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Integral membrane proteins are usually very hydrophobic and insoluble in water. It is therefore not trivial to establish a working model system for the study of membrane protein insertion and folding. Whether it is possible at all, depends largely on the properties of the membrane protein. In comparison to the α-helical bundle proteins, the outer membrane proteins that form transmembrane β-barrels have a low average hydrophobicity. Some of these proteins can be denatured to a state of very little or no secondary structure in a solution of 8 M urea. We use these to explore the principles of membrane protein insertion and folding from a state of disordered secondary structure. Insertion and folding often can be accomplished by a strong dilution of the denaturant in the presence of preformed lipid bilayers. In our research on the mechanism of insertion and folding of β-barrel membrane proteins we use outer membrane proteins of bacteria and mitochondria and their chaperones.

 

Kleinschmidt*, J.H. Bulieris, P.V., Qu, J., Dogterom, M., den Blaauwen, T. 2011, Association of neighboring β-strands of outer membrane protein A in lipid bilayers revealed by site directed fluorescence quenching J. Mol. Biol. 407, 316-332

Patel, G. J., Behrens-Kneip, S., Holst, O., and Kleinschmidt, J. H., 2009, The periplasmic chaperone Skp facilitates targeting, insertion and folding of OmpA into lipid membranes with a negative membrane surface potential. Biochemistry, 48, 10235-10245

Shanmugavadivu, B., Apell, H.-J., Meins, T., Zeth, K., and Kleinschmidt, J. H., 2007, Correct folding of the β-barrel of the human membrane protein VDAC requires a lipid bilayer. J. Mol. Biol. 368, 66-78

Pocanschi, C.L., Apell, H.-J., Puntervoll, P., Høgh, B., Jensen, H. B., Welte, W. and Kleinschmidt, J. H.*, 2006. The Major Outer Membrane Protein of Fusobacterium Nucleatum (FomA) Folds and Inserts into Lipid Bilayers via Parallel Folding Pathways. J. Mol. Biol., 355, 548-561

Pocanschi, C.L., Patel, G. J., Marsh, D., Kleinschmidt, J.H., 2006, Curvature elasticity and refolding of OmpA in large unilamellar vesicles. Biophys. J., 91, L75-L78

Bulieris, P.V., Behrens, S., Holst, O., Kleinschmidt, J. H., 2003. Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide. J. Biol. Chem. 278, 9092-9099.

Outer membrane proteins are synthesized in the cytosol. They are translocated across a membrane in unfolded form before they insert into outer membranes from a complex with a molecular chaperone. We examine the molecular interactions of these chaperones with outer membrane proteins and their role in the insertion process into a lipid bilayer.

 

Qu, J., Behrens-Kneip, S., Holst, O., and Kleinschmidt, J. H., 2009, Binding Regions of Outer Membrane Protein A in Complexes with the Periplasmic Chaperone Skp. A Site-Directed Fluorescence Study. Biochemistry, 48, 4926-4936

Patel, G. J., Behrens-Kneip, S., Holst, O., and Kleinschmidt, J. H., 2009, The periplasmic chaperone Skp facilitates targeting, insertion and folding of OmpA into lipid membranes with a negative membrane surface potential. Biochemistry, 48, 10235-10245

Qu, J., Mayer, C., Behrens, S., Holst, O., and Kleinschmidt, J. H., 2007, The trimeric periplasmic chaperone Skp of E. coli forms 1:1 complexes with outer membrane proteins via hydrophobic and electrostatic interactions. J. Mol. Biol. 374, 91-105.

Bulieris, P.V., Behrens, S., Holst, O., Kleinschmidt, J. H., 2003. Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide. J. Biol. Chem. 278, 9092-9099.

We also actively develop new spectroscopic methodology, e.g.Inter- and Intramolecular site-directed fluorescence quenching, see

Kleinschmidt*, J.H. Bulieris, P.V., Qu, J., Dogterom, M., den Blaauwen, T. 2011, Association of neighboring β-strands of outer membrane protein A in lipid bilayers revealed by site directed fluorescence quenchingJ. Mol. Biol. 407, 316-332

Kleinschmidt, J. H., Tamm, L. K., 1999. Time-Resolved Distance Determination by Tryptophan Fluorescence Quenching (TDFQ): Probing Intermediates during Membrane Protein Folding. Biochemistry 38, 4996-5005

Kleinschmidt, J. H., den Blaauwen, T., Driessen, A., Tamm, L. K., 1999. Outer Membrane Protein A of E. coli Inserts and Folds into Lipid Bilayers by a Concerted Mechanism. Biochemistry 38, 5006-5016

and methods to renature membrane proteins from a denatured state:

Pocanschi, C. L., Dahmane, T., Gohon, Y., Rappaport, F., Apell, H.-J., Kleinschmidt, J. H.*, Popot, J.-L.*, 2006, Amphipathic Polymers: Tools to Fold Integral Membrane Proteins to their Active Form. Biochemistry 45, 13954-13961

Keywords that characterize the topics and methods in this laboratory: Biomembranes, Membrane Proteins, Lipid-Bilayers, Protein Expression and Purification, Site-directed Mutagenesis, Membrane Protein Folding and Insertion (Structural changes, Thermodynamics and Kinetics), Membrane Structure, Lipid-Protein Interactions, Protein-Protein Interactions, Molecular Chaperones, Protein-Detergent Interactions, Detergent Micelles, Fluorescence Spectroscopy, Fluorescence Quenching, Circular Dichroism Spectrocopy, Electron Spin Resonance Spectroscopy, Site Directed Protein-Labelling, Single-Channel Conductance Recordings on Membrane Proteins in Black Lipid Bilayers.

 

Kleinschmidt, J.H., 2007, Assembly of Integral Membrane Proteins from the Periplasm into the Outer Membrane. In: The Periplasm, Ed.: Ehrmann, M., ASM Press Washington D.C., p30-66, (ISBN-10: 1-55581-398-4)

Kleinschmidt, J.H., 2006. Folding kinetics of the outer membrane proteins OmpA and FomA into phospholipid bilayers. Chem. Phys. Lipids, 141, 30-47

Kleinschmidt, J.H., 2005, Folding and Stability of monomeric β-barrel Membrane proteins, In: Protein-Lipid Interactions: From Membrane Domains to Cellular Networks, Ed. Tamm, L.K. Wiley-VCH Weinheim, p27-56, (ISBN 3-527-31151-3)

Kleinschmidt, J.H.*, 2003. Membrane protein folding on the example of outer membrane protein A (OmpA) of Escherischia coli. Cell. Mol. Life Sci., 60, 1547-1558.

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