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|Paul Czyzykowski||-22817 (office) -24813 (lab)|
Membrane remodeling by proteins
Eukaryotic cells have a very complex organization, featuring various, membrane-enclosed internal compartments. As a consequence, ‘cargo’ molecules need to be sorted and transported between these compartments. To facilitate transport, the formation of small vesicles from a donor lipid bilayer is mediated by coat proteins, which interact with the membrane. We are interested in how coat proteins accomplish the process of remodeling a lipid bilayer into a bud and finally into a separate vesicle. Important aspects of budding processes can be artificially reconstituted and studied in vitro using purified proteins and artificial membrane systems.
Advantages of in vitro reconstitution
In vitro reconstitution represents a ‘bottom-up’ approach. A complicated biological process is taken apart and the various reactants can subsequently be examined by putting them together in any desired combination. To accomplish this, we express the proteins recombinantly, purify them and add them to artificial lipid bilayers. Among the advantages of using artificial membrane systems compared to biological cells is that we can control the composition of the lipids and choose among different types of spatial configurations. Moreover, artificial systems allow for a more flexible choice of biochemical, biophysical and microscopy techniques for characterizing the proteins and the lipid environment.
By studying the physicochemical properties of the proteins and their lipid environment and by exploring new avenues to reconstitution, we aim to make membrane proteins more accessible to high resolution structure determination by NMR and crystallography.
Artificial membrane systems
Liposomes can be prepared in various sizes, ranging from tens of nanometers to tens of micrometers. Reconstitution of integral membrane proteins into liposomes (i.e. the formation of proteoliposomes) as well as the peripheral binding of coat proteins to liposomes is analyzed using both biochemical and biophysical assays.
We prepare very large liposomes, so-called Giant Unilamellar Vesicles (GUVs) by the electroformation method. GUVs are on the order of 10 µm in size, making them well-suited for studies by confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS, see below).
Supported bilayers can be assembled on flat glass or mica surfaces for fluorescence imaging or AFM. We also prepare lipid bilayers on curved substrates to obtain membranes with a defined curvature. Langmuir monolayers are prepared at the buffer/air interface. In collaboration with the Blume lab, the interaction of proteins with the monolayers can be studied using infrared reflection absorption spectroscopy (IRRAS).
Lipid phase behavior, protein-lipid interactions and Fluorescence Correlation Spectroscopy (FCS)
We are interested in the influence of the lipid environment on membrane proteins and vice versa. Dynamic lateral heterogeneities may have a role in membrane remodeling processes. This is a very attractive concept, but it has proven difficult to investigate experimentally. We use a combination of imaging techniques and single-molecule sensitive fluorescence spectroscopy to study membrane proteins and lipids in artificial systems. Using these methods we want to assess membrane protein reconstitution and learn about the intricate connection between lateral intramembrane sorting and vesicular budding.
Fluorescence correlation spectroscopy (FCS) is a very useful tool for examining mobility and interactions in a variety of systems including membranes. FCS is highly sensitive to small differences in the diffusion rates of proteins and lipids, which allows for instance to characterize differences in phase behavior of lipid bilayers. FCS is used to analyze the binding of diffusible ligands to membrane receptors, such as membrane proteins or glycolipids. Changes in the fluorescence brightness parameter reveal membrane protein oligomerization. Moreover, the use of dual-color fluorescence cross-correlation (dcFCCS) allows to assess protein-protein binding in cases, where binding does not lead to significant changes in diffusion rates. The dual-color cross-correlation technique can also be employed to detect dynamic co-localization of labeled cargo molecules in small, mobile carriers, such as transport vesicles. Owing to the use of fluorescent labels, FCS is highly specific and can be applied both to artificial, reconstituted systems and directly to living cells.
FCS is typically performed on a setup that is similar to a confocal microscope. One or more laser lines are focused in the sample and the fluorescence is collected through the same objective. A pinhole serves to delimit the detection volume. The fluorescence emission(s) from the label(s) are selected by means of emission filter(s) and the fluorescence intensity as a function of time is recorded by avalanche photodiode detectors. Different methods of analysis are available to extract information from the fluorescence fluctuations, which occur as labeled molecule diffuse through the focus. Correlation analysis yields an autocorrelation curve, whose amplitude is inversely related to the concentration of the fluorescent particles. The decay time of the correlation curve reflects the diffusional mobility of the particles. In dual-color FCCS, the relative amplitude of the cross-correlation curve depends on the fraction of double-labeled (i.e., bound) particles.
Direction and goals
We aim to develop methods for efficient reconstitution and enrichment of functional membrane protein for structural studies. We are especially interested in pharmaceutically relevant membrane receptors and enzymes.
