Cholesterol controls the activity of a wide range of membrane receptors through specific interactions and identifying cholesterol recognition motifs is therefore critical for understanding signaling receptor function. The membrane-spanning domains of the paradigm neurotransmitter receptor for acetylcholine (AChR) display a series of cholesterol consensus domains (referred to as “CARC”). Here we use a combination of molecular modeling, lipid monolayer/mutational approaches and NMR spectroscopy to study the binding of cholesterol to a synthetic CARC peptide. The CARC-cholesterol interaction is of high affinity, lipid-specific, concentration-dependent, and sensitive to single-point mutations. The CARC motif is generally located in the outer membrane leaflet and its reverse sequence CRAC in the inner one. Their simultaneous presence within the same transmembrane domain obeys a “mirror code” controlling protein-cholesterol interactions in the outer and inner membrane leaflets. Deciphering this code enabled us to elaborate guidelines for the detection of cholesterol-binding motifs in any membrane protein. Several representative examples of neurotransmitter receptors and ABC transporters with the dual CARC/CRAC motifs are presented. The biological significance and potential clinical applications of the mirror code are discussed.

Cholesterol is a ubiquitous neutral lipid, which finely tunes the activity of a wide range of membrane proteins, including neurotransmitter and hormone receptors and ion channels. Given the scarcity of available X-ray crystallographic structures and the even fewer in which cholesterol sites have been directly visualized, application of in silico computational methods remains a valid alternative for the detection and thermodynamic characterization of cholesterol-specific sites in functionally important membrane proteins. The membrane-embedded segments of the paradigm neurotransmitter receptor for acetylcholine display a series of cholesterol consensus domains (which we have coined "CARC"). The CARC motif exhibits a preference for the outer membrane leaflet and its mirror motif, CRAC, for the inner one. Some membrane proteins possess the double CARC-CRAC sequences within the same transmembrane domain. In addition to in silico molecular modeling, the affinity, concentration dependence, and specificity of the cholesterol-recognition motif-protein interaction have recently found experimental validation in other biophysical approaches like monolayer techniques and nuclear magnetic resonance spectroscopy. From the combined studies, it becomes apparent that the CARC motif is now more firmly established as a high-affinity cholesterol-binding domain for membrane-bound receptors and remarkably conserved along phylogenetic evolution.

Lipidic bicelles have been widely used for the analysis of integral membrane proteins where their spontaneous alignment in an magnetic field has been exploited for oriented sample solidstate NMR studies. Many of their physical properties however make them well suited to the analysis of membrane proteins by circular dichroism (CD) and synchrotron radiation circular dichroism (SRCD). In this paper we have identified bicelle compositions that permit comparable studies of integral membrane proteins by solid-state NMR, CD, and SRCD; complementary methods that can provide insights into protein structure and the interactions with drugs and other small molecules. Furthermore we have been able to identify bicelle compositions that allow the magnetic alignment of the bicelles at fields routinely available in magnetic CD instruments. This potentially provides a route to the preparation of samples for oriented CD that mitigates many of the issues associated with the preparation of mechanically aligned samples, although we demonstrate that the dynamics present within the system can complicate the analysis of such spectra.

ClC-ec1 is a bacterial Cl -/H þ antiporter that exists as a homodimer with each subunit containing a structurally independent pathway for ion transport. Mutations at the dimerization interface I201W/I422W (WW) destabilize the dimer in detergent while preserving function. In this study, we have applied single fluorescent molecule imaging and photobleaching to study the dimerization of an intermediately stable mutant; I422W (IW) in phospholipid membranes. WT CLC-ec1 and its mutants were purified from E. coli into detergent and labelled with Cy5-maleimide at a unique surface exposed cysteine residue. The dye/protein ratio for all samples were~0.66. Cy5 labelled proteins were reconstituted in Alexa Fluor 488 SDP-ester labelled 2:1 POPE/POPG liposomes at 0.03 % fluorophore/lipid. Dialyzed liposomes were then freeze-thawed 7 times to form large multilamellar membranes, and then incubated at 25 C to facilitate protein mixing. Prior to imaging on a multi-color TIR-FM these membranes were extruded through a 400 nm filter. Protein reconstitution in liposomes follows a Poisson distribution resulting in liposomes containing either 0, 1, 2 or more protein molecules. We counted the overall occupancy of liposomes using two-color colocalization and counting the number of labelled protein particles in each liposome using step-wise photobleaching of Cy5. We determined the photo-bleaching distribution for dimeric WT, monomeric WW and intermediately stable IW. Upon increasing protein/lipid mole fraction from 1.5 x 10-9 to 7.5 x 10-4, we find that IW evolves from a monomer to dimer distribution. By carrying out a least-squares fit to the monomer and expected dimer distribution, we calculate Fdimer, I422W(X) that shows a density dependent increase that fits to a dimerization isotherm with KD=2x10-6 protein/lipid. The dimers dissociate into monomers upon diluting proteoliposomes with empty membranes suggesting that we are observing the reversible dimerization reaction in membranes.

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