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Problems in Obtaining Diffraction-Quality Crystals of Integral Membrane Proteins

Problems in Obtaining Diffraction-Quality Crystals of Integral Membrane Proteins. Discussed in the context of 2 recently obtained structures of integral membrane protein (IMP) complexes:

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Problems in Obtaining Diffraction-Quality Crystals of Integral Membrane Proteins

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  1. Problems in Obtaining Diffraction-Quality Crystals of Integral Membrane Proteins • Discussed in the context of 2 recently obtained structures of integral membrane protein (IMP) complexes: • (I) Hetero-oligomeric cytochrome b6f complex of oxygenic photosynthesis (8 gene products; dimer; 26 TM a-helices; MW = 220 kDa); 3.0 Å. • (II) Complex between the 22 strand b-barrel E. coli outer membrane vitamin B12 receptor (BtuB) and the colicin E3 receptor (R) binding-domain; 2. 75 Å. (II) The complex between the 22 strand b-barrel vitamin B12 receptor and the colicin E3 R-domain.

  2. (I) Cytochrome b6f complex: functions in membrane energy transduction

  3. (I) The Cytochrome b6f Complexwith H. Zhang, G. Kurisu, & J. L. Smith

  4. (II) Structure of the complex between BtuB and R135, which functions in protein import 447 313 438 323 40º LPS LPS OM

  5. (II) complex of the vitamin B12 receptor and the colicin E3 R-domainwith Genji Kurisu, Stas Zakharov, Masha Zhalnina, &M. Wiener, S. Bano, Y. Antonenko (not shown)

  6. Challenge for Membrane Protein Structure Determination Presently, in the protein data bank, there are >22,000 protein structures. Among these, and 20 years after determination of the first integral membrane protein structure, there are 46 independent IMP structures, and 10 hetero-oligomeric IMP at a resolution  3.0 Å (http://www.mpibp.frankfurt. mpg.de/michel/public/memprotstruct.html).

  7. Some problems in the crystallization of IMP1 • Use of thermophilic sources • Detergents: (i) undecyl-maltoside (a); (ii) LDAO (b) • Purity; don’t over-purify! lipid depletion (part I). • Activity • Stability (oligomeric state; integral proteases) • Ligands for soluble domains (part II) • Problem of storage. • 1 Iwata, S. (Ed.) [2003] Methods and Results in Crystallization of Membrane Proteins., IUL, pp. 355

  8. Electron transport complexes in oxygenic photosynthesis: cytochrome b6f complex provides electron connection between photosystem II & photosystem I reaction centers and translocates H+ across the membrane. NADPH Fd FNR Cyclic e-pathway n (stromal) -side PQ p (lumen) -side 2H2O O2 + 4H+ 4H+ PC (cyt c6) PSII: Zouni et al (2001) Nature 409,739 PSI: Jordan et al (2001) Nature 411, 909 Cyt b6f

  9. Cells ofthe Thermophilic Cyanobacterium,Mastigocladus laminosus

  10. Cross-Section of the Protein-Detergent Micelle Complex Michel, H. (1990) Crystallization of Membrane Proteins; Pebay-Peyroula, et al., (1995) Structure, 3: 1051-1059

  11. Electron Transfer Activity of Cytochrome b6f ComplexEfficiency of Action of Inhibitor

  12. Masses of Eight Polypeptide Subunits of b6f Complex from the Thermophilic Cyanobacterium, Mastigocladus laminosus SubunitMeasured Mass (Da) Cyt f 32,270 Cyt b6 24,710 (calc., 24, 268) Rieske ISP 19,295 Sub IV 17,529 PetG 4057 PetM 3841 PetL 3530 PetN 3304 Dimer MW = 217,057 Da Whitelegge et al. Molec. Cell Proteomics (2002),1: 816-826

  13. Two problems:(i) It turned out that the protein was very pure, except for the possibility of trace protease (see below), and in fact was over-purified because the lipid was depleted (< 1 lipid/monomer);(ii) the protease activity has not, until now, been inhibitable.

