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. 2017 Jun 7;17(6):3502-3511.
doi: 10.1021/acs.cgd.7b00458. Epub 2017 May 12.

Chemically Stable Lipids for Membrane Protein Crystallization

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Free PMC article

Chemically Stable Lipids for Membrane Protein Crystallization

Andrii Ishchenko et al. Cryst Growth Des. .
Free PMC article

Abstract

Lipidic cubic phase (LCP) has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment. Application of this material to a wide variety of membrane proteins, however, is hindered due to a limited number of available host lipids, mostly monoacylglycerols (MAGs). Here, we designed, synthesized and characterized a series of chemically stable lipids resistant to hydrolysis, with properties complementary to the widely used MAGs. In order to assess their potential to serve as host lipids for crystallization, we characterized the phase properties and lattice parameters of mesophases made of two most promising lipids at a variety of different conditions by polarized light microscopy and small-angle X-ray scattering. Both lipids showed remarkable chemical stability and an extended LCP region in the phase diagram covering a wide range of temperatures down to 4 °C. One of these lipids has been used for crystallization and structure determination of a prototypical membrane protein bacteriorhodopsin at 4 °C and 20 °C.

Figures

Figure 1
Figure 1
Stability of LCP made of GlyNCOC15+4 and monoolein under a range of pH values (2.2-11) over time. Both lipids were mixed with water to produce LCP, and the LCP boluses were covered with solutions containing 150 mM NaCl, 30% PEG400 and 100 mM of the respective buffer. The buffer solutions were taken from the StockOptions pH screen (Hampton).
Figure 2
Figure 2
Crystals of bacteriorhodopsin obtained by LCP crystallization using GlyNCOC15+4 as the host lipid. (a) Crystals grown at 20 °C. (b) Crystals grown at 4 °C.
Figure 3
Figure 3
Crystal packing of bR crystallized using monoolein, PDB ID 1M0L (a, d, g) and GlyNCOC15+4 as a host lipid for LCP at 4 °C (b, e, h) and 20 °C (c, f, i) as viewed (a, b, c) in the membrane plane, (d, e, f) along the membrane, (g, h, i) in the membrane plane with B factors mapped as both thickness of the lines and as a gradient coloring with the blue color corresponding to the lowest values and the red color corresponding to the highest values. The core bR trimer for the 20 °C structure is shown in green and the peripheral protomers are shown in cyan (panels c and f). Unit cells are outlined with solid black lines.
Figure 4
Figure 4
Crystal contacts between bR molecules crystallized at 20 °C. A bR protomer from the trimeric core (shown in green) was superimposed with a peripheral protomer (shown in dark blue). The structure of the BC loop is clearly different in the respective protomers. Molecules of the core bR trimer show a typical configuration of the BC loop containing a β-hairpin motif. On the other hand, the peripheral bR protomers exhibit an extended loop conformation without the β-hairpin, which would clash with its crystal neighbor, and instead with F71 positioned to potentially stabilize the packing by a hydrophobic interaction with the next membrane layer. The symmetry mate molecules are shown in light grey and the membrane boundary is shown as a dashed line. The membrane boundary was calculated using the Orientations of Proteins in Membranes Server (http://opm.phar.umich.edu/).
Figure 5
Figure 5
Crystal hits obtained from crystallization of A2AAR using GlyNCOC15+4 as the LCP host lipid. Brightfield mode Cross-polarized light Two-photon UV fluorescence mode SHG (second harmonic generation) mode
Scheme 1
Scheme 1
Chemical structures of monoolein (9.9 MAG) and two new lipids (GlyNCOC15+4 and GlyNMeCOC15+4) characterized in this study.

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