Abstract. (1) The system. The cytochrome b6f complex is one of only 60 independent hetero-oligomeric membrane proteins in the data bank. (2) Recent achievements, structure-function: (a) a newly recognized unique heme in the complex was characterized;(b) the quinone dependent pathway for proton translocation was described at a resolution of 2.7 ? (c) a unique function of the bound chlorophyll in occluding the quinone entry portal was demonstrated;(d) detailed lipid analysis was undertaken, conservation shown of many lipid functions between the b6f complex and the mitochondrial cytochrome bc1 complex, and (e) conformational changes shown to be induced by anionic lipid. (3) Proposed studies: (a) Using His-tagged cytochrome b6f complex derived from C. reinhardtii, (b) crystal structures of higher order (""""""""super"""""""")-complexes of the electron transport complexes in the photosynthetic electron transport chain will be sought, following the precedent set for such super-complexes in the respiratory chain. A starting point is utilization of an isolated complex of the 230 kDa cytochrome b6f complex with the 1 MDa trimeric photosystem I reaction center. Elucidation of the structure of such a complex would likely describe new pathways for electronic communication between the complexes via quinones and soluble electron transport proteins, and be used to define the interactions and functions of the lipids internal to the cytochrome complex. (c) The stoichiometry and arrangement of the lipids, and their effects on the dynamics of the cytochrome complex, will be analyzed by native and hydrogen-deuterium mass spectroscopy. (d) Assembly;role of lipids. To test the role of the lipids in separating different domains of the cytochrome complex, mass spectroscopic analysis, particularly native MS, will be applied to analysis of lipid interactions at different stages of assembly of the b6f complex. (e) Dielectric heterogeneity of the b6f complex;dependence of structure on electric field. Our studies imply that the internal dielectric constant within the b6f complex is heterogeneous, i. e., different between the different heme pairs within the complex. Virtually all membrane proteins, certainly those involved in energy-transduction and active transport, function in the presence of large electrical fields. With the long-term goal of determining the effect of electric fields on membrane protein structures, the effect of such fields on cytochrome spectra, redox properties, and secondary structure assayed by far-UV circular dichroism spectra, will be studied. (f) Trans- membrane signaling;structure of the signaling complex. The redox signaling system in the cytochrome b6f complex uses a unique trans-membrane signaling mechanism dependent upon oxidation of plastoquinol on the electro-positive side of the complex. A crystal structure will be sought of the trans-membrane signaling complex consisting of the b6f complex reconstituted with the protein kinase. These studies are directed not only to better crystallographic resolution and understanding of the structure of the cytochrome complex as a hetro- oligomeric membrane lipoprotein that is relevant to its function of trans-membrane signaling.

Public Health Relevance

It is well known that an understanding of membrane proteins in fundamental to an understanding of many diseases, thereby providing the majority of known drug targets. The hetero-oligomeric membrane protein described in this proposal is a representative of the group of membrane proteins that has the greatest degree of complexity and, partly for this reason, only 16% of the solved membrane protein structures are hetero- oligomeric. The understanding of the role of internal lipid in the assembly and function of these proteins is in an early stage, with a significant fraction of these data arising from the backgroun studies described in this proposal.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM038323-25
Application #
8640616
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Anderson, Vernon
Project Start
1987-04-01
Project End
2017-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
25
Fiscal Year
2014
Total Cost
$343,615
Indirect Cost
$118,615
Name
Purdue University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Celia, Hervé; Noinaj, Nicholas; Zakharov, Stanislav D et al. (2016) Structural insight into the role of the Ton complex in energy transduction. Nature 538:60-65
Singh, Sandeep K; Hasan, S Saif; Zakharov, Stanislav D et al. (2016) Trans-membrane Signaling in Photosynthetic State Transitions: REDOX- AND STRUCTURE-DEPENDENT INTERACTION IN VITRO BETWEEN STT7 KINASE AND THE CYTOCHROME b6f COMPLEX. J Biol Chem 291:21740-21750
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Zakharov, Stanislav D; Wang, Xin S; Cramer, William A (2016) The Colicin E1 TolC-Binding Conformer: Pillar or Pore Function of TolC in Colicin Import? Biochemistry 55:5084-94
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Hasan, S Saif; Zakharov, Stanislav D; Chauvet, Adrien et al. (2014) A map of dielectric heterogeneity in a membrane protein: the hetero-oligomeric cytochrome b6f complex. J Phys Chem B 118:6614-25
Hasan, S Saif; Proctor, Elizabeth A; Yamashita, Eiki et al. (2014) Traffic within the cytochrome b6f lipoprotein complex: gating of the quinone portal. Biophys J 107:1620-8
Agarwal, Rachna; Zakharov, Stanislav; Hasan, S Saif et al. (2014) Structure-function of cyanobacterial outer-membrane protein, Slr1270: homolog of Escherichia coli drug export/colicin import protein, TolC. FEBS Lett 588:3793-801
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