Overexpression and stability of helical membrane proteins

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Presentation transcript:

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

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

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)

(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

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 ?

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

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

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

+ FRET Fragment 1 Fragment 2 His-tail Calorimetric measurements Intact DsbB His-tail FRET Activity Fluorescence [Fragment 1] or Time Calorimetric measurements 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)