2 research outputs found
New Tethered Phospholipid Bilayers Integrating Functional G‑Protein-Coupled Receptor Membrane Proteins
Membrane
proteins exhibiting extra- and intracellular domains require
an adequate near-native lipid platform for their functional reconstitution.
With this aim, we developed a new technology enabling the formation
of a peptide-tethered bilayer lipid membrane (pep-tBLM), a lipid bilayer
grafted onto peptide spacers, by way of a metal–chelate interaction.
To this end, we designed an original peptide spacer derived from the
natural α-laminin thiopeptide (P19) possessing a cysteine residue
in the N-terminal extremity for grafting onto gold and a C-terminal
extremity modified by four histidine residues (P19-4H). In the presence
of nickel, the use of this anchor allowed us to bind liposomes of
variable compositions containing a 2% molar ratio of a chelating lipid,
1,2-dioleoyl-<i>sn</i>-glycero-3-[(<i>N</i>-(5-amino-1-carboxypentyl)iminodiacetic
acid)succinyl] so-called DOGS-NTA, and to form the planar bilayer
by triggering liposome fusion by an α-helical (AH) peptide derived
from the N-terminus of the hepatitis C virus NS5A protein. The formation
of pep-tBLMs was characterized by surface plasmon resonance imaging
(SPRi), and their continuity, fluidity, and homogeneity were demonstrated
by fluorescence recovery after photobleaching (FRAP), with a diffusion
coefficient of 2.5 × 10<sup>–7</sup> cm<sup>2</sup>/s,
and atomic force microscopy (AFM). By using variable lipid compositions
including phosphatidylcholine (PC), phosphatidylserine
(PS), phosphatidylethanolamine (PE),
phosphatidylinositol 4,5-bisphosphate (PIP<sub>2</sub>), sphingomyelin (SM), phosphatidic
acid (PA), and cholesterol (Chol) in various ratios, we show that
the membrane can be formed independently from the lipid composition.
We made the most of this advantage to reincorporate a transmembrane
protein in an adapted complex lipid composition to ensure its functional
reinsertion. For this purpose, a cell-free expression system was used
to produce proteoliposomes expressing the functional C-X-C
motif chemokine receptor 4 (CXCR4), a seven-transmembrane protein
belonging to the large superfamily of G-protein-coupled receptors
(GPCRs). We succeeded in reinserting CXCR4 in pep-tBLMs formed on
P19-4H by the fusion of tethered proteoliposomes. AFM
and FRAP characterization allowed us to show that pep-tBLMs inserting
CXCR4 remained fluid, homogeneous, and continuous. The value of the
diffusion coefficient determined in the presence of reinserted CXCR4
was 2 × 10<sup>–7</sup> cm<sup>2</sup>/s. Ligand binding
assays using a synthetic CXCR4 antagonist, T22 ([Tyr5,12, Lys7]-polyphemusin
II), revealed that CXCR4 can be reinserted in pep-tBLMs with functional
folding and orientation. This new approach represents a method of
choice for investigating membrane protein reincorporation and a promising
way of creating a new generation of membrane biochips adapted for
screening agonists or antagonists of transmembrane proteins
Red Emitting Neutral Fluorescent Glycoconjugates for Membrane Optical Imaging
A family of neutral fluorescent probes
was developed, mimicking
the overall structure of natural glycolipids in order to optimize
their membrane affinity. Nonreducing commercially available di- or
trisaccharidic structures were connected to a push–pull chromophore
based on dicyanoisophorone electron-accepting group, which proved
to fluoresce in the red region with a very large Stokes shift. This
straightforward synthetic strategy brought structural variations to
a series of probes, which were studied for their optical, biophysical,
and biological properties. The insertion properties of the different
probes into membranes were evaluated on a model system using the Langmuir
monolayer balance technique. Confocal fluorescence microscopy performed
on muscle cells showed completely different localizations and loading
efficiencies depending on the structure of the probes. When compared
to the commercially available ANEPPS, a family of commonly used membrane
imaging dyes, the most efficient probes showed a similar brightness,
but a sharper pattern was observed. According to this study, compounds
bearing one chromophore, a limited size of the carbohydrate moiety,
and an overall rod-like shape gave the best results