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Problems in Obtaining Diffraction-Quality Crystals of Integral Membrane Proteins Discussed in the context of 2 recently obtained structures of integral.

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Presentation on theme: "Problems in Obtaining Diffraction-Quality Crystals of Integral Membrane Proteins Discussed in the context of 2 recently obtained structures of integral."— Presentation transcript:

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 b 6 f complex of oxygenic photosynthesis (8 gene products; dimer; 26 TM -helices; MW = 220 kDa); 3.0 Å. (II) Complex between the 22 strand -barrel E. coli outer membrane vitamin B 12 receptor (BtuB) and the colicin E3 receptor (R) binding-domain; Å. (II) The complex between the 22 strand -barrel vitamin B 12 receptor and the colicin E3 R-domain.

2 (I) Cytochrome b 6 f complex: functions in membrane energy transduction

3 (I) The Cytochrome b 6 f Complex with H. Zhang, G. Kurisu, & J. L. Smith

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

5 (II) complex of the vitamin B 12 receptor and the colicin E3 R-domain with 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 IMP 1 Use of thermophilic sources Detergents: (i) undecyl-maltoside ( ); (ii) LDAO ( ) Purity; dont 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 b 6 f complex provides electron connection between photosystem II & photosystem I reaction centers and translocates H + across the membrane. p (lumen) -side n (stromal) -side 2H2O2H2OO 2 + 4H + PQ 4H+4H+ PC (cyt c 6 ) FdFNR Cyclic e-pathway NADPH PSII: Zouni et al (2001) Nature 409,739 PSI: Jordan et al (2001) Nature 411, 909 Cyt b6f

9 Cells of the 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:

11 Electron Transfer Activity of Cytochrome b 6 f Complex Efficiency of Action of Inhibitor

12 Masses of Eight Polypeptide Subunits of b 6 f Complex from the Thermophilic Cyanobacterium, Mastigocladus laminosus Whitelegge et al. Molec. Cell Proteomics (2002),1: Subunit Measured Mass (Da) Cyt f 32,270 Cyt b 6 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

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 b 6 f Complex

15 Proteolysis of Cytochrome b 6 f Complex in Different Detergents Protease activity could not inhibited.

16 Crystals of cytochrome b 6 f complex from M. laminosus made after augmentation with the lipid, DOPC (10:1, DOPC: Cytochrome f ) Hexagonal crystals, 78 % solvent content [Zhang, H. et al. (2003) PNAS, 100: ]

17 SDS-PAGE of Cytochrome b 6 f Crystals Lane 1, fresh cyt b 6 f complex Lane 2, new crystal Lane 3, old crystal Cyt f Cyt b 6 Sub IV ISP Cyt f proteolysed Cyt b 6 proteolysed ISP and Sub IV

18 Structure of Cytochrome b 6 f Complex 2 b-type Hemes, 1 c-type Heme, 1 [2Fe-2S] 1 new heme, chlorophyll a, -carotene p-side n-side DOPC +10 kT-10 kT

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

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:

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

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-membrane How are proteins imported across double membranes? Colicins as test molecules

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

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 40º LPS OM Kurisu et al., Nat Struct Biol, 10: , 2003;pdb: 1UJW)

27 Two receptor translocon for colicin import across the E. coli outer membrane Colicin E3 Cork domain

28 Acknowledgments Cytochome complexBtuB/R135 Complex J. T. BolinY. Eroukova (Moscow St.) A. FriedmanM. Lindeberg D. W. Krogmann S. Schendel* M. Ponamarev R. Taylor* G. M. Soriano L. A. Sherman Discussions M. G. RossmannK. 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 of Science & Education (GK); DOE, NIH (APS)


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