1. Overexpression and stability of helical membrane proteins
Daniel Otzen
Department of Life Sciences
Aalborg University
Denmark

2. Can be purified in large amounts Easily crystallized
Water soluble
Can be purified in large amounts
Easily crystallized
Biophysical characterization “simple”
Cytosol
Soluble in lipids/detergent
Difficult to produce and purify
Very difficult to crystallize
Difficult to handle in general

3. 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)

4. (beta-barrel proteins)
How folding of helical membrane proteins occurs in E. coli
Inner
membrane
Bacteriophage membrane proteins
SRP
Ffh+4.5S RNA
FtsY
GTP
SecB
Outer membrane
(beta-barrel proteins)
Inner
membrane
SecYE translocon
Leader
sequence

5. Short-circuiting the membrane transport system
Strategy:
Short-circuiting the membrane transport system
with inclusion body formation
Express as fusion proteins
Water
soluble
Membrane
protein
Formation of inclusion bodies (insoluble, high yield?)
Reconstitute as active protein in vitro using SRP, FtsY, SecYE
Redissolved
but denatured
membrane
protein
SecYE translocon ?

6. Membrane protein stability: The two-stage model
1. Insertion of
individual helices
into the bilayer
2. Association of
helices
Probably the major
determinants of
membrane protein
stability
3. Association of
helix hairpins

7. Native proteinnon-ionic Denatured proteinanionic
How can we measure this stability?
Problem: Not straightforward to measure stability in lipid environment.
Unfolding requires high
temperatures and is generally
irreversible
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 in
lipid

8. DsbB (disulfide bond formation protein B) from E. coli
Our model system:
DsbB (disulfide bond formation protein B) from E. coli
Cytosol
Periplasmic space
DsbB (176 residues)
Inner
membrane
Enzymatic activity
DsbBox
DsbBred
DsbAred
DsbAox
Transfers electrons
to the electron-transport
chain
Oxidizes protein
disulfide bridges
in the periplasm

9. + FRET Fragment 1 Fragment 2 His-tail Calorimetric measurements
Intact DsbB
His-tail
FRET
Activity
Fluorescence
[Fragment 1] or Time
Calorimetric
measurements
NMR studies

10. 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)