Rhodopsin Reconstituted into a Planar-Supported Lipid Bilayer Retains Photoactivity after Cross-Linking Polymerization of Lipid Monomers

Abstract

Transmembrane proteins (TMPs), particularly ion channels and receptors, play key roles in transport and signal transduction. Many of these proteins are pharmacologically important and therefore targets for drug discovery. TMPs can be reconstituted in planar-supported lipid bilayers (PSLBs), which has led to development of TMP-based biosensors and biochips. However, PSLBs composed of natural lipids lack the high stability desired for many technological applications. One strategy is to use synthetic lipid monomers that can be polymerized to form robust bilayers. A key question is how lipid polymerization affects TMP structure and activity. In this study, we have examined the effects of UV polymerization of bis-Sorbylphosphatidylcholine (bis-SorbPC) on the photoactivation of reconstituted bovine rhodopsin (Rho), a model G-protein-coupled receptor. Plasmon-waveguide resonance spectroscopy (PWR) was used to compare the degree of Rho incorporation and activation in fluid and poly(lipid) PSLBs. The results show that reconstitution of Rho into a supported lipid bilayer composed only of bis-SorbPC, followed by photoinduced lipid cross-linking, does not measurably diminish protein function.

Unique agonist-bound cannabinoid CB1 receptor conformations indicate agonist specificity in signaling

“…To evaluate our hypothesis, we have used a sensitive biophysical technique, PWR spectroscopy. This technique has already provided novel insights into the interaction of other G-protein coupled receptorssuch as the human δ-opioid (Salalmon et al, 2000;Alves et al, 2003;Alves et al, 2004a;Alves et al, 2004b) and the β-adrenergic (Devanathan et al, 2004) receptors and the visual receptor rhodopsin (Subramaniam et al, 2005) -with ligands, G proteins, GTPγS and/or lipids.…”

“…Addition of a GTP molecule results in the dissociation of the high affinity ternary complex and the activation of the G protein. Therefore, measuring the affinity of a non-hydrolyzable GTPanalogue (GTPγS) to the agonist/receptor/G protein ternary complex can provide information about the potency of the given agonist-bound receptor conformation to activate G protein types (Alves et al, 2003;Alves et al, 2004b;Subramaniam et al, 2005). As seen in Fig 10A, addition of GTPγS to WIN 55,212-2 bound human cannabinoid CB 1 -myc-His 6 receptor led to a decrease in the resonance angle, indicating a decrease in the proteolipid mass, presumably due to the dissociation of the agonist/ human cannabinoid CB 1 -myc-His 6 receptor /G iα1 ternary complex and release of the alpha subunit from the bilayer.…”

250GHz CW gyrotron oscillator for dynamic nuclear polarization in biological solid state NMR

“…Two research areas in which high resolution MAS experiments have proved especially successful are studies of amyloid fibrils [37,[51][52][53][54][55][56][57] and membrane proteins [42,[58][59][60][61][62][63][64][65][66][67][68][69][70][71][72][73][74][75]. However, in both of these cases, low sensitivity currently limits the information that can be gleaned from the spectra.…”

Ultra-High Vacuum Surface Analysis Study of Rhodopsin Incorporation into Supported Lipid Bilayers

“…14,15,31 Rho was reconstituted into PSLBs before polymerization using a surfactant dilution method. 1 An aliquot of Rho (2 mg/mL) solublized in 30 mM octyl glucoside (OG) was injected into the aqueous buffer (10 mM phosphate, pH 7.2) above an unpolymerized PSLB in a custommade cell. Injection caused dilution of the aliquot to final bulk protein and OