding. Project 5 will utilize powerful new rapid kinetics techniques to obtain a better understanding of energy coupling mechanisms in oxidative phosphorylation.
Keywords: protein structure function
Project start date: 2000-09-30
Project end date: 2005-09-29
5P20RR015569-05 (2004): $1934441
5P20RR015569-04 (2003): $1940472
5P20RR015569-03 (2002): $1942807
5P20RR015569-02 (2001): $1939159
5P20RR015569-08 (2007): $2004487
5P20RR015569-07 (2006): $2068071
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 5P20RR015569-059001 from National Center For Research Resources IRG: ZRR1
Keywords: biomedical facility, protein transport
Project start date: 2004-07-01
Project end date: 2005-06-30
Grants awarded to Francis S Millett
STUDENT AND/OR TEACHER TRAINING OPPORTUNITIES
Francis S Millett, Distinguished Professor
University Of Arkansas At Fayetteville, 120 Ozark Hall, Fayetteville, Ar 72701
Abstract: This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Institution will hire a student and/or teacher as part of the more than $33 million in ARRA funds allotted to create research experiences over the next two summers for over 3,700 high school and college students and science teachers at NIH-funded laboratories across the country
Keywords: CRISP; Computer Retrieval of Information on Scientific Projects Database; Country; Funding; Grant; Institution; Investigators; Laboratories; NIH; National Institutes of Health; National Institutes of Health (U.S.); Research; Research Personnel; Research Resources; Researchers; Resources; Science; Source; Students; Training; United States National Institutes of Health; college student; experience; high school; protein structure function; teacher; university student
Project start date: 2009-07-13
Project end date: 2010-09-30
Budget start date: 13-JUL-2009
Budget end date: 30-SEP-2010
PFA/PA: RFA-RR-04-008
3P20RR015569-10S1_6789 (2009): $656040
CENTER FOR PROTEIN STRUCTURE AND FUNCTION
Francis S Millett, Distinguished Professor
University Of Arkansas At Fayetteville, 120 Ozark Hall, Fayetteville, Ar 72701
Grant 3P20RR015569-10S2 from National Center For Research Resources
Abstract: This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. For research into new antiviral medications via consideration of opposing actions of virus-specific and host proteins that function as part of a body´s viral defense mechanism
Keywords: No Project Terms available
Project start date: 2009-08-18
Project end date: 2011-08-17
Budget start date: 18-AUG-2009
Budget end date: 17-AUG-2011
PFA/PA: RFA-RR-04-008
3P20RR015569-10S2 (2009): $854803
3P20RR015569-10S2_6800 (2009): $854803
RENOVATION SUPPLEMENT FOR COBRE
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 3P20RR015569-03S1 from National Center For Research Resources IRG: STRB
Abstract: This is an application for the alteration and renovation of laboratories and core facilities that are urgently needed for the COBRE Center for Protein Structure and Function (CPSF) at the UA. The CPSF was established in October 2000 with (P20 RR15569-01). Protein structure and function is a central biomedical research area that has great potential for leading to improvements in human health. The Center supports five multidisciplinary, collaborative research projects involving eight junior faculty and six new faculty who receive direct National Institutes of Health (NIH) COBRE research support, and nine senior faculty who provide expertise in a broad range of techniques needed to study protein structure and function. All five research projects involve a multidisciplinary, collaborative approach to obtaining a better understanding of the structure and function of biomedically important proteins. Project 1 focuses on protein folding and orientation within membranes, as well as peptide transport via ABC-type permeases. Applications include a better understanding of Clostridium infections and development of anti-Clostridium drugs. Project 2 involves the development of new families of specific protein inhibitors as candidates for new drugs. Current targets include nucleic acid analogues that can bind to the NS3 helicase of the hepatitis C virus. Project 3 explores new approaches to determining the structures of signal recognition particles, which facilitate protein targeting. Project 4 focuses on new experimental and theoretical approaches to understanding the principles governing protein folding. Project 5 utilizes powerful new rapid kinetics techniques to obtain a better understanding of energy coupling mechanisms in oxidative phosphorylation. The Center provides the junior investigators with the support and mentoring necessary to develop nationally competitive, NIH-supported biomedical research careers. The specific aims of this supplemental application are the alteration and renovation of rooms currently used for teaching activities. The room would be converted into four new research laboratories needed for the Center in the following areas 1) protein preparation, protein folding, drug design, and membrane protein Interaction.
