Problems in obtaining diffraction quality crystals of integral membrane proteins
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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:

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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:

  • (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.


I cytochrome b 6 f complex functions in membrane energy transduction
(I) Cytochrome Integral Membrane Proteinsb6f complex: functions in membrane energy transduction


I the cytochrome b 6 f complex with h zhang g kurisu j l smith
(I) The Cytochrome Integral Membrane Proteinsb6f Complexwith H. Zhang, G. Kurisu, & J. L. Smith


(II) Structure of the complex between BtuB and R135, which functions in protein import

447

313

438

323

40º

LPS

LPS

OM


(II) complex of the vitamin B functions in protein import12 receptor and the colicin E3 R-domainwith Genji Kurisu, Stas Zakharov, Masha Zhalnina, &M. Wiener, S. Bano, Y. Antonenko (not shown)


Challenge for membrane protein structure determination
Challenge for Membrane Protein Structure Determination functions in protein import

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).


Some problems in the crystallization of imp 1
Some problems in the crystallization of IMP functions in protein import1

  • 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


Electron transport complexes in oxygenic photosynthesis: functions in protein importcytochrome 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


Cells of the thermophilic cyanobacterium mastigocladus laminosus
Cells of functions in protein importthe Thermophilic Cyanobacterium,Mastigocladus laminosus


Cross section of the protein detergent micelle complex
Cross-Section of the Protein-Detergent Micelle Complex functions in protein import

Michel, H. (1990) Crystallization of Membrane Proteins;

Pebay-Peyroula, et al., (1995) Structure, 3: 1051-1059


Electron transfer activity of cytochrome b 6 f complex efficiency of action of inhibitor
Electron Transfer Activity of Cytochrome functions in protein importb6f ComplexEfficiency of Action of Inhibitor


Masses of Eight Polypeptide Subunits of functions in protein importb6f 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


Two problems: functions in protein import(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.


Proteolysis problem in first crystals of the cytochrome b 6 f complex
Proteolysis Problem in First Crystals of the Cytochrome functions in protein importb6f Complex


Proteolysis of cytochrome b 6 f complex in different detergents
Proteolysis of Cytochrome functions in protein importb6f Complex in Different Detergents

Protease activity could not inhibited.


Crystals of cytochrome functions in protein importb6f 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]


Sds page of cytochrome b 6 f crystals
SDS-PAGE of Cytochrome functions in protein importb6f 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


Structure of cytochrome b 6 f complex
Structure of Cytochrome functions in protein importb6f 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


Crystal Structure of the Complex between BtuB functions in protein import

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


Problem of protein protein contacts for squat imp in detergent increase soluble domain with mab
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.


Cytotoxic colicins: colicin E3, a ribosomal RNAase; detergent; increase soluble domain with mabn. 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


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.


The purified complex was augmented with pure synthetic lipid.E. coli Cell Envelope: receptor-containing outer-membrane, periplasmic space, & metabolically active inner-membraneHow are proteins imported across double membranes? Colicins as test molecules


E. coli purified complex was augmented with pure synthetic lipid. 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


Colicin E3 receptor-binding domain (R135); purified complex was augmented with pure synthetic lipid.Crystallization strategy: use R135 as soluble ligand of BtuB colicin receptor


Crystal Structure of the Complex between BtuB purified complex was augmented with pure synthetic lipid.

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


Two receptor translocon for colicin import across the purified complex was augmented with pure synthetic lipid.E. coli outer membrane

447

313

438

323

Colicin E3

3-4

5-6

7-8

7-8

Cork domain


Acknowledgments
Acknowledgments purified complex was augmented with pure synthetic lipid.

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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