Publication

Article Metrics

Citations


Online attention

The cubicon method for concentrating membrane proteins in the cubic mesophase

DOI: 10.1038/nprot.2017.057 DOI Help

Authors: Pikyee Ma (Trinity College Dublin) , Dietmar Weichert (Trinity College Dublin) , Luba A. Aleksandrov (University of North Carolina at Chapel Hill) , Timothy J. Jensen (University of North Carolina at Chapel Hill) , John R. Riordan (University of North Carolina at Chapel Hill) , Xiangyu Liu (Tsinghua University) , Brian K. Kobilka (Stanford University) , Martin Caffrey (Trinity College Dublin)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature Protocols , VOL 12 , PAGES 1745 - 1762

State: Published (Approved)
Published: August 2017
Diamond Proposal Number(s): 11890 , 10357 , 9406

Abstract: The lipid cubic phase (in meso) method is an important approach for generating crystals and high-resolution X-ray structures of integral membrane proteins. However, as a consequence of instability, it can be impossible—using traditional methods—to concentrate certain membrane proteins and complexes to values suitable for in meso crystallization and structure determination. The cubicon method described here exploits the amphiphilic nature of membrane proteins and their natural tendency to partition preferentially into lipid bilayers from aqueous solution. Using several rounds of reconstitution, the protein concentration in the bilayer of the cubic mesophase can be ramped up stepwise from less than a milligram per milliliter to tens of milligrams per milliliter for crystallogenesis. The general applicability of the method is demonstrated with five integral membrane proteins: the β2-adrenergic G protein-coupled receptor (β2AR), the peptide transporter (PepTSt), diacylglycerol kinase (DgkA), the alginate transporter (AlgE) and the cystic fibrosis transmembrane conductance regulator (CFTR). In the cases of β2AR, PepTSt, DgkA and AlgE, an effective 20- to 45-fold concentration was realized, resulting in a protein-laden mesophase that allowed the formation of crystals using the in meso method and structure determination to resolutions ranging from 2.4 Å to 3.2 Å. In addition to opening up in meso crystallization to a broader range of integral membrane protein targets, the cubicon method should find application in situations that require membrane protein reconstitution in a lipid bilayer at high concentrations. These applications include functional and biophysical characterization studies for ligand screening, drug delivery, antibody production and protein complex formation. A typical cubicon experiment can be completed in 3–5 h.

Journal Keywords: Membrane biophysics; Membrane proteins; X-ray crystallography

Subject Areas: Biology and Bio-materials, Technique Development


Instruments: I24-Microfocus Macromolecular Crystallography

Other Facilities: Swiss Light Source