HALOmem is equipped with state-of-the art equipment for protein expression and purification (e.g. Äkta FPLC systems), protein analysis, crystallization (liquid handling robot) and physico-chemical analysis (differential scanning and isothermal calorimetry, FT-IR spectroscopy, dynamic light scattering, Langmuir balance). Fluorescence correlation spectroscopy (FCS) is used for single-molecule-sensitive diffusion and interaction analysis of membrane proteins and lipids in reconstituted membrane systems. Fluorescence auto- and cross-correlation measurements in combination with high sensitivity confocal fluorescence imaging can be performed on our Zeiss ConfoCor3/LSM710 setup. High speed confocal imaging is possible on our spinning-disk system. Cryo electron microscopy is carried out together with the group of Annette Meister and the university imaging facility.
Achilles A, Baerenwald R, Lechner BD, Werner S, Ebert H, Tschierske C, Blume A, Bacia K, Saalwaechter K (2016), Self-Assembly of X-Shaped Bolapolyphiles in Lipid Membranes: Solid-State NMR Investigations, Langmuir, 32(3), 673-682, DOI 10.1021/acs.langmuir.5b03712
Werner S, Ebert H, Lechner BD, Lange F, Achilles A, Bärenwald R, Poppe S, Blume A, Saalwächter K, Tschierske C, Bacia K (2015), “Dendritic Domains with Hexagonal Symmetry Formed by X-Shaped Bolapolyphiles in Lipid Membranes”, Chem. Eur. J., 21: 8840-8850, DOI 10.1002/chem.201405994
Lechner BD, Ebert H, Prehm M, Werner S, Meister A, Hause G, Beerlink A, Saalwächter K, Bacia K, Tschierske C, Blume A (2015), “Temperature-Dependent In-Plane Structure Formation of an X-Shaped Bolapolyphile within Lipid Bilayers”, Langmuir, 31(9):2839-2850, DOI 10.1021/la504903d
Schwieger C, Achilles A, Scholz S, Rüger J, Bacia, K, Saalwächter K, Kressler J, Blume A (2014), ” Binding of amphiphilic and triphilic block copolymers to lipid model membranes: the role of perfluorinated moieties”, Soft Matter, 10(33): 6147-6160, DOI 10.1039/C4SM00830H
Daum S, Krüger D, Meister A, Auerswald J, Prinz S, Briggs JA, Bacia K (2014), “Insights from reconstitution reactions of COPII vesicle formation using pure components and low mechanical perturbation”, Biol Chem, 395(7-8):801-812, DOI 10.1515/hsz-2014-0117
Schulz M, Olubummo A, Bacia K, Binder WH (2014), “Lateral surface engineering of hybrid lipid–BCP vesicles and selective nanoparticle embedding”, Soft Matter, 10(6): 831-839, DOI 10.1039/C3SM52040D
Bacia K (2013) “Intracellular transport mechanisms (Highlight: Nobel Prize for Medicine 2013).”, Angew Chem Int Ed, DOI 10.1002/anie.201308937, Intrazelluläre Transportmechanismen (Highlight: Nobelpreis für Medizin 2013), Angew Chem DOI 10.1002/ange.201308937
Simeonov P, Werner S, Haupt C, Tanabe M, Bacia K (2013), “Membrane Protein Reconstitution into Liposomes Guided by Dual-Color Fluorescence Cross-Correlation Spectroscopy”, Biophys. Chem., DOI 10.1016/j.bpc.2013.08.003, open access
Zanetti G, Prinz S, Daum S, Meister A, Schekman R, Bacia K, Briggs JAG (2013), “The structure of the COPII transport-vesicle coat assembled on membranes”, eLife, DOI 10.7554/eLife.00951, open access
Schulz M, Werner S, Bacia K, Binder WH (2012), “Controlling Molecular Recognition with Lipid/Polymer Domains in Vesicle Membranes”, Angewandte Chemie Intern. Ed., DOI 10.1002/anie.201204959
Faini M, Prinz S, Beck R, Schorb M, Riches JD, Bacia K, Brügger B, Wieland FT, Briggs JAG (2012), “The Structures of COPI-Coated Vesicles Reveal Alternate Coatomer Conformations and Interactions”, Science, DOI 10.1126/science.1221443
Olubummo A, Schulz M, Lechner BD, Scholtysek P, Bacia K, Blume A, Kressler J, Binder WH (2012), “Controlling the Localization of Polymer-Functionalized Nanoparticles in Mixed Lipid/Polymer Membranes”, ACS Nano, DOI 10.1021/nn3023602
