Chemically Stable Lipids for Membrane Protein Crystallization

The 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 and 20 °C

FMN binding site of holoNqrC'.

<p>The protein is shown as a space-filling model at (a). H-bonds stabilizing the conformation of FMN residue are shown at (b). The intensity of orange color represents the 4 levels of amino acid conservation in agreement with <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118548#pone.0118548.g005" target="_blank">Fig. 5</a>. Green color represents nonconservative amino acids.</p

Ellipsoids of atomic-displacement parameters of the FMN residue in NqrC' drawn at 50% probability.

<p>Numeration of the isoalloxazine atoms is shown according to the IUPAC nomenclature.</p

HoloNqrC' structure.

<p>(a) and (b)—Overall view with 90°-rotation. Different secondary structure elements are shown in colors: β-sheets in yellow, α-helices in cyan, 3₁₀-helix in green. β-strands and helices are designated with numbers and Latin latters, respectively. Secondary structure was assigned with DSSP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118548#pone.0118548.ref029" target="_blank">29</a>].</p

Sequence alignment of the NqrC subunits of NQR from different bacteria (<i>V</i>. <i>harveyi</i> (<i>Vh</i>_NqrC), <i>Yersinia pestis</i> (<i>Yp</i>_NqrC), <i>Haemophilus influenza</i> (<i>Hi</i>_NqrC), <i>Pasteurella multocida</i> (<i>Pm</i>_NqrC), <i>Neisseria meningitidis</i> (<i>Nm</i>_NqrC) and <i>Pseudomonas aeroginosa</i> (<i>Pa</i>_NqrC)) as well as paralogous RnfG subunits of the RNF complex from <i>E</i>. <i>coli</i> (<i>Ec</i>_RnfG) and <i>V</i>. <i>cholerae</i> (<i>Vc</i>_RnfG).

<p>The intensity of orange color represents the 4 levels of amino acid conservation (calculated in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118548#pone.0118548.ref030" target="_blank">30</a>]).</p

Crystal packing of the holoNqrC' protein.

<p>FMN residue is shown in orange. (a) and (b)—along <i>a</i> and <i>c</i> axis, respectively.</p

Crystallographic table.

<p>Crystallographic table.</p