Research

Background

Biomembranes form the essential interfaces between functional biological units (cells, cell organelles) and their environment. They consist of a lipid bilayer and intrinsic and peripheral membrane proteins, which are responsible for exchange of signals and solutes across the hydrophobic region of the bilayer and for membrane fusion processes. The integral membrane proteins fall into two major classes that are characterized by their transmembrane structure, β-barrel and α-helical transmembrane proteins. How membrane proteins insert and fold into membranes is not well understood.

The reserach of this group is focussed on the biochemistry and biophysics of membrane proteins and membranes. Our main interest is to examine insertion, folding and stability of membrane proteins in membranes, i.e. the mechanisms and physical principles of membrane protein folding. We also study interactions of unfolded membrane proteins with other proteins in aqueous solution and in membranes.

For these studies, we use an array of molecular biological, biochemical and biophysical techniques. We express and isolate membrane proteins of interest to investigate their structural properties and their function in model membranes, i.e. lipid bilayers of selected lipid and protein composition. We use a wide range of biochemical and biophysical methods, like site-directed spectroscopic labeling of membrane proteins and spectroscopy, to examine structure, kinetics, and thermodynamics of proteins. Fluorescence, electron spin resonance, or circular dichroism methods are used to examine proteins, membrane protein folding, protein-protein interactions and protein-lipid interactions. Single channel conductance recordings are used to examine the function of transmembrane proteins as transporters for solutes. We also develop new methods for these studies.

Projects in the lab include:

A. Membrane protein folding and stability

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.

See, e.g. articles

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.

B. Interactions of Molecular Chaperones with Membrane Proteins

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.

Representative articles

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.

C. Method development

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 quenching J. 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.

For reviews, see e.g.

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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These pages were last edited Saturday, December 3, 2011, 3:31:34 PM, Jörg Kleinschmidt.

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