Keywords: protein structure function
Project start date: 2000-09-30
Project end date: 2005-08-31
3P20RR015569-03S1 (2002): $500000
Center For Protein Structure And Function
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 2P20RR015569-06 from National Center For Research Resources IRG: ZRR1
Abstract: This is a proposal to renew the NCRR COBRE Center for Protein Structure and Function at the University of Arkansas, which was established in October, 2000 with NIH COBRE Grant 1 P20 RR15569-01. Protein structure and function is a central biomedical research area that has great potential for leading to improvements in human health. The goals of the COBRE Center are to strengthen collaboration between investigators and allow them to develop promising new approaches to biomedical research in protein structure and function. The Center is building upon the unique research expertise of our faculty in the areas of X-ray crystallography, NMR spectroscopy, mass spectrometry, and drug discovery. The center will support five thematically-linked multidisciplinary research projects involving 8 junior faculty and 5 new faculty who receive direct NIH COBRE grant support, and 8 senior faculty who provide expertise in a broad range of techniques needed to study protein structure and function. The Center provides the junior investigators with the support and mentoring necessary to develop nationally competitive biomedical research careers. All five research projects will involve a multidisciplinary, collaborative approach to obtaining a better understanding of the structure and function of biomedically important proteins. Project 1 will focus on the interaction of the human fibroblast growth factor protein with its receptor, which regulates a wide array of key physiological processes including embryogenesis, cell growth, differentiation, and wound healing. Project 2 will explore new approaches to determining the structures of signal recognition particles, which facilitate protein targeting. Project 3 will involve a study of proteins of the extracellular matrix, including collagen binding proteins. Project 4 will involve the development of new families of specific protein inhibitors as candidates for new drugs. Project 5 will focus on studies of the structure and function of membrane proteins. All of the projects are focused on problems that could lead to improvements in human health. For example, project 1 could lead to new therapeutic strategies against a variety of fibroblast growth factor-mediated disorders and wound healing, project 3 is directed towards development of a novel drug-delivery vehicle for the extracellular matrix which could lead to improved nerve regeneration, and project 4 is focused on the development of improved pharmaceuticals to treat Hepatitis C and HIV.
Keywords: protein structure function, structural biology
Project start date: 2000-09-30
Project end date: 2010-04-30
2P20RR015569-06 (2005): $1776805
1P20RR015569-01 (2000): $1874433
1P30RR031154-01 (2010): $1091700
3P20RR015569-10S1 (2009): $656040
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 5R01GM020488-34 from National Institute Of General Medical Sciences IRG: BMT
Abstract: The long range goals of our research program supported by this grant are to develop and apply new techniques to study biological electron transfer reactions. These reactions play essential roles in numerous biological processes important to human health, including oxidative phosphorylation in mitochondria. Despite the importance of these reactions, relatively few techniques are available to measure the actual rate of electron transfer between two redox centers in a protein complex. A new method to study biological electron transfer has been introduced which utilizes a covalently attached tris(bipyridine)ruthenium group [Ru(ll)]. Several strategies have been developed for the design and synthesis of ruthenium-labeled redox proteins that are optimized for the measurement of interprotein electron transfer. Photoexcitation of Ru(ll) to a metal-to-ligand charge-transfer state, Ru(ll*), leads to rapid reduction or oxidation of a redox center within 16 Angstroms. This new technique is being used to measure intracomplex electron transfer between cytochrome c and its physiological partners, cytochrome c oxidase, cytochrome bc1, and cytochrome c peroxidase. A new ruthenium dimer has recently been developed which binds with high affinity to cytochrome bc1 and can photooxidize cyt c1 within 1 microsecond. This new technique has been used to measure the rate constant for electron transfer between the Rieske iron-sulfur center and cyt c1 for the first time. The specific aims for the next grant period are to 1) Carry out a detailed study of electron transfer within cytochrome bc1. A major goal will be to determine what factors control the conformational changes in the Rieske iron-sulfur protein as it transfers electrons from QH2 in the Qo site to cyt c1. 2) Characterize electron transfer between cyt c and cyt c1 in the cytochrome bc1 complex. 3) Characterize electron transfer between cytochrome c and cytochrome c oxidase using rapid kinetics and site-directed mutagenesis. Major goals will be to determine the pathway and kinetics of electron transfer from cytochrome c through CuA and heme a to the heme a3--CuB binuclear center, as well as coupled proton uptake and release.
