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Principal Investigators

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P02  Volker Dötsch
Professor

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Institute of Biophysical Biochemistry, Biocenter
Goethe University Frankfurt a.M.
Max-von-Laue-Str. 9
60438 Frankfurt am Main, Germany

Phone +49 (0)69 798-29631
Fax +49 (0)69 798-29632

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P02  Frank Bernhard
Group Leader

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Institute of Biophysical Biochemistry, Biocenter
Goethe University Frankfurt a.M.
Max-von-Laue-Str. 9
60438 Frankfurt am Main, Germany

Phone +49 (0)69 798-29620
Fax +49 (0)69 798-29632

> send email
> homepage

> publications

P02 Cell-free synthesized G-protein coupled receptors in synthetic membranes and other hydrophobic environments


Cell-free expression and the characterization of membrane proteins in nanodiscs are emerging key technologies in the field of synthetic biology. Our combination of both strategies enables rapid access to membrane proteins in individually designed membrane environments and without any detergent contacts. We focus on G-protein coupled receptors (GPCRs) as crucial regulators of cellular physiology and playing central roles in current and future medical research. Despite some exceptions, GPCRs are mostly difficult to obtain in conventional cell-based expression systems that is often due to their high detergent sensitivity.

During the last funding period, the cell-free production of functionally folded endothelin A, endothelin B, and beta1-adrenergic receptors was established by their co-translational insertion into defined membrane environments of preformed nanodiscs. Characteristic differential binding specificities with sets of ligands were obtained with the GPCR/nanodisc complexes. Solubilization, functional folding and affinities to specific ligands of the GPCRs were found to depend on charge, flexibility, and lipid chain length of the nanodisc membranes. Parameters identified for the improved folding of a thermostabilized beta1-adrenergic receptor could successfully be transferred to non-stabilized GPCRs providing a basic platform for pharmacological and structural studies with full-length and hardly engineered GPCRs. Dynamics and formation of membrane protein/nanodisc complexes were analyzed in collaboration with Glaubitz (P06) and Wachtveitl (P12). Structural details of cell-free synthesized membrane proteins were obtained together with Abele (P09) by NMR. A further milestone was the first crystallization of a post-translationally solubilized cell-free synthesized membrane protein. 

In the proposed funding period, we intend to extend our studies to the human beta1-3-adrenergic receptors and potentially to other GPCRs. The increased target variety is important for further method development and will help to identify specific as well as rather general parameters affecting GPCR stabilization, functional folding, and their insertion mechanisms into artificial membranes. We expect synergistic effects by comparison of the endothelin and beta-adrenergic systems in view of sample optimization, structural insights and identification of mechanisms responsible for ligand selectivity and ligand binding. A key objective will be the functional characterization of cell-free synthesized GPCRs and the molecular mapping of ligand interactions in defined nanodisc lipid environments in collaboration with Glaubitz (P06), Tampé (P16), Heilemann (P20), and Morgner (P24). The modulation of GPCR/ligand interactions upon contact with interacting proteins will further be considered. Structural details of GPCRs and other cell-free synthesized membrane proteins will be approached in collaboration with Glaubitz (P06), Abele (P09), and Pos (P18). 

 

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Fig. 1: CF expression modes for high level membrane protein production. Schematic view of cell-free approaches for the production of membrane proteins. P-CF: Membrane proteins are synthesized in the absence of supplied hydrophobic compartments and precipitate after translation. D-CF: Membrane proteins are kept in soluble form by insertion into provided detergent micelles. L-CF: Membrane proteins can become inserted into preformed liposomes, bicelles or nanodiscs of defined composition.

Fig. 2: Throughput design for the optimization of cell-free membrane protein production. An integrated process for pipetting, incubation, separation, purification, quantification and parameter selection of cell-free membrane protein expression conditions has been developed. The expression screening is demonstrated with an example of Mg2+/K+ ion concentration optimization.

Fig. 3: Transmembrane segment (TMS) labelling of membrane proteins by cell-free expression. Specific labelling of  amino acid types prevalent in transmembrane regions significantly reduces the complexity of NMR spectra and facilitates the structural analysis of these areas. [15N, 1H] TROSY spectra of A: Uniformly 15N-labelled presenilin-1-CTF and B: TMS-labelled presenilin-1-CTF.

 

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Publications

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Merk H, Rues RB, Gless C, Beyer K, Dong F, Dötsch V, Gerrits M, Bernhard F (2015) Biosynthesis of translocation dependent proteins in insect cell lysates: Identification of limiting parameters for folding and processing. Biol Chem 396, 1097-107.

Orbán E, Proverbio D, Haberstock S, Dötsch V, Bernhard F (2015) Cell-free expression of G-protein coupled receptors. Meth Mol Biol 1261, 171-195.

Kai L, Orbán E, Henrich E, Proverbio D, Dötsch V, Bernhard F (2015) Cotranslational stabilization of insoluble proteins in cell-free expressin systems. Meth Mol Biol 1258, 125-43.

Rues RB, Orbán E, Dötsch V, Bernhard F (2014) Cell-free expression of G-protein coupled receptors: New pipelines for challenging targets. Biol Chem 395, 1425-1434.

Boland C, Li D, Shah STA, Haberstock S, Dötsch V, Bernhard F, Caffrey M (2014) Cell-free expression and in meso crystallization of an integral membrane kinase for structure determination. Cell Mol Life Sci 71, 4895-4910.

Roos C, Kai L, Haberstock S, Proverbio D, Ghoshdastider U, Ma Y, Filipek S, Wang X, Dötsch V, Bernhard F (2014) High level cell-free production of membrane proteins with nanodiscs. Meth Mol Biol 1118, 109-130.

Hein C, Henrich E, Orbán E, Dötsch V, Bernhard F (2014) Hydrophobic environments in cell-free systems: Designing artificial environments for membrane proteins. J Eng Life Sci 14, 365-379.

Tumulka F, Roos C, Löhr F, Bock C, Bernhard F, Dötsch V (2013) Conformational stabilization of the membrane embedded targeting domain of the lysosomal peptide transporter TAPL for solution NMR. J Biomol NMR 57, 141-154.

Mörs K, Roos C, Scholz F, Wachtveitl J, Dötsch V, Bernhard F, Glaubitz C (2013) Modified lipid and protein dynamics in nanodiscs. Biochim Biophys Acta 1828, 1222-1229.

Kai L, Dötsch V, Kaldenhoff R, Bernhard F (2013) Artificial environments for the co-translational stabilization of cell-free expressed proteins. Plos One 8, e56637.

Proverbio D, Roos C, Beyermann M, Orbán E, Dötsch V, Bernhard F (2013) Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments. Biochim Biophys Acta 1828, 2182-2192.

Bernhard F, Tozawa Y (2013) Cell-free expression - making a mark. Cur Opin Struct Biol 23, 374-380.

Roos C, Kai L, Proverbio D, Ghoshdastider U, Filipek S, Dötsch V, Bernhard F (2013) Co-translational association of cell-free expressed membrane proteins with supplied lipid bilayers. Mol Membr Biol 30, 75-89.

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Collaborations

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Glaubitz (P06), Abele (P09), Wachtveitl/Bamann (P12), Schwalbe (P13), Tampé (P16),
Pos (P18), Heilemann (P20), Ernst (P21), Gottschalk (P11), Morgner (P24)