Refolding of membrane proteins for structural studies
description
Transcript of Refolding of membrane proteins for structural studies
Refolding of membrane proteins for structural studies
Lars Linden * RAMC 2005
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%
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
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
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
Percentage of structures from proteins produced in E. coli
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
Does in vitro refolding of membrane proteins work ?
Critical issues:• Energy landscape in
micelles?• Non-vectorial insertion• Local vs. Global
minimum?
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
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
How to identify refolding conditions
Inclusion Bodies(Aggregated Protein)
Refolded & nativeMisfolded Re-aggregated
Solubilisation
Solubilised, butmisfolded protein
Detergent exchange
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)
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
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
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
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]
Refolded GPCRs form crystals
CrystallizedPipelineCrystallized
Pipeline
Rhodopsin family
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
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
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
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
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
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)
K channel crystals
Ion channel crystals diffract to 12 Å
Acknowledgement