Martin-Luther-Universität Halle-Wittenberg

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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. Schematic view of the formation of a protein-coated vesicle from a lipid bilayer

Schematic view of the formation of a protein-coated vesicle from a lipid bilayer

Schematic view of the formation of a protein-coated vesicle from a lipid bilayer

Advantages of in vitro reconstitution

(Left) Protein purification. (Middle) Confocal microscopy of the COPII coat reconstituted on giant liposomes (from Bacia et al., Sci. Rep. 2013, doi:10.1038/srep00017). Scale bar = 10 µm. (Right) COPII coat structure from cryo electron microscopy (from Zanetti et al., eLife 2013, doi: 10.7554/eLife.00951). Scale bar = 100 nm.

(Left) Protein purification. (Middle) Confocal microscopy of the COPII coat reconstituted on giant liposomes (from Bacia et al., Sci. Rep. 2013, doi:10.1038/srep00017). Scale bar = 10 µm. (Right) COPII coat structure from cryo electron microscopy (from Zanetti et al., eLife 2013, doi: 10.7554/eLife.00951). Scale bar = 100 nm.

(Left) Protein purification. (Middle) Confocal microscopy of the COPII coat reconstituted on giant liposomes (from Bacia et al., Sci. Rep. 2013, doi:10.1038/srep00017). Scale bar = 10 µm. (Right) COPII coat structure from cryo electron microscopy (from Zanetti et al., eLife 2013, doi: 10.7554/eLife.00951). Scale bar = 100 nm.

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 advanced light and electron microscopy techniques for characterizing the proteins and the lipid environment.


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.
(A) FCS (fluorescence correlation spectroscopy) analysis of membrane protein reconstitution. The protein carries a fluorescent label. Free protein in solution diffuses fast (red curve), whereas proteoliposomes diffuse much more slowly (purple curve). (B) Biochemical assay for membrane protein reconstitution.

(A) FCS (fluorescence correlation spectroscopy) analysis of membrane protein reconstitution. The protein carries a fluorescent label. Free protein in solution diffuses fast (red curve), whereas proteoliposomes diffuse much more slowly (purple curve). (B) Biochemical assay for membrane protein reconstitution.

(A) FCS (fluorescence correlation spectroscopy) analysis of membrane protein reconstitution. The protein carries a fluorescent label. Free protein in solution diffuses fast (red curve), whereas proteoliposomes diffuse much more slowly (purple curve). (B) Biochemical assay for membrane protein reconstitution.

Using dual-color fluorescence cross-correlation spectroscopy (FCCS), the success of membran protein reconstitution can be assessed within minutes. (from Simeonov et al., Biophys. Chem. 2013, doi: 10.1016/j.bpc.2013.08.003)

Using dual-color fluorescence cross-correlation spectroscopy (FCCS), the success of membran protein reconstitution can be assessed within minutes. (from Simeonov et al., Biophys. Chem. 2013, doi: 10.1016/j.bpc.2013.08.003)

Using dual-color fluorescence cross-correlation spectroscopy (FCCS), the success of membran protein reconstitution can be assessed within minutes. (from Simeonov et al., Biophys. Chem. 2013, doi: 10.1016/j.bpc.2013.08.003)

Dual-color FCCS (see below) allows to obtain quantitative binding curves of protein (or ligand) binding to freely diffusing liposomes (from Kruger et al., BioRxiv 2017, DOI 10.1101/146464 , now published in Biophys. J. 2017, doi: 10.1016/j.bpj.2017.06.023)

Dual-color FCCS (see below) allows to obtain quantitative binding curves of protein (or ligand) binding to freely diffusing liposomes (from Kruger et al., BioRxiv 2017, DOI 10.1101/146464 , now published in Biophys. J. 2017, doi: 10.1016/j.bpj.2017.06.023)

Dual-color FCCS (see below) allows to obtain quantitative binding curves of protein (or ligand) binding to freely diffusing liposomes (from Kruger et al., BioRxiv 2017, DOI 10.1101/146464 , now published in Biophys. J. 2017, doi: 10.1016/j.bpj.2017.06.023)

Very large liposomes, so-called Giant Unilamellar Vesicles (GUVs), prepared by the electroformation method 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).

(A) Electroformation setup for producing Giant Unilamellar Vesicle; (B) Dye-labeled Giant Unilamellar Vesicles in a test tube; (C) Confocal slice image of Giant Unilamellar Vesicles

(A) Electroformation setup for producing Giant Unilamellar Vesicle; (B) Dye-labeled Giant Unilamellar Vesicles in a test tube; (C) Confocal slice image of Giant Unilamellar Vesicles

(A) Electroformation setup for producing Giant Unilamellar Vesicle; (B) Dye-labeled Giant Unilamellar Vesicles in a test tube; (C) Confocal slice image of Giant Unilamellar Vesicles

Langmuir monolayers are prepared at the buffer/air interface. The interaction of proteins with the monolayers can be studied using infrared reflection absorption spectroscopy (IRRAS).

Determination of the orientation of the COPII-protein Sar1p at a lipid monolayer by IRRAS (from Schwieger et al., Polymers 2017, doi: 10.3390/polym9110612)

Determination of the orientation of the COPII-protein Sar1p at a lipid monolayer by IRRAS (from Schwieger et al., Polymers 2017, doi: 10.3390/polym9110612)

Determination of the orientation of the COPII-protein Sar1p at a lipid monolayer by IRRAS (from Schwieger et al., Polymers 2017, doi: 10.3390/polym9110612)

Lipid phase behavior

We are interested in dynamic lateral heterogeneities in membranes from natural components (proteins, lipids) and synthetic components (artificial amphiphiles and polyphiles).
Dendritic, star-shaped domains with sixfold symmetry formed in membranes from DPPC and X-shaped bolapolyphiles (from Werner et al., Polymers 2017, doi:10.3390/polym9100476, see also Werner et al., Chem. Eur. J. 2015, doi 10.1002/chem.201405994). The molecular structure of the bolapolyphile influences the domain shape.

Dendritic, star-shaped domains with sixfold symmetry formed in membranes from DPPC and X-shaped bolapolyphiles (from Werner et al., Polymers 2017, doi:10.3390/polym9100476, see also Werner et al., Chem. Eur. J. 2015, doi 10.1002/chem.201405994). The molecular structure of the bolapolyphile influences the domain shape.

Dendritic, star-shaped domains with sixfold symmetry formed in membranes from DPPC and X-shaped bolapolyphiles (from Werner et al., Polymers 2017, doi:10.3390/polym9100476, see also Werner et al., Chem. Eur. J. 2015, doi 10.1002/chem.201405994). The molecular structure of the bolapolyphile influences the domain shape.

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.

Parameters accessible by FCS (for details see: Bacia et al., Nat. Methods 2006)

Parameters accessible by FCS (for details see: Bacia et al., Nat. Methods 2006)

Parameters accessible by FCS (for details see: Bacia et al., Nat. Methods 2006)

Schematic view of an FCS setup with dual color FCCS capability

Schematic view of an FCS setup with dual color FCCS capability

Schematic view of an FCS setup with dual color FCCS capability

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.

Principle of FCS and dcFCCS measurements, for details see Bacia et al., Nat. Methods 2006

Principle of FCS and dcFCCS measurements, for details see Bacia et al., Nat. Methods 2006

Principle of FCS and dcFCCS measurements, for details see Bacia et al., Nat. Methods 2006

Equipment

HALOmem is equipped with state-of-the art equipment for protein expression and purification (e.g. Äkta FPLC systems), protein analysis and physico-chemical analysis (FT-IR spectroscopy, dynamic light scattering, Langmuir film balances). Fluorescence correlation spectroscopy (FCS) and related techniques are 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 are performed on our Zeiss ConfoCor3/LSM710 setup. TIRF and super-resolution microscopy are carried out on our Zeiss Elyra microscope. Samples for cryo electron microscopy are prepared using our Leica grid plunger and imaged in collaboration with the university imaging facility.

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