refolding of membrane proteins for structural studies
DESCRIPTION
Refolding of membrane proteins for structural studies. Lars Linden * RAMC 2005. Membrane proteins as drug targets. 25% of the human genes encode for membrane proteins. The human genome:. 25%. 75%. 67% of the known drug targets are membrane proteins. 67%. 33%. The known drug targets:. - PowerPoint PPT PresentationTRANSCRIPT
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Refolding of membrane proteins for structural studies
Lars Linden * RAMC 2005
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Membrane proteins as drug targets
m-phasys is the only company focussed exclusively on 3D structures of membrane protein targets
m-phasys is the only company focussed exclusively on 3D structures of membrane protein targets
The human genome: 25% of the human genes
encode for membrane proteins
75%25%
membrane proteins
soluble proteins
67% of the known drug targets are membrane proteins
The known drug targets:67% 33%
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All known protein structures:~ 28,000
No human GPCR structure solved
GPCR structures: 1(Rhodopsin from bovine retina)
Membrane protein structures: ~ 100(mostly bacterial proteins)
Why ?Why ?
Human GPCR structures: 0
PDB
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Barriers in membrane protein structural analysis...
... and how to get around them
... and how to get around them
Expression system
Purification & Crystallization
3D structureDNA
Refolded protein
Expression in Inclu-sion bodies
Detergent Solubilized
protein
crystal
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E. coli ?
• Fast• Cheap• High yields• Multiple strains available• Multiple plasmids available• Selenomethionine derivatives
• Less time for expression = more time for crystallization!
• In 2004, 67% of all structures deposited in the PDB were from proteins expressed in E. coli
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Percentage of structures from proteins produced in E. coli
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Expression of membrane proteins in E. coli can be toxic
• Eukaryotic membrane proteins are not readily inserted into bacterial membranes
• Bacterial insertion machinery becomes jammed
• Protein production stops after 1 min
• Low yields
Possible solution: Prevent membrane insertion
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Does in vitro refolding of membrane proteins work ?
Critical issues:• Energy landscape in
micelles?• Non-vectorial insertion• Local vs. Global
minimum?
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Does in vitro refolding of membrane proteins work ?
Yes!
• Bacteriorhodopsin• Light harvesting complex LHC2• Mitochondrial transporters• Diacyl glycerol kinase• Olfactory receptor OR5• Potassium channel KcsA• DsbB• Leukotriene receptor BLT1
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pGEX2a-GPCR-His
~6000 bp
APrGST
lac I
HisTag
Ptac
ori
rrBT1T2
Protease cleavage site
GPCR
Expression vector for GST-GPCR-(His)6 fusions
•Expression in E.coli•Preparation of inclusion bodies•Typical yields: 2-50 mg / l
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How to identify refolding conditions
Inclusion Bodies(Aggregated Protein)
Refolded & nativeMisfolded Re-aggregated
Solubilisation
Solubilised, butmisfolded protein
Detergent exchange
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Purification and quality control of GPCRs
Principal analysis Threshold
Purity (SDS-PAGE): > 90%
Monodispersity (SEC) > 90%
Specific activity (arrestin assay*)
> 70%
Concomitant analysis
Light scattering (DLS)
Ligand binding measurement
G protein activation
GPCRs are rigorously testedfor activity and homogeneitybefore crystallization
GPCRs are rigorously testedfor activity and homogeneitybefore crystallization
*) proprietary functional assay applicableto all GPCRs (including orphans)
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Arrestin activity assay
• Arrestin mutant binds to GPCRs constitutively • Doesn't require phosphorylation• Affinity depends on ligand binding• Requires folded GPCR
RA A
RA
RA
AR
1. Bind & wash 2. Detect bound arrestin
GPCR properly folded
GPCR not properly folded
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G protein activation