Keywords: chemical synthesis, cytochrome b, cytochrome c, cytochrome c peroxidase, cytochrome oxidase, electron transport, enzyme complex, membrane transport protein, ruthenium, bioenergetics, hemoprotein structure, iron sulfur protein, metalloenzyme, oxidation reduction reaction, protein structure function, Rhodopseudomonas, X ray crystallography, chemical kinetics, laser spectrometry, site directed mutagenesis
Project start date: 1976-06-01
Project end date: 2008-07-31
5R01GM020488-34 (2006): $294752
3R01GM020488-33S1 (2005): $65640
Sponsored Links Excellgen http://Excellgen.com
5R01GM020488-33 (2005): $302579
5R01GM020488-32 (2004): $307453
2R01GM020488-31 (2003): $307740
5R01GM020488-36 (2010): $292655
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 5R01GM020488-22 from National Institute Of General Medical Sciences IRG: PB
Abstract: The long range goals of our research program supported by NIH GM20488 are to develop and apply new techniques to study biological electron transfer reactions. Despite the importance of these reactions, relatively few techniques are available to measure the actual rate of electron transfer between redox centers within a protein complex. New approaches to this problem are being explored in a multi-disciplinary project directed by an inorganic chemist and physical biochemist. A new class of ruthenium- labeled proteins have been developed that allow biological electron transfer reactions to be initiated by a laser pulse and detected on a nanosecond time scale. A novel two-step procedure was developed to label cytochrome c at specific lysine amino groups with ruthenium bis(bipyridine) dicarboxybipyridine. More than ten singly labeled derivatives have now been purified and characterized. One of the most remarkable properties of this ruthenium complex is that it can be photoexcited to a mental-to-ligand charge-transfer state, RuII*, which is a strong reducing agent and can transfer an electron to the heme group FeIII in cytochrome c. The electron transfer rate constants were found to be 14-30 X 106 S-1 for derivatives modified at lysines 13, 27, and 72, which have a separation of 8 to 12 A between the ruthenium and heme groups. These are the largest electron transfer rate constants ever measured for a native heme group in a cytochrome. The rate constants were significantly smaller for derivatives with a larger separation between the ruthenium and heme groups. A new method was also developed to measure the rates of electron transfer within complexes between the ruthenium-cytochrome c derivatives and other proteins. The specific aims for the next granting period will be 1) to measure the photoinduced electron transfer reactions between the ruthenium and heme groups as a function of temperature and driving force to determine the reorganization barrier of the reaction. 2) to study the dependence of electron transfer on distance and protein medium by comparing the rate constants of derivatives labeled at different lysines on the surface of cytochrome c. 3) to study the intra-complex electron transfer reaction between the ruthenium cytochrome c derivatives and cytochrome oxidase. 4) to study the intra-complex, cytochrome c peroxidase, and cytochrome b5. 5) to develop new chemical modification methods to extend the ruthenium labeling technique to other electron transfer proteins. 6) to study the interaction of Rps. viridis cytochrome c2 with the Rps. viridis photosynthetic reaction center.
Keywords: cytochrome c, cytochrome oxidase, electron transport, enzyme complex, hemoprotein structure, membrane protein, ruthenium, bioenergetics, chemical reaction, cytochrome b, cytochrome b5 reductase, enzyme mechanism, ligand, nuclear magnetic resonance spectroscopy, oxidation /reduction, photosynthesis, photosynthetic reaction center, protein reconstitution, protein sequence, protein structure function, Rhodopseudomonas, electron spin resonance spectroscopy, fluorescent dye /probe, high performance liquid chromatography, laser spectrometry, lysine, temperature
Project start date: 1976-06-01
Project end date: 1995-07-31
5R01GM020488-22 (1994): $210626
STUDIES OF ELECTRON TRANSFER PROTEINS
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 5R01GM020488-21 from National Institute Of General Medical Sciences IRG: PB
Abstract: The long range goals of our research program supported by NIH GM20488 are to develop and apply new techniques to study biological electron transfer reactions. Despite the importance of these reactions, relatively few techniques are available to measure the actual rate of electron transfer between redox centers within a protein complex. New approaches to this problem are being explored in a multi-disciplinary project directed by an inorganic chemist and physical biochemist. A new class of ruthenium- labeled proteins have been developed that allow biological electron transfer reactions to be initiated by a laser pulse and detected on a nanosecond time scale. A novel two-step procedure was developed to label cytochrome c at specific lysine amino groups with ruthenium bis(bipyridine) dicarboxybipyridine. More than ten singly labeled derivatives have now been purified and characterized. One of the most remarkable properties of this ruthenium complex is that it can be photoexcited to a mental-to-ligand charge-transfer state, RuII*, which is a strong reducing agent and can transfer an electron to the heme group FeIII in cytochrome c. The electron transfer rate constants were found to be 14-30 X 106 S-1 for derivatives modified at lysines 13, 27, and 72, which have a separation of 8 to 12 A between the ruthenium and heme groups. These are the largest electron transfer rate constants ever measured for a native heme group in a cytochrome. The rate constants were significantly smaller for derivatives with a larger separation between the ruthenium and heme groups. A new method was also developed to measure the rates of electron transfer within complexes between the ruthenium-cytochrome c derivatives and other proteins. The specific aims for the next granting period will be 1) to measure the photoinduced electron transfer reactions between the ruthenium and heme groups as a function of temperature and driving force to determine the reorganization barrier of the reaction. 2) to study the dependence of electron transfer on distance and protein medium by comparing the rate constants of derivatives labeled at different lysines on the surface of cytochrome c. 3) to study the intra-complex electron transfer reaction between the ruthenium cytochrome c derivatives and cytochrome oxidase. 4) to study the intra-complex, cytochrome c peroxidase, and cytochrome b5. 5) to develop new chemical modification methods to extend the ruthenium labeling technique to other electron transfer proteins. 6) to study the interaction of Rps. viridis cytochrome c2 with the Rps. viridis photosynthetic reaction center.
Keywords: cytochrome c, cytochrome oxidase, electron transport, enzyme complex, hemoprotein structure, membrane protein, ruthenium, bioenergetics, chemical reaction, cytochrome b, cytochrome b5 reductase, enzyme mechanism, ligand, nuclear magnetic resonance spectroscopy, oxidation /reduction, photosynthesis, photosynthetic reaction center, protein reconstitution, protein sequence, protein structure function, Rhodopseudomonas, electron spin resonance spectroscopy, fluorescent dye /probe, high performance liquid chromatography, laser spectrometry, lysine, temperature
Project start date: 1976-06-01
Project end date: 1995-07-31
5R01GM020488-21 (1993): $207511
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 5R01GM020488-20 from National Institute Of General Medical Sciences IRG: PB
Abstract: The long range goals of our research program supported by NIH GM20488 are to develop and apply new techniques to study biological electron transfer reactions. Despite the importance of these reactions, relatively few techniques are available to measure the actual rate of electron transfer between redox centers within a protein complex. New approaches to this problem are being explored in a multi-disciplinary project directed by an inorganic chemist and physical biochemist. A new class of ruthenium- labeled proteins have been developed that allow biological electron transfer reactions to be initiated by a laser pulse and detected on a nanosecond time scale. A novel two-step procedure was developed to label cytochrome c at specific lysine amino groups with ruthenium bis(bipyridine) dicarboxybipyridine. More than ten singly labeled derivatives have now been purified and characterized. One of the most remarkable properties of this ruthenium complex is that it can be photoexcited to a mental-to-ligand charge-transfer state, RuII*, which is a strong reducing agent and can transfer an electron to the heme group FeIII in cytochrome c. The electron transfer rate constants were found to be 14-30 X 106 S-1 for derivatives modified at lysines 13, 27, and 72, which have a separation of 8 to 12 A between the ruthenium and heme groups. These are the largest electron transfer rate constants ever measured for a native heme group in a cytochrome. The rate constants were significantly smaller for derivatives with a larger separation between the ruthenium and heme groups. A new method was also developed to measure the rates of electron transfer within complexes between the ruthenium-cytochrome c derivatives and other proteins. The specific aims for the next granting period will be 1) to measure the photoinduced electron transfer reactions between the ruthenium and heme groups as a function of temperature and driving force to determine the reorganization barrier of the reaction. 2) to study the dependence of electron transfer on distance and protein medium by comparing the rate constants of derivatives labeled at different lysines on the surface of cytochrome c. 3) to study the intra-complex electron transfer reaction between the ruthenium cytochrome c derivatives and cytochrome oxidase. 4) to study the intra-complex, cytochrome c peroxidase, and cytochrome b5. 5) to develop new chemical modification methods to extend the ruthenium labeling technique to other electron transfer proteins. 6) to study the interaction of Rps. viridis cytochrome c2 with the Rps. viridis photosynthetic reaction center.
Keywords: cytochrome c, cytochrome oxidase, electron transport, enzyme complex, hemoprotein structure, membrane protein, ruthenium, bioenergetics, chemical reaction, cytochrome b, cytochrome b5 reductase, enzyme mechanism, ligand, nuclear magnetic resonance spectroscopy, oxidation /reduction, photosynthesis, photosystem, protein reconstitution, protein sequence, protein structure function, Rhodopseudomonas, electron spin resonance spectroscopy, fluorescent dye /probe, high performance liquid chromatography, laser spectrometry, lysine, temperature
Project start date: 1976-06-01
Project end date: 1995-07-31
5R01GM020488-20 (1992): $203233
STUDIES OF ELECTRON TRANSFER PROTEINS
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 5R01GM020488-19 from National Institute Of General Medical Sciences IRG: PB
Abstract: The long range goals of our research program supported by NIH GM20488 are to develop and apply new techniques to study biological electron transfer reactions. Despite the importance of these reactions, relatively few techniques are available to measure the actual rate of electron transfer between redox centers within a protein complex. New approaches to this problem are being explored in a multi-disciplinary project directed by an inorganic chemist and physical biochemist. A new class of ruthenium- labeled proteins have been developed that allow biological electron transfer reactions to be initiated by a laser pulse and detected on a nanosecond time scale. A novel two-step procedure was developed to label cytochrome c at specific lysine amino groups with ruthenium bis(bipyridine) dicarboxybipyridine. More than ten singly labeled derivatives have now been purified and characterized. One of the most remarkable properties of this ruthenium complex is that it can be photoexcited to a mental-to-ligand charge-transfer state, RuII*, which is a strong reducing agent and can transfer an electron to the heme group FeIII in cytochrome c. The electron transfer rate constants were found to be 14-30 X 106 S-1 for derivatives modified at lysines 13, 27, and 72, which have a separation of 8 to 12 A between the ruthenium and heme groups. These are the largest electron transfer rate constants ever measured for a native heme group in a cytochrome. The rate constants were significantly smaller for derivatives with a larger separation between the ruthenium and heme groups. A new method was also developed to measure the rates of electron transfer within complexes between the ruthenium-cytochrome c derivatives and other proteins. The specific aims for the next granting period will be 1) to measure the photoinduced electron transfer reactions between the ruthenium and heme groups as a function of temperature and driving force to determine the reorganization barrier of the reaction. 2) to study the dependence of electron transfer on distance and protein medium by comparing the rate constants of derivatives labeled at different lysines on the surface of cytochrome c. 3) to study the intra-complex electron transfer reaction between the ruthenium cytochrome c derivatives and cytochrome oxidase. 4) to study the intra-complex, cytochrome c peroxidase, and cytochrome b5. 5) to develop new chemical modification methods to extend the ruthenium labeling technique to other electron transfer proteins. 6) to study the interaction of Rps. viridis cytochrome c2 with the Rps. viridis photosynthetic reaction center.
Keywords: cytochrome c, cytochrome oxidase, electron transport, enzyme complex, hemoprotein structure, membrane protein, ruthenium, bioenergetics, chemical reaction, cytochrome b, cytochrome b5 reductase, enzyme mechanism, ligand, nuclear magnetic resonance spectroscopy, oxidation /reduction, photosynthesis, protein sequence, protein structure function, Rhodopseudomonas, electron spin resonance spectroscopy, fluorescent dye /probe, high performance liquid chromatography, laser spectrometry, lysine, temperature
Project start date: 1976-06-01
Project end date: 1995-07-31
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 2R01GM020488-27 from National Institute Of General Medical Sciences IRG: BMT
Abstract: The long range goals of our research program supported by NIH GM20488 are to develop and apply new techniques to study biological electron transfer reactions. Despite the importance of these reactions to numerous biological processes, relatively few techniques are available to measure the actual rate of electron transfer between two redox centers in a protein complex. We have recently introduced a new method to study biological electron transfer that utilizes a covalently attached tris(bipyridine)ruthenium group [Ru(ll)]. Several strategies have been developed for the design and synthesis of ruthenium-labeled redox proteins that are optimized for the measurement of inter protein electron transfer. Over twenty singly labeled derivatives of cytochrome c have now been prepared and characterized. One of the most