Journal
CELLULAR AND MOLECULAR LIFE SCIENCES
Volume 71, Issue 24, Pages 4895-4910Publisher
SPRINGER BASEL AG
DOI: 10.1007/s00018-014-1655-7
Keywords
Cell-free; Crystallisation; In meso; Lipid cubic phase; Macromolecular crystallography; Membrane protein structure
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Funding
- Science Foundation Ireland [07/IN.1/B1836, 12/IA/1255]
- FP7 COST Action [CM0902]
- National Institutes of Health [P50GM073210, U54GM094599]
- Collaborative Research Center of the German Research Foundation (DFG) [(SFB) 807]
- European Instruct consortium
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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- 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.
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