title: Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination.
abstract: Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.
authors.affiliation: Membrane Structural and Functional Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Ireland.
authors.forename: Coilín
authors.initials: C
authors.lastname: Boland
authors.affiliation: Membrane Structural and Functional Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Ireland.
authors.forename: Dianfan
authors.initials: D
authors.lastname: Li
authors.affiliation: Membrane Structural and Functional Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Ireland.
authors.forename: Syed Tasadaque Ali
authors.initials: STA
authors.lastname: Shah
authors.affiliation: Institute of Biophysical Chemistry, Centre of Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University of Frankfurt, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Haberstock
authors.affiliation: Institute of Biophysical Chemistry, Centre of Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University of Frankfurt, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.affiliation: Institute of Biophysical Chemistry, Centre of Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University of Frankfurt, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
authors.affiliation: Membrane Structural and Functional Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Ireland.
authors.forename: Martin
authors.initials: M
authors.lastname: Caffrey
mesh_terms: D002462:Cell Membrane / Q000201:enzymology*; D002474:Cell-Free System; D002942:Circular Dichroism; D003460:Crystallization; D018360:Crystallography, X-Ray; D019852:Diacylglycerol Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays; D015971:Gene Expression Regulation, Enzymologic; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D055503:Protein Multimerization
chemical_list: D008055:Lipids; D008565:Membrane Proteins; D019852:Diacylglycerol Kinase