remarkable properties of Ru(ll) is that it can be photoexcited to a metal-to-ligand charge-transfer state, Ru(lI*), which is a strong reducing agent and can rapidly transfer an electron to the heme group Fe(llI) in cytochrome c. Rate constants up to 3 x 10(7) s-1 are observed for derivatives with separations of about 12 Angstroms between the ruthenium and heme groups. We are using this new technique to measure intracomplex electron transfer between cytochrome c and its physiological partners, cytochrome c oxidase, cytochrome c1, cytochrome c peroxidase. The rate constants for these reactions range from 10(4) to over 10(6) s-1 , and are up to three orders of magnitude larger than previous estimates. The specific aims for the next grant period are to 1) Carry out a detailed study of the electron transfer reaction between cytochrome c and cytochrome c peroxidase that brings together rapid kinetics, site-directed mutagenesis, and X-ray crystallography. The rate constant, reorganization energy, interaction domain, and pathway of each electron transfer step in the mechanism will be determined. 2) Carry out a detailed study of the electron transfer reaction between cytochrome c and cytochrome c oxidase. A major goal will be to determine the pathway and kinetics of electron transfer from cytochrome c through Cu-a and heme a to the heme a3--Cu-B binuclear center under coupled turnover conditions. 3) Carry out a detailed study of the electron transfer reaction between cytochrome c and the cytochrome bc1 complex. A major goal will be to determine the pathway and kinetics of electron transfer from the Rieske iron-sulfur center to cytochrome c1 and to cytochrome c.
Keywords: chemical synthesis, cytochrome b, cytochrome c, cytochrome c peroxidase, cytochrome oxidase, electron transport, enzyme complex, membrane transport protein, ruthenium, bioenergetics, cytochrome b5 reductase, hemoprotein structure, hydrogen channel, iron sulfur protein, metalloenzyme, oxidation /reduction, protein structure /function, Rhodopseudomonas, X ray crystallography, laser spectrometry, site directed mutagenesis
Project start date: 1976-06-01
Project end date: 2003-07-31
2R01GM020488-27 (1999): $296823
5R01GM020488-30 (2002): $297025
Sponsored Links Excellgen http://Excellgen.com
5R01GM020488-29 (2001): $288375
5R01GM020488-28 (2000): $279974
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 5R01GM020488-26 from National Institute Of General Medical Sciences IRG: PB
Abstract: The long range goals of our research program supported by NIH GM20488 are to develop and apply new techniques to study biological electron transfer reactions. Despite the importance of these reactions to numerous biological processes, relatively few techniques are available to measure the actual rate of electron transfer between two redox centers in a protein complex. We have recently introduced a new method to study biological electron transfer that utilizes a covalently attached tris(bipyridine)ruthenium group [Ru(ll)]. Several strategies have been developed for the design and synthesis of ruthenium-labeled redox proteins that are optimized for the measurement of inter protein electron transfer. Over twenty singly labeled derivatives of cytochrome c have now been prepared and characterized. One of the most remarkable properties of Ru(ll) is that it can be photoexcited to a metal-to-ligand charge-transfer state, Ru(lI*), which is a strong reducing agent and can rapidly transfer an electron to the heme group Fe(llI) in cytochrome c. Rate constants up to 3 x 10(7) s-1 are observed for derivatives with separations of about 12 Angstroms between the ruthenium and heme groups. We are using this new technique to measure intracomplex electron transfer between cytochrome c and its physiological partners, cytochrome c oxidase, cytochrome c1, cytochrome c peroxidase. The rate constants for these reactions range from 10(4) to over 10(6) s-1 , and are up to three orders of magnitude larger than previous estimates. The specific aims for the next grant period are to 1) Carry out a detailed study of the electron transfer reaction between cytochrome c and cytochrome c peroxidase that brings together rapid kinetics, site-directed mutagenesis, and X-ray crystallography. The rate constant, reorganization energy, interaction domain, and pathway of each electron transfer step in the mechanism will be determined. 2) Carry out a detailed study of the electron transfer reaction between cytochrome c and cytochrome c oxidase. A major goal will be to determine the pathway and kinetics of electron transfer from cytochrome c through Cu-a and heme a to the heme a3--Cu-B binuclear center under coupled turnover conditions. 3) Carry out a detailed study of the electron transfer reaction between cytochrome c and the cytochrome bc1 complex. A major goal will be to determine the pathway and kinetics of electron transfer from the Rieske iron-sulfur center to cytochrome c1 and to cytochrome c.
