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WILLIAM A. CRAMER

Henry Koffler Distinguished Professor of Biological Sciences
HOCK 325
Phone: 765-494-4956

Personal Site: Link

Structure-Function of Membrane Proteins

Although there are more than 100,000 independent structures of soluble proteins in the Protein Data Bank, there are only approximately 500 such structures of integral membrane proteins in the PDB, and only a fraction are structures of hetero-oligomeric membrane proteins solved to a resolution ≤ 3.0 Å.  These membrane proteins are of great interest in health and disease-related studies.  In addition, they provide the major problem in understanding the assembly of membrane proteins.

            A 2.5 Å structure has been obtained of the hetero-oligomeric cytochrome b6f complex, the electron and proton-transferring complex in photosynthetic electron transport (protein data bank deposition, PDB 4OGQ) The dimeric 250-kDa complex contains 8 polypeptide subunits, 13 trans-membrane helices, 7 prosthetic groups (4 hemes, 1 FeS cluster, 1 Chl a, 1 β-carotene), and 23 lipid binding sites per monomer. Such energy-transducing membrane proteins have the special property that their function can be studied by spectrophotometry. This property was used to determine that the dielectric constants between the 4 hemes in the dimer are heterogeneous, partly a consequence of integration of lipid in the protein.

            Some problems of current interest are (i) further analysis of the role of lipid in stabilizing the b6complex, (ii) effect of the trans-membrane electrical potential on the atomic structure, (iii) engineering the electron transport chain with a possible application to modifying the rate-limiting step of photosynthesis, (iv) understanding the mechanism of the elevated rate of superoxide production, (v) understanding the unique mechanism of redox-dependent cross-membrane information transfer in which oxidation of plastoquinol on one side of the membrane actives a kinase on the other side.

I. Photosynthetic Energy Transduction: Structure-Function of the Hetero-Oligomeric Membrane Lipoprotein, the Electron Transport Proton Translocating Cytochrome b6f Complex

 

Fig 1.  Cytochrome b6f complex; electron transport pathway and site of superoxide generation.  (A) Trans-membrane quinol-mediated electron transfer pathway in dimeric cytochrome b6f complex, crystallized from cyanobacteria, showing site of superoxide generation on electro-chemically positive (p, lumen side) of the membrane.  (B ) View of b6f complex along membrane normal, showing 26 trans-membrane helices, 2-fold symmetry axis, lipids and bound detergent. (C) p-side binding site of quinone analogue, tri-decyl-stigmatellin

II.  Hypothesis for trans-membrane (TM) signaling and activation of Stt7 kinase.

TM signaling involving a serine-threonine kinase (Stt7 in Chlamydomonas reinhardtii) directs light energy distribution between the 2 photosystems of oxygenic photosynthesis.  Oxidation of plastoquinol mediated by the cyt b6f complex on the electrochemically positive (p) side of the thylakoid membrane, activates the kinase domain of Stt7 on the trans (negative (n)) side, leading to phosphorylation and redistribution (“state transition”) of the light-harvesting chlorophyll proteins between the 2 photosystems. Stt7 kinase was cloned, expressed, and purified, (a) found to be active in vitro in the presence of reductant, and (b) purified as a tetramer, determined by analytical ultracentrifugation, EM, and electrospray-ionization mass spectrometry, with a molecular weight of 332 kDa, consisting of an 83.41 k. (c) Far-UV circular dichroism spectra showed Stt7 to be mostly α-helical, and document a physical interaction with the b6f complex.  O2•-, generated in the b6f complex via plastosemiquinone, proposed to reduce S-S bond between 2 Cys in Stt7, causing a conformation change in the Stt7 TM domain, which is transmitted to the n-side through a proline hinge in TM domain, thus activating the kinase. In cartoon mode, p-side domain containing Cys 68 and 73 shown as blue oval, TM domain as brown cylinders, and the proline hinge as a gray (A) and yellow (B) pentagon. n-side Stt7 kinase domain shown in gray in inactive state (labeled A) and, when activated, as red rectangle (labeled B) C-terminal disordered domain, orange rectangle. Cyt b6f complex (PDB ID 4H13) shown in surface representation, with the p-side plastoquinol (Qp) binding site marked by the quinol-analog tridecyl stigmatellin (purple sticks). Lipid bilayer, light green. (A) Qp-site of cyt b6f complex unoccupied, cysteines 68 and 73 of Stt7 form disulfide bond, and kinase remains inactive. (B) Upon binding and oxidation of plastoquinol at the Qp-site, PQ•- is generated which, serving as reductant for O2, generating O2•-, is oxidized to PQ.   

 III. Structure-Function of Integral Membrane Proteins: (ll) β-barrel proteins, protein import, the colicin transloco

Like many bacteriophage, for their import into the bacterial cell, colicins parasitize outer membrane proteins normally used for uptake of metabolites (e. g., sugars, metals, vitamins).

            Hypotheses for the pathway and mechanisms of import into E. coli of the cytotoxic E colicins are based on crystal structures of the colicins, and of complexes of the colicins with their outer membrane receptors, for example, the vitamin B12 receptor (BtuB), [panels A, B] which the colicins parasitize for import. (A) 1.95 Å structure of the BtuB vitamin B12 receptor (J. Mol. Biol, 364, 716, 2006). (B) (side view). (C) A 2.75 Å structure of the complex of receptor-binding domain of the endoribonucleolytic colicin E3 showed its elongate 100 Å long colicin domain to be bound obliquely, in which it appears to be 'fishing' for a second outer membrane receptor-translocator (Nature Struct. Biol., 10, 948-, 2003) (D). A similar structure has been obtained with the receptor-binding domain of colicin E2 (Sharma et al., JBC, 2007).  

            Single molecule fluorescence studies show a rapid diffusion of the colicin-BtuB receptor complex in the outer membrane as it diffuses rapidly in the outer membrane (Biophys. J., 99: 3880-3886, 2010) and searches for its outer membrane OmpF translocator protein in what has been described as a “fishing pole” mechanism. Thus, colicin import across the outer membrane involves formation of a ‘Translocon' between BtuB and OmpF.  A 3.0 Å crystal structure was obtained of a complex between the translocation domain of colicin E3 and OmpF, and 1.6 Å of OmpF alone (EMBO J., 27, 2171, 2008), and summarized in a review co-authored with K. Jakes (Annual Reviews of Genetics, 46, 209-231, 2012).

            Applications of these approaches and concepts to membrane-mediated action of proteins involved in other infectious diseases: (a) The membrane-mediated interaction of the alpha-synuclein protein, implicated in the etiology of Parkinson’s Disease, in which the 140 residue disordered polypeptide was found to assume a significantly ordered helical structure upon binding to anionic liposome membranes (Biochemistry, 46:14369-14379). (b) Development of vaccines against bacterial proteins containing the Omp antigen to treat infectious diseases, in progress.

Education

B. S., Physics, Massachusetts Institute of Technology
Ph. D., Biophysics, University of Chicago
Postdoctoral, University of California/San Diego