  14. Proteolysis Problem in First Crystals of the Cytochrome b6f Complex

  15. Proteolysis of Cytochrome b6f Complex in Different Detergents Protease activity could not inhibited.

  16. Crystals of cytochrome b6f complex from M. laminosus made afteraugmentation withthe lipid,DOPC(10:1, DOPC: Cytochrome f ) Hexagonal crystals, 78 % solvent content [Zhang, H. et al. (2003) PNAS, 100: 5160-5163]

  17. SDS-PAGE of Cytochrome b6f Crystals 1 2 3 Lane 1, fresh cytb6f complex Lane 2, new crystal Lane 3, old crystal Cyt f Cyt f Cyt b6 proteolysed Cyt b6 ISP Sub IV proteolysed ISP and Sub IV

  18. Structure of Cytochrome b6f Complex 2 b-type Hemes,1 c-type Heme,1 [2Fe-2S] 1 new heme,chlorophyll a,b-carotene p-side DOPC n-side -10 kT +10 kT

  19. Crystal Structure of the Complex between BtuB and R135 at 2.75 Å Resolution 447 313 438 Kurisu et al., Nat Struct Biol, 10: 948-954, 2003;pdb: 1UJW) 323 40º LPS LPS OM

  20. Problem of protein-protein contacts for “squat“ IMP in detergent; increase soluble domain with mab. Hunte, C., H. Michel (2002) Curr Opin Struct Biol, 12: 503-508.

  21. Cytotoxic colicins: colicin E3, a ribosomal RNAase; n. b., coiled-coil motif N Domains: Translocation Receptor- binding Activity Colicin N Colicin Ia Nature, 385, 461, 1997 Colicin E3 C Mol Cell,8, 1053, 2001

  22. To try to solve the problem of the lipid depletion, the purified complex was augmented with pure synthetic lipid. • The result: the rate of formation of crystals of intact complex increased greatly; i. e., crystals appeared over-night! • Thus, the protease problem could be solved, but only by winning the race against it.

  23. The E. coli Cell Envelope: receptor-containing outer-membrane, periplasmic space, & metabolically active inner-membraneHow are proteins imported across double membranes? Colicins as test molecules

  24. E. coli outer membrane protein BtuB, cobalamin translocator, 22-antiparallel b-barrel(Chimento et al., Nat Struct Biol, 10, 394-401, 2003) How does colicin bind to, or insert into receptor? n. b., N-terminal cork (green) domain blocks insertion

  25. Colicin E3 receptor-binding domain (R135); Crystallization strategy: use R135 as soluble ligand of BtuB colicin receptor

  26. Crystal Structure of the Complex between BtuB and R135 at 2.75 Å Resolution 447 313 438 Kurisu et al., Nat Struct Biol, 10: 948-954, 2003;pdb: 1UJW) 323 40º LPS LPS OM

  27. Two receptor translocon for colicin import across the E. coli outer membrane 447 313 438 323 Colicin E3 3-4 5-6 7-8 7-8 Cork domain

  28. Acknowledgments Cytochome complexBtuB/R135 Complex J. T. Bolin Y. Eroukova (Moscow St.) A. Friedman M. Lindeberg D. W. Krogmann S. Schendel* M. Ponamarev R. Taylor* G. M. Soriano L. A. Sherman Discussions M. G. Rossmann K. Jakes (AECOM) W. Minor (Virginia) M. Shoham (CWRU) Synchrotron Lines & Staff APS SBC-19 (N. Duke, F. Rotella); BioCARS 14 [Argonne NL] Spring-8 (Hyogo, Japan) Grant Support NIH-GMS (WAC); *NIH-GMS Biophysics Training Grant; Japan Ministry ofScience & Education (GK); DOE, NIH (APS)

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