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Crystallizing Membrane Proteins for Structure-Function Studies Using Lipidic Mesophases

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Advancing Methods for Biomolecular Crystallography

Abstract

The lipidic cubic mesophase or in meso method for crystallizing membrane proteins has posted some high profile successes recently. This is especially true in the area of G protein-coupled receptors with over a dozen new crystallographic structures emerging in the past 5years. Slowly, it is becoming an accepted method with a proven record and convincing generality. However, it is not a method that is used in every membrane structural biology laboratory and that is unfortunate. The reluctance in adopting it is attributable, in part, to the anticipated difficulties associated with handling the sticky, viscous cubic mesophase in which crystals grow. Harvesting and collecting diffraction data with the mesophase-grown crystals is also viewed with some trepidation. It is acknowledged that there are challenges associated with the method. However, over the years we have worked to make the method user-friendly. To this end, tools for handling the mesophase in the pico- to nanolitre volume range have been developed for efficient crystallization screening in manual and robotic modes. Glass crystallization plates have been built that provide unparalleled optical quality and sensitivity to nascent crystals. Lipid and precipitant screens have been implemented for a more rational approach to crystallogenesis such that the method can now be applied to a wide variety of membrane protein types and sizes. These assorted advances are outlined here along with a summary of the membrane proteins that have yielded to the method. The challenges that must be overcome to further develop the method are described.

Parts of this article have been adapted from Caffrey [6] and Caffrey et al. [9].

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Notes

  1. 1.

    The recent successes in using experimental phasing for structure determination have occurred with channelrhodopsin from C. reinhardtii (PDB entry 3UG9; mercury-MAD), the Na+-Ca2+ exchanger from M. jannaschii (PDB entry 3V5U; samarium-SAD), β-barrels from E. coli (PDB entry 4E1S; seleno-methionine-SAD), and Y. pseudotuberculosis (PDB entry 4E1T; seleno-methionine-SAD) and with a membrane kinase from E. coli (D. Li, J. Lyons, V. Pye, D. Aragao, and M. Caffrey, in preparation; seleno-methionine-SAD).

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Acknowledgements

There are many who contributed to this work and most are from the MS&FB Group, both past and present members. To all I extend my warmest thanks and appreciation. This work was supported in part by grants from Science Foundation Ireland (07/IN.1/B1836, 12/IA/1255), FP7 COST Action CM0902, Marie Curie Actions (PIEF-GA-2009-254103) and the National Institutes Health (GM75915, P50GM073210, U54GM094599).

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Authors and Affiliations

  1. Membrane Structural and Functional Biology Group, School of Biochemistry and Immunology, and School of Medicine, Trinity College Dublin, Dublin, Ireland

    Martin Caffrey

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  1. Martin Caffrey
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Correspondence to Martin Caffrey .

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Editors and Affiliations

  1. , Department of Haematology, University of Cambridge, Hills Road, Cambridge, CB2 0XY, United Kingdom

    Randy Read

  2. Fac.des Sciences et des Technologies, Physics Department, Université de Lorraine, Blvd. des Aiguillettes 1, Vandoeuvre-lès-Nancy, 54506, France

    Alexandre G. Urzhumtsev

  3. , Institute of Mathematical Problems of Bi, Russian Academy of Sciences, Institutskaya st. 4, Pushchino, 142290, Russia

    Vladimir Y. Lunin

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Caffrey, M. (2013). Crystallizing Membrane Proteins for Structure-Function Studies Using Lipidic Mesophases. In: Read, R., Urzhumtsev, A., Lunin, V. (eds) Advancing Methods for Biomolecular Crystallography. NATO Science for Peace and Security Series A: Chemistry and Biology. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-6232-9_4

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