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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 membrane protein (IMP) complexes:
• (I) Hetero-oligomeric cytochrome b6f 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 B12 receptor (BtuB) and the colicin E3 receptor (R) binding-domain; 2. 75 Å.
(II) The complex between the 22 strand -barrel vitamin B12 receptor and the colicin E3 R-domain.
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(I) Cytochrome b6f complex: functions in membrane energy transduction
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(I) The Cytochrome b6f Complexwith H. Zhang, G. Kurisu, & J. L. Smith
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(II) Structure of the complex between BtuB and R135, which functions in protein import
40º
323313 438
447
LPSLPS
OM
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(II) complex of the vitamin B12 receptor and the colicin E3 R-domain
with Genji Kurisu, Stas Zakharov, Masha Zhalnina, &M. Wiener, S. Bano, Y. Antonenko (not shown)
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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 thefirst 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).
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Some problems in the crystallization of IMP1
• Use of thermophilic sources• Detergents: (i) undecyl-maltoside (); (ii) LDAO ()
• 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
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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.
p (lumen) -side
n (stromal) -side
2H2O O2 + 4H+
PQ
4H+ PC (cyt c6)
Fd FNR Cyclic e-pathway
NADPH
PSII: Zouni et al (2001) Nature 409,739
PSI: Jordan et al (2001) Nature 411, 909
Cyt b6f
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Cells of the Thermophilic Cyanobacterium, Mastigocladus laminosus
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Cross-Section of the Protein-Detergent Micelle Complex
Michel, H. (1990) Crystallization of Membrane Proteins;Pebay-Peyroula, et al., (1995) Structure, 3: 1051-1059
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Electron Transfer Activity of Cytochrome b6f Complex
Efficiency of Action of Inhibitor
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
0 10 20 30 40 50 60 70
Voltage (V)
Time (sec)
MLbf + DOPC
MLbf+DBMIB
MLbf
MLbf+DBMIB+DOPC
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Masses of Eight Polypeptide Subunits of b6f Complex from the Thermophilic
Cyanobacterium, Mastigocladus laminosus
Whitelegge et al. Molec. Cell Proteomics (2002),1: 816-826
Subunit Measured Mass (Da)
Cyt f 32,270 Cyt b6 24,710 (calc., 24, 268)Rieske ISP 19,295Sub IV 17,529PetG 4057PetM 3841PetL 3530PetN 3304
Dimer MW = 217,057 Da
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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.
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Proteolysis Problem in First Crystals of the Cytochrome b6f Complex
1 2 3 4
Lane 1, fresh cytochrome b6f complexLane 2, after 7 days at room temperatureLane 3, crystalLane 4, thermolysin treated complex
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Proteolysis of Cytochrome b6f Complex in Different Detergents
NG OG UDM DM DG DDM
t = 0
NG OG UDM DM DG DDM
t = 14 days
NG OG UDM DM DG DDM
t =7 days
Protease activity could not inhibited.
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Crystals of cytochrome b6f 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: 5160-5163]
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SDS-PAGE of Cytochrome b6f Crystals
1 2 3 Lane 1, fresh cyt b6f complex
Lane 2, new crystalLane 3, old crystal
Cyt f
Cyt b6
Sub IVISP
Cyt f
proteolysed Cyt b6
proteolysed ISP and Sub IV
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Structure of Cytochrome b6f 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
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Crystal Structure of the Complex between BtuB and R135 at 2.75 Å Resolution
40º
323313 438
447
LPSLPS
OM
Kurisu et al., Nat Struct Biol,
10: 948-954, 2003;pdb: 1UJW)
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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.
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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
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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.
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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
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How does colicin bind to, or insert into receptor?How does colicin bind to, or insert into receptor?n. bn. b., N-terminal cork (green) domain ., N-terminal cork (green) domain
blocks insertionblocks insertion
E. coliE. coli outer membrane protein BtuB, cobalamin outer membrane protein BtuB, cobalamin translocator, 22-antiparallel translocator, 22-antiparallel -barrel-barrel
((Chimento et al., Nat Struct Biol, 10, 394-401, 2003)
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Colicin E3 receptor-binding domain (R135); Crystallization strategy: use R135 as soluble ligand of BtuB colicin receptor
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Crystal Structure of the Complex between BtuB and R135 at 2.75 Å Resolution
40º
323313 438
447
LPSLPS
OM
Kurisu et al., Nat Struct Biol,
10: 948-954, 2003;pdb: 1UJW)
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7-8
5-6
7-8
3-4
323
313 438447
Two receptor translocon for colicin import across the E. coli outer membrane
Colicin E3
Cork domain
Acrobat Reader 5.0.lnk
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AcknowledgmentsCytochome complex BtuB/R135 ComplexJ. T. Bolin Y. Eroukova (Moscow St.)A. Friedman M. LindebergD. W. Krogmann S. Schendel*M. Ponamarev R. Taylor*G. M. Soriano L. A. Sherman
DiscussionsM. G. Rossmann K. Jakes (AECOM)W. Minor (Virginia) M. Shoham (CWRU)
Synchrotron Lines & StaffAPS SBC-19 (N. Duke, F. Rotella); BioCARS 14 [Argonne NL]Spring-8 (Hyogo, Japan)
Grant SupportNIH-GMS (WAC); *NIH-GMS Biophysics Training Grant; Japan
Ministry of Science & Education (GK); DOE, NIH (APS)