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Overexpression and stability of helical membrane proteins

Overexpression and stability of helical membrane proteins. Daniel Otzen Department of Life Sciences Aalborg University Denmark. Cytosol. Water soluble Can be purified in large amounts Easily crystallized Biophysical characterization “simple”. Soluble in lipids/detergent

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Overexpression and stability of helical membrane proteins

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

  2. 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

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

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

  6. 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 Membrane protein stability: The two-stage model

  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 • Split protein up into fragments and measure their association tendency in • lipid

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

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

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