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)