Bacia K, Petrasek Z, Schwille P (2012), “Correcting for Spectral Cross-Talk in Dual-Color Fluorescence Cross-Correlation Spectroscopy”, ChemPhysChem, DOI 10.1002/cphc.201100801, open access
Schulz M, Glatte D, Meister A, Scholtysek P, Kerth A, Blume A, Bacia K, Binder W (2011), “Hybrid lipid/polymer giant unilamellar vesicles: effects of incorporated biocompatible PIB–PEO block copolymers on vesicle properties”, Soft Matter, DOI 10.1039/C1SM05725A
Bacia K, Futai E, Prinz S, Meister A, Daum S, Glatte D, Briggs JAG, Schekman R (2011), “Multibudded tubules formed by COPII on artificial liposomes”, Sci Rep. 1, 17 nature.com/srep open access
Ewers H, Römer W, Smith AE, Bacia K, Dmitrieff S, Chai W, Mancini R, Kartenbeck J, Chambon V, Berland L, Oppenheim A, Schwarzmann G, Feizi T, Schwille P, Sens P, Helenius A & Johannes L (2010), “GM1 structure determines SV40-induced membrane invagination and infection” Nat Cell Biol. 12(1), 11-18 Pubmed
Bacia K, Haustein E & Schwille P, “Fluorescence Correlation Spectroscopy: Principles and Applications”, Chapter 39 (p. 609) in: Imaging: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2010, ed. Rafael Yuste, ISBN 978-0879699352
Schön P, García-Sáez AJ, Malovrh P, Bacia K, Anderluh G & Schwille P (2008), “Equinatoxin II permeabilizing activity depends on the presence of sphingomyelin and lipid phase coexistence.” Biophys J. 95(2),691-698 Pubmed
Bacia K & Schwille P (2007), “Fluorescence correlation spectroscopy.” Methods Mol Biol. 398, 73-84 Pubmed
Bacia K & Schwille P (2007), “Practical guidelines for dual-color fluorescence cross-correlation spectroscopy.” Nat Protoc. 2(11), 2842-2856 Pubmed
Becker CF, Seidel R, Jahnz M, Bacia K, Niederhausen T, Alexandrov K, Schwille P, Goody RS& Engelhard M (2006), “C-terminal fluorescence labeling of proteins for interaction studies on the single-molecule level.” Chembiochem. 7(6), 891-895 Pubmed
Bacia K, Kim SA & Schwille P (2006), “Fluorescence cross-correlation spectroscopy in living cells.” Nat Methods. 3(2), 83-89 Pubmed
Kim SA, Heinze KG, Bacia K, Waxham MN & Schwille P (2005), “Two-photon cross-correlation analysis of intracellular reactions with variable stoichiometry.” Biophys J. 88(6), 4319-4336 Pubmed
Bacia K, Schwille P & Kurzchalia T (2005), “Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes.” Proc Natl Acad Sci U S A. 102(9), 3272-3277 Pubmed
Schwille P, Kahya N & Bacia K, “Protein and Lipid Partitioning in Locally Heterogeneous Model Membranes”, Chapter 14 (p. 337) in: Protein-Lipid Interactions, Wiley-VCH, 2005, ed. Lukas K. Tamm, ISBN 978-3527311514
Bacia K, Scherfeld D, Kahya N & Schwille P (2004), “Fluorescence correlation spectroscopy relates rafts in model and native membranes.” Biophys J. 87(2), 1034-1043 Pubmed
Bacia K, Schuette CG, Kahya N, Jahn R & Schwille P (2004), “SNAREs prefer liquid-disordered over “raft” (liquid-ordered) domains when reconstituted into giant unilamellar vesicles.” J Biol Chem. 279(36), 37951-37955 Pubmed
Kahya N, Scherfeld D, Bacia K & Schwille P (2004), “Lipid domain formation and dynamics in giant unilamellar vesicles explored by fluorescence correlation spectroscopy.” J Struct Biol. 147(1), 77-89 Pubmed
Kahya N, Scherfeld D, Bacia K, Poolman B & Schwille P (2003), “Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy.” J Biol Chem. 278(30), 28109-28115 Pubmed
Bacia K & Schwille P (2003), “A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy.” Methods. 29(1), 74-85 Pubmed
Bacia K, Majoul IV & Schwille P (2002), “Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis.” Biophys J. 83(2), 1184-1193 Pubmed
Keyhani NO, Bacia K & Roseman S (2000), “The transport/phosphorylation of N,N’-diacetylchitobiose in Escherichia coli. Characterization of phospho-IIB(Chb) and of a potential transition state analogue in the phosphotransfer reaction between the proteins IIA(Chb) AND IIB(Chb).” J Biol Chem. 275(42), 33102-33109 Pubmed