Teaching 

 Recent Outside Lectures

  • "Structure-Function of, and Trans-Membrane Signaling by, a Large Hetero-Oligomeric Membrane Protein Complex," and "Mechanisms of Membrane Import of a Bacterial Cytotoxin,"  Orton K. Stark Lectures, Dept. of Microbiology, Miami University, Oxford, OH, April 18-20, 2017
  • "Structure-Function of the Cytochrome b6f Lipoprotein Complex; Interaction with the Trans-Membrane Stt7 Ser-Thr Kinase that Mediates State Transitions." Keynote Lecture, 26th Western Photosynthesis Conf., Pt. Reyes, CA, Jan 5-8, 2017.
  • "Redox-Dependent Trans-Membrane Signaling in Photosynthetic State Transitions: Interaction of a Serine-Threonine Kinase with the Cytochrome
    b6f Complex," 7th International Conference, Photosynthesis Research for Sustainability,
    Pushchino, Moscow Region, June 19-26, 2016.
  • "A Potential Problem with the Accuracy of Membrane Protein Structures”, NIH Roadmap/Argonne National Laboratory Meeting on Membrane Protein Structures, April 11, 2015
  • "Mapping the Dielectric Heterogeneity of the Cytochrome b6f Membrane Lipoprotein Complex," Symposium on Membrane Proteins, University of Delaware, Newark, Deleware, May 12, 2014.
  • "Structure-Function of Two Membrane Proteins: helical and β-Barrel," Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, April 3, 2014.
  • "Mapping of the Heterogenous Dielectric Constant of the Cytochrome b6f Complex," Department of Biochemistry, UC Davis, Davis, CA, March 7, 2014.
  • "Mapping Dielectric Heterogeneity in a Membrane Lipoprotein: the Cytochrome b6f Complex," Department of Chemistry, UCLA, Los Angeles, CA, February 19, 2014.
  • "Dielectric Heterogeneity in the Cytochrome b6f Complex," Annual Meeting of the German Society of Biochemistry and Molecular Biology, Frankfurt, Germany, October 3, 2013.
  • "Dielectric Heterogeneity in the Cytochrome b6f Complex," International Congress of Photosynthesis, St. Louis, MO, August 12, 2013.
  • "Properties of the Hetero-Oligomeric Cytochrome b6f-Lipidic Charge Transfer Complex of Oxygenic Photosynthesis," Department of Molecular Biology, University of Geneva, Geneva, Switzerland, June 17, 2013.
  • “Transfer of electrons, Protons, and Information in the Cytochrome b6f Lipidic Complex of Oxygenic Photosynthesis,” NIH Roadmap Meeting to Membrane Protein Technologies, University of California, San Francisco, CA, November 27-30, 2012.
  • "Structure-Function of the Cytochrome b6f Complex of Oxygenic Photosynthesis," Department of Chemistry and Biochemisty, Arizona State University, November 15, 2012.“Lipid Functions in Cytochrome bc Complexes: an unusual event in evolution,” Conference, “How bugs kill bugs, progress and challenges in bacteriocin research,” British Biochemical Society, Nottingham, England. July 16 – 18, 2012.
  • "Lipid Functions in cytochrome bc complexes: an unusual event in evolution," May 13-17, 2012. Membrane Conference, Arizona State University, Phoenix, AZ.
  • “The Cytochrome b6f Complex: an Odd Lipid Effect in Evolution; An Electrostatically Constrained Electron Transport Pathway,” May 3 – 4, 2012. Royal Society Discussion on “Structure and Dynamics of the Thylakoid Membrane,” Kavi Royal Society International Centre, Buckinghamshire, UK.
  • "Lipid Functions in cytochrome bc complexes: an unusual event in evolution," May 3, 2012. Royal Society Center, Chicheley Buckinghamshire, UK.
  • “Oligomeric Membrane Protein Complexes;" Symposium on "Structure-Function of Hetero-Oligomeric Membrane Proteins," February 25, 2012. Bioenergetics Subgroup, Biophysical Society, San Diego, CA.
  • "Impediments in Travel through Membrane Proteins: Problems in Bioenergetics and Protein Import;" February 13, 2012. Department of Biochemistry and Biophysics, University of Stockholm, Sweden.
  • "Traveling Inside the Cytochrome bc Complex of Energy-Transducing Membranes," January 5, 2012. Department of Biophysics, Albert Einstein College of Medicine, Bronx, NY.
  • Seminar: “Structure-Function of Cytochrome bf Complex,” December 5, 2011, the Biophysics and Computational Biology Department, University of Illinois-Champaign/Urbana.
  •  “Impediments in traveling through membrane proteins: problems in bioenergetics and protein import.” March 31, 2011, Dept. of Biochemistry & Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL.
  • Organization meeting of the X-ray free electron laser. January 18 – 21, 2011, University of California/Berkley, Berkley, CA.
  • “Problems and Strategies in the Crystallization and Structure Solution of the Helical Cyanobacterial Cytochrome E. coli β-barrel Omps, btuB b6f complex,” November 16-18, 2010, 3rd annual NIH Roadmap Meeting on Membrane Protein Technologies, La Jolla, CA.
  • “The cytochrome b6f complex of oxygenic photosynthesis,” September 28, 2010, Danforth Institute Symposium on Plant Structural Biology, St. Louis, MO.
  • "The Q Cycle of the Cytochrome b6f Complex in a Structural Perspective." Chair's address, Bioenergetics Symposium, August 23, 2010, XV International Photosynthesis Congress, Beijing, China.
  • "The Q Cycle in a Structural Perspective." Symposium on "Molecular Basis of Photosynthetic Energy and Electron Transfer and Related Respiratory Processes" August 18-19, 2010, Institute of Advanced Studies, Nanyang Technological University, Singapore.
  • "The Q Cycle of Cytochrome bc Complexes Considered in a Structural Perspective." Symposium on "Quinone and Oxygen in Energy Coupling and Catalysis" July 23, 2010, Jagiellonian University, Kraków, Poland.
  •  "Labyrinths and Barrels: Inside Membrane Proteins," at the Cambridge Healthtech Institute PepTalk on Membrane Proteins, Jan. 14, 2010, San Diego, CA.
  • "Towards the Structure of the Ion Channel Formed by Alpha-Synuclein," May12, 2010, M. J. Fox Foundation, New York, NY.
  • "Traffic Problems in Membrane Proteins," Skirball Institute of Biomolecular Medicine, May 14, 2010,New York University School of Medicine  New York, NY.
  •  "Membrane Proteins; Life in an Oily Environment." Short Course, " Membrane Proteins; The Essential Protein Engineering Summit," May 16, 2010, Cambridge HealthCare Institute,  Boston, MA.
  • Keynote address: November 14, 2009, "Traveling Inside Membrane Proteins," Symposium for 50th Anniversary, Institute for Protein Research, Osaka University, Japan.

Awards/Honors

National Science Foundation Postdoctoral Fellowship, Univ. Calif/San Diego 1965-67;

NIH(NIGMS) Research Career Development Award, 1970-75;

Senior Fellow, European Molecular Biology Org., Univ. of Amsterdam, The Netherlands, 1974-75;

McCoy Award, Purdue University, Achievements in Science, 1988;

Plenary Lectures, International Congress of Photosynthesis, 1989, 2004;

Fellow, Alexander von Humboldt Foundation, 1992;

Fellow, John Simon Guggenheim Foundation, 1992-93;

C.F. Kettering Award, Amer. Soc. Plant Physiology, 1996;

Rosetta Briegel Barton Lecture, University of Oklahoma, 2001;

Maurice Hilleman Lecture, Montana State University, 2004;

Daniel Arnon Lecture, University of California/Berkeley, 2006;

Fellow, Biophysical Society, 2007;

Eminent Scholar Lecture, Oklahoma State University, 2008.

 


Professional Faculty Research

Structure/function of alpha-helical and beta-barrel membrane proteins involved in photosynthetic electron transport and energy transduction; structure-function of receptors involved in protein (specifically colicins) import.

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