Overexpression and stability of helical membrane proteins Daniel Otzen Department of Life Sciences...
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Transcript of Overexpression and stability of helical membrane proteins Daniel Otzen Department of Life Sciences...
Overexpression and stabilityof helical membrane proteins
Daniel OtzenDepartment of Life Sciences
Aalborg UniversityDenmark
Cytosol
• Water soluble• Can be purified in large amounts• Easily crystallized• Biophysical characterization “simple”
• Soluble in lipids/detergent• Difficult to produce and purify• Very difficult to crystallize• Difficult to handle in general
Membrane proteins make out 30% of all proteins(and 70% of all pharmaceutical targets) ...
... but only about 1% of all known structures.
Project objective• Overproduction of membrane proteins
• Factors influencing their stability and folding
Model proteins for overexpression• Serotonin transporter (Drs. Ove Wiborg and Poul Nissen, Aarhus University)• G-protein coupled receptors (Dr. Hans Kiefer, m-phasys GmbH)
How folding of helical membrane proteins occurs in E. coli
FtsY
GTP
SRPFfh+4.5S RNA
Innermembrane
SecYE translocon
Innermembrane
Bacteriophage membrane proteins
Leader sequence
SecB
Outer membrane(beta-barrel proteins)
Strategy: Short-circuiting the membrane transport system
with inclusion body formation
Express as fusion proteins
Watersoluble
Membraneprotein
Reconstitute as active protein in vitro using SRP, FtsY, SecYE
SecYE translocon
?
Formation of inclusion bodies (insoluble, high yield?)
Redissolvedbut denatured
membraneprotein
Membrane protein stability: The two-stage model
1. Insertion of individual helicesinto the bilayer
2. Association ofhelices
3. Association ofhelix hairpins
Probably the majordeterminants of membrane proteinstability
How can we measure this stability?
Problem: Not straightforward to measure stability in lipid environment.
Unfolding requires hightemperatures and is generallyirreversible
Alternative approaches:• Use mixture of stabilizing (non-ionic) and destabilizing (ionic) detergents
Native proteinnon-ionic Denatured proteinanionic
• Split protein up into fragments and measure their association tendency inlipid
Our model system: DsbB (disulfide bond formation protein B) from E. coli
Cytosol
Periplasmic space
DsbB (176 residues)
Inner membrane
DsbBox
DsbBred
DsbAred
DsbAox
Oxidizes proteindisulfide bridgesin the periplasm
Transfers electronsto the electron-transport
chain
Enzymatic activity
85
90
95
100
0 0.1 0.2 0.3 0.4 0.5
Flu
ores
cenc
e
Mole fraction SDS
-2.2
-2
-1.8
-1.6
-1.4
-1.2
0 200 400 600 800 1000
Flu
ores
cens
Tid (s)
Dead-timejump influorescence
+
Fragment 1 Fragment 2Intact DsbB
His-tail
ActivityFluorescence
[Fragment 1] or Time
FRET
Calorimetricmeasurements
NMR studies
Perspectives
• Systematic replacement of amino acids at interface to map out which interactions stabilize/destabilize protein.• Explore how much can stabilize protein and analyze consequences for expression levels• Gain greater understanding of interplay between structure and stability• Contribute to greater expression levels of membrane proteins for structural studies (X-ray crystallography, NMR)