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 quinone (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 ubiquinone-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 menaquinone. (4) Quinone transfer though the narrow p-side portal. Electrons and protons are carried across an inter-monomer quinone exchange cavity in bc1 and b6f complexes by lipophilic ubi- and plastoquinone (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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038323-24
Application #
8225190
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Anderson, Vernon
Project Start
1987-04-01
Project End
2013-12-31
Budget Start
2012-02-01
Budget End
2013-12-31
Support Year
24
Fiscal Year
2012
Total Cost
$332,442
Indirect Cost
$111,919
Name
Purdue University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
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
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
Saif Hasan, S; Yamashita, Eiki; Cramer, William A (2013) Transmembrane signaling and assembly of the cytochrome b6f-lipidic charge transfer complex. Biochim Biophys Acta 1827:1295-308
Nowaczyk, Marc M; Wulfhorst, Hannes; Ryan, Christopher M et al. (2011) NdhP and NdhQ: two novel small subunits of the cyanobacterial NDH-1 complex. Biochemistry 50:1121-4
Yamashita, E; Zhang, H; Cramer, W A (2007) Structure of the cytochrome b6f complex: quinone analogue inhibitors as ligands of heme cn. J Mol Biol 370:39-52
Yan, Jiusheng; Kurisu, Genji; Cramer, William A (2006) Intraprotein transfer of the quinone analogue inhibitor 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone in the cytochrome b6f complex. Proc Natl Acad Sci U S A 103:69-74
Cramer, William A; Zhang, Huamin; Yan, Jiusheng et al. (2006) Transmembrane traffic in the cytochrome b6f complex. Annu Rev Biochem 75:769-90
Cramer, William A; Zhang, Huamin (2006) Consequences of the structure of the cytochrome b6f complex for its charge transfer pathways. Biochim Biophys Acta 1757:339-45
Cramer, William A; Yan, Jiusheng; Zhang, Huamin et al. (2005) Structure of the cytochrome b6f complex: new prosthetic groups, Q-space, and the 'hors d'oeuvres hypothesis' for assembly of the complex. Photosynth Res 85:133-43

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