Keywords: cytochrome c, cytochrome c peroxidase, cytochrome oxidase, electron transport, enzyme complex, enzyme mechanism, membrane transport protein, ruthenium, bioenergetics, cytochrome b, cytochrome b5 reductase, hemoprotein structure, hydrogen channel, iron sulfur protein, metalloenzyme, oxidation /reduction, protein reconstitution, protein structure /function, Rhodopseudomonas, X ray crystallography, high performance liquid chromatography, laser spectrometry, protein sequence, site directed mutagenesis
Project start date: 1976-06-01
Project end date: 1999-07-31
5R01GM020488-26 (1998): $257579
5R01GM020488-25 (1997): $247672
CHEMICAL MODIFICATION STUDIES OF MEMBRANE BOUND PROTEINS
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 5R01GM020488-17 from National Institute Of General Medical Sciences IRG: BIO
Abstract: The broad objective of the proposed research is to develop new chemical modification and cross-linking techniques to study protein structure and function in biological membranes. We have recently prepared 17 different derivatives of cytochrome c in each of which a single lysine amino group has been modified to form an uncharged CF3CO- or CF3PhNHCO-lysine. Enzyme kinetic studies with these derivatives have shown that the interaction domain for both cytochrome c1 and cytochrome oxidase involves the ring of positively charged lysine amino groups surrounding the heme crevice of cytochrome c. This result suggests that cytochrome c must undergo some type of diffusion as it transports electrons from cytochrome c1 to cytochrome oxidase. Our studies are now focused on identifying which specific residues on cytochromes oxidase and c1 are involved in binding cytochrome c. We have recently used the water-soluble carbodiimide EDC to modify three carboxyl groups on cytochrome oxidase that are required for cytochrome c binding. HPLC peptide mapping was used to identify the labeled carboxylates as Asp 112, Glu 114, and Glu 198 of subunit II. Glu 198 is located between cysteine residues 196 and 200 which have been proposed to ligand the EPR-detectable copper. This copper atom thus appears to be located close enough to the cytochrome c binding site to be reduced directly. Chemical modification techniques are proposed to determine where the other electron acceptor, cytochrome a, is located in the oxidase structure. We have recently developed a new general procedure to cross-link any lysine amino group on cytochrome c to its complementary carboxyl group on the redox partner. HPLC peptide mapping will be used to identify the specific carboxyl group that interacts with each lysine. This should help refine our understanding of the surface topology and folding patterns of these proteins. The type of diffusional motion cytochrome c must undergo will be evaluated from the effect of different specific cross-links on the rate of electron transport from cytochrome c1 to cytochrome oxidase. These studies should also indicate whether cytochrome c1 must have some rotational flexibility to transfer electrons from the iron-sulfur protein in the cytochrome bc1 complex to cytochrome c.
Keywords: CHEMICAL TRANSFER, ELECTRON TRANSPORT, ENZYME COMPLEXES, HEMOPROTEINS STRUCTURE, HEMOPROTEINS, CYTOCHROME C, OXIDOREDUCTASES, CYTOCHROME OXIDASES, PROTEINS, MEMBRANE PROTEINS, CELL COMPONENTS, MITOCHONDRIAL MEMBRANE, CHEMICAL BONDS, BINDING AND BINDING SITES, CHEMICAL BONDS, CROSSLINKS, CHEMICAL REACTIONS (DYNAMICS), CHEMICAL STRUCTURE--BIOLOGICAL ACTIVITY, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, NMR, ENZYME MECHANISMS, HEMOPROTEINS, CYTOCHROME B, MEMBRANE, MEMBRANE (BIOLOGICAL) STRUCTURE, METALS, HEAVY METALS, CHROMIUM (COMPOUNDS), OXIDATION-REDUCTION, OXIDOREDUCTASES, CYTOCHROME B5 REDUCTASES, PEROXIDASES, PORPHYRINS, CHLOROPHYLL, PROTEIN (PEPTIDE) SEQUENCE, PROTEINS, BINDING PROTEINS, SULFUR AMINO ACIDS, CYSTEINE, ANIMALS, CHORDATES, MAMMALS, UNGULATES, CATTLE, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, ESR, DIAMINO ACIDS, LYSINE, DYES, FLUORESCENT DYES AND PROBES
Project start date: 1976-06-01
Project end date: 1990-07-31
NMR STUDIES OF MEMBRANE BOUND PROTEINS