Log Interleukin-8 [M]
-2,0 -1,5 -1,0 -0,5 0,0
5000
10000
15000
20000
25000
30000
35000
40000
45000
g
EC50
= 0.1 nM
Bound G
TPS
[dpm
]
Ligand binding
-2 -1 0 1 2 3
1500
2000
2500
3000
3500
Interleukin 8K
D = 5 nM
Log Interleukin-8 [M]
Bound lig
and [
dpm
]
Refolded GPCRs are functionalExample: CXCR1
Refolded GPCR binds ligand and couples to G protein
Refolded GPCR binds ligand and couples to G protein
Conclusion:• Ligand affinity (KD) like
native receptor• > 80% refolded (Bmax)
Conclusion:• Couples to Gi/o
• EC50 like native receptor
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Refolded GPCRs are homogenousExample: CXCR1
SDS-PAGE
1 2 3
- GPCR dimer
- GST-GPCR fusion
- GPCR monomer
1. Inclusion body fraction2. Ni chelate purified3. SEC purified
Refolded CXCR1 is >90% pure and monodisperse
Refolded CXCR1 is >90% pure and monodisperse
Conclusion:• 95 % pure on SDS gel
Conclusion:• 85 % pure by SEC
analysis
SEC
Abso
rpti
on
Volume [ml]
8 10 12 14 16-0,02
0,00
0,02
0,04
0,06
0,08
0,10
0,12
Superdex 200
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Refolded GPCRs are homogenousExample: GPR3
Analysis Result
Purity (SDS-PAGE): 95 %
Monodispersity (SEC) 90 %
Specific activity (arrestin assay)
80 %
8 10 12 14 16-0,02
0,00
0,02
0,04
0,06
0,08
0,10
0,12
0,14
0,16
A
bso
rpti
on
Volume [ml]
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Refolded GPCRs form crystals
CrystallizedPipelineCrystallized
Pipeline
Rhodopsin family
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Optimization of crystallization conditions: strategy
• Truncated mutants (N- and C-termini, long loops)
• Co-crystallization with ligands (agonists, antagonists, inverse agonists)
• Co-crystallization with binding proteins (ß-arrestin, G proteins, antibody fragments)
• Stabilization with lipids
• Variation of crystallization method: vapour diffusion, microbatch, lipidic cubic phases, free interface diffusion
• Selection for more thermostable mutants
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Anti-GPCR monoclonal antibodies
• Successful programs with antibody companies and academic
groups
• Refolded GPCRs used as immunogen or panning target
• Antibodies obtained from mice (IgG) and phage display systems
(scFvs and Fabs)
• Antibodies recognize native GPCRs (FACS)
• Affinity from 1 nM to 1 µM
• Some are antagonistic
• Some have conformation-specific epitopes
Apart from their use in co-crystallization, antibodies might be
used as diagnostic tools or therapeutics
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m-fold CXCR1-antibody complex formation
• Immunization with CXCR1 Liposomes• Monoclonal IgG, FACS and ELISA positiv
• Ligand (IL-8) is displaced by antibody (IC50 = 0,33 nM)
• CXCR1 receptor and 9D1 antibody form a stable complex• scFv cloned, expressed and purified -> Co-crystallisation
0.0
10.0
20.0
30.0
mAU
6.0
8.010.0
12.014.0
ml
CXCR1
B: anti-CXCR1 mAB 9D1A: CXCR1-receptor
0
20
40
60
80
100
mAU
6.0
8.010.0
12.0 14.0 ml
9D1
BSA
Aggregate
C: co-complex
0
20
40
60
mAU
6.0 8.0 10.0 12.0 14.0 ml
CXCR1 + 9D1
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Bacterial and human ion channels
• Potassium Channels :
voltage gated KvLQT4
hERG
Kv1.3
VIC (Salmonella t.)
MJKch (Methanococcus j.)
Ca2+ activated KCa4
• Cloning and expression of different constructs of hERG, Kv1.3, KCa4 transmembrane region S1-S6
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Bacterial and human ion channels
• Ion channels are easily purified
• Refoldung screen for hERG, Kv1.3, KCa4, VIC and MJKch
• Tetramerisation can be detected on modified SDS or blue native Gels
VIC
tetramer
monomer
hERG
tetramer
66
132
11666
4535
25
116
66
45
35
25
1818
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Potassium channel can be produced with M-FOLD™
Refolding works for K channels
Refolding works for K channels
116
66
4535
25
18
unfo
lded
116
66
4535
25
18re
fold
ed
Conclusion:• Refolded K channel
forms tetramer• > 95 % refolded
Refolded K channels reconstituted into planar bilayer (BLM)
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K channel crystals
Ion channel crystals diffract to 12 Å
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Acknowledgement
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