Refolding of membrane proteins
for structural studies
Lars Linden * RAMC 2005
Membrane proteins as
drug targets
The human genome:
25%
67%
The known drug targets:
75%
33%
 25% of the human genes
encode for membrane
proteins
 67% of the known drug
targets are membrane
proteins
membrane proteins
soluble proteins
m-phasys is the only company
focussed exclusively on 3D structures
of membrane protein targets
No human GPCR structure solved
Membrane protein
structures: ~ 100
(mostly bacterial proteins)
GPCR structures: 1
(Rhodopsin from bovine retina)
PDB
All known protein structures:
~ 28,000
Human GPCR
structures: 0
Why ?
Barriers in membrane protein
structural analysis...
Expression
system
DNA
Expression
in Inclusion bodies
Detergent
Solubilized
protein
Purification &
Crystallization
3D structure
... and how to get
around them
crystal
Refolded
protein
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
100
Fraction produced in E. coli
90
80
70
60
50
40
30
20
10
0
1980 1985 1990 1995 2000 2005 2010 2015 2020 2025 2030
Year of PDB Deposition
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
Expression vector for GST-GPCR(His)6 fusions
HisTag
GPCR
rrBT1T2
Protease cleavage site
GST
APr
pGEX2a-GPCR-His
Ptac
•Expression in E.coli
•Preparation of inclusion bodies
•Typical yields: 2-50 mg / l
~6000 bp
ori
lac I
How to identify refolding
conditions
Inclusion Bodies
(Aggregated Protein)
Solubilisation
Solubilised, but
misfolded protein
Detergent exchange
Misfolded
Refolded & native
Re-aggregated
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
*) proprietary functional assay applicable
to all GPCRs (including orphans)
GPCRs are rigorously tested
for activity and homogeneity
before crystallization
Arrestin activity assay
•
•
•
•
Arrestin mutant binds to GPCRs constitutively
Doesn't require phosphorylation
Affinity depends on ligand binding
Requires folded GPCR
1. Bind & wash
2. Detect bound arrestin
 GPCR properly
folded
 GPCR not properly
folded
Refolded GPCRs are functional
Example: CXCR1
Ligand binding
Interleukin 8
KD = 5 nM
3000
2500
2000
1500
Bound GTPgS [dpm]
45000
3500
Bound ligand [dpm]
G protein activation
40000
EC50 = 0.1 nM
35000
30000
25000
20000
g
15000
10000
5000
-2
-1
0
1
2
3
Log Interleukin-8 [M]
Conclusion:
• Ligand affinity (KD) like
native receptor
• > 80% refolded (Bmax)
-2,0 -1,5 -1,0 -0,5 0,0
Conclusion:
• Couples to Gi/o
• EC50 like native receptor
Log Interleukin-8 [M]
Refolded GPCR binds
ligand and couples
to G protein
Refolded GPCRs are homogenous
Example: CXCR1
SDS-PAGE
1
2
SEC
3
Conclusion:
• 95 % pure on SDS gel
0,12
- GPCR dimer
- GST-GPCR fusion
- GPCR monomer
Absorption
0,10
0,08
0,06
Conclusion:
• 85 % pure by SEC
analysis
0,04
0,02
0,00
-0,02
8
10
12
14
16
Volume [ml]
1. Inclusion body fraction
2. Ni chelate purified
3. SEC purified
Superdex 200
Refolded CXCR1 is
>90% pure and
monodisperse
Refolded GPCRs are homogenous
Example: GPR3
Analysis
Result
Purity (SDS-PAGE):
95 %
Monodispersity (SEC)
90 %
Specific activity
(arrestin assay)
80 %
0,16
0,14
Absorption
0,12
0,10
0,08
0,06
0,04
0,02
0,00
-0,02
8
10
12
14
16
Volume [ml]
Refolded GPCRs form crystals
Rhodopsin family
b
g
a
d
Crystallized
Pipeline
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
A: CXCR1-receptor
B: anti-CXCR1 mAB 9D1
C: co-complex
BSA
mAU
mAU
mAU
CXCR1
Aggregate
100
30.0
9D1
CXCR1 + 9D1
60
80
20.0
40
60
40
10.0
20
20
0
0.0
6.0
10.0
8.0
14.0
12.0
ml
6.0
8.0
10.0
12.0
14.0
ml
0
6.0
8.0
10.0
12.0
14.0
ml
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
hERG
116
116
66
45
35
tetramer
66
45
35
monomer
25
25
18
18
tetramer
132
66
Potassium channel can be
produced with M-FOLD™
66
45
35
116
refolded
unfolded
116
66
45
35
25
25
18
18
Conclusion:
• Refolded K channel
forms tetramer
• > 95 % refolded
Refolded K channels reconstituted
into planar bilayer (BLM)
Refolding works for
K channels
Ion channel crystals diffract
to 12 Å
K channel crystals
Acknowledgement