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 5R01GM020488-12 from National Institute Of General Medical Sciences IRG: BBCB
Abstract: The broad objective of the proposed research is to develop and exploit nuclear magnetic resonance (NMR) techniques for the study of protein structure and function in biological membranes. We have prepared seventeen different cytochrome c derivatives in each of which a single lysine group has been specifically modified with a fluorine containing reagent. All surface areas on cytochrome c have been labelled with these F-19 NMR probes. F-19 NMR methods will be used to accurately locate the binding sites on cytochrome c for cytochrome oxidase, cytochrome c1, and phospholipid membranes. We will investigate possible conformational changes at these binding sites due to electron transfer, and also attempt to measure distances between the F-19 labels on cytochrome c and paramagnetic metals in the oxidase and cytochrome c1. This information is very important for an understanding of what types of electron transfer mechanisms might be feasible. F-19 NMR methods will be used to study the interaction of the cytochrome c derivatives with intact mitochrondria. These studies should allow us to determine what components of the mitochondrial membrane cytochrome c is binding to under different physiological conditions. A central question is whether cytochrome c can bind to cytochrome oxidase and cytochrome reductase simultaneously, thus transporting electrons directly, or whether the binding sites are competitive and cytochrome c must undergo rotational and translational motion to transport an electron from cytochrome reductase to cytochrome oxidase. We will also carry out a detailed study of the cytochrome b 5-cytochrome c complex, which is the only such system in which both components have been characterized by x-ray crystallography.
Keywords: BIOPHYSICAL CHEMISTRY STUDY SECTION, CELL COMPONENTS, MITOCHONDRIAL MEMBRANE, ENZYME COMPLEXES, HEMOPROTEINS STRUCTURE, HEMOPROTEINS, CYTOCHROME B, HEMOPROTEINS, CYTOCHROME C, PROTEINS, MEMBRANE PROTEINS, BIOMEDICAL ENGINEERING, INSTRUMENTATION NOT CLINICALLY ORIENTED, CHEMICAL BONDS, VIBRATIONS, CHEMICAL REACTION RATES, CHEMICAL REACTIONS (DYNAMICS), CHEMICAL REACTIONS, DISSOCIATION-ASSOCIATION, CHEMICAL TRANSFER, ELECTRON TRANSPORT, CHEMISTRY, ANALYTICAL METHODS, SPECTROMETRY, NMR, MATHEMATICS, BIOMATHEMATICS (GENERAL), MEMBRANE, MEMBRANE (BIOLOGICAL) STRUCTURE, METALLOPROTEINS, ADRENAL FERREDOXIN, OXIDATION-REDUCTION, OXIDOREDUCTASES, CYTOCHROME B5 REDUCTASES, OXIDOREDUCTASES, CYTOCHROME OXIDASES, PEROXIDASES, PHOSPHOLIPIDS, PROTEIN BINDING, ionic strength, molecular rearrangement, phosphorylation, DIAMINO ACIDS, LYSINE, DYES, FLUORESCENT DYES AND PROBES, FUNGI, YEASTS, MAMMALS, ARTIODACTYLA, CATTLE, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, ADP, PURINE NUCLEOTIDES, ADENINE NUCLEOTIDES, ATP, phosphorus
Project start date: 1976-06-01
Project end date: 1985-07-31
Sponsored Links Excellgen http://Excellgen.com
Francis S Millett
Chemistry And Biochemistry (chbc)university Of Arkansas At Fayetteville
Grant 2R01GM020488-35A1 from National Institute Of General Medical Sciences IRG: BBM
Project start date: 1976-06-01
Project end date: 2011-08-31
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 2P20RR015569-069001 from National Center For Research Resources IRG: ZRR1
Keywords: X ray crystallography, biomedical facility, nuclear magnetic resonance spectroscopy, protein structure function, structural biology
Project start date: 2005-09-30
Project end date: 2006-04-30
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 5R01GM020488-24 from National Institute Of General Medical Sciences IRG: PB
Project start date: 1976-06-01
Project end date: 1999-07-31
5R01GM020488-24 (1996): $238147
Francis S Millett, Professor Of Chemistry
University Of Arkansas At Fayetteville 120 Ozark Hall Fayetteville, Ar 72701
Grant 1S15GM050477-01 from National Institute Of General Medical Sciences IRG: NSS
Keywords: biomedical equipment purchase
Project start date: 1993-06-04
Project end date: 1994-12-31
1S15GM050477-01 (1993): $6371
Sponsored Links Excellgen http://Excellgen.com