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Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination

DOI: 10.1007/s00018-014-1655-7 DOI Help

Authors: Coiiín Boland (Trinity College, Dublin) , Dianfan Li (Membrane Structural and Functional Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Ireland) , Syed Tasadaque Ali Shah (Membrane Structural and Functional Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Ireland) , Stefan Haberstock (Centre of Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry, Johann Wolfgang Goethe-University of Frankfurt, Germany) , Volker Dötsch (Centre of Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry, Johann Wolfgang Goethe-University of Frankfurt, Germany) , Frank Bernhard (Centre of Biomolecular Magnetic Resonance, Institute of Biophysical Chemistry, Johann Wolfgang Goethe-University of Frankfurt, Germany) , Martin Caffrey (Trinity College, Dublin)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Cellular and Molecular Life Sciences

State: Published (Approved)
Published: July 2014
Diamond Proposal Number(s): 8224 , 8518 , 9406

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.

Subject Areas: Biology and Bio-materials


Instruments: I24-Microfocus Macromolecular Crystallography

Other Facilities: APS, SLS

Added On: 30/09/2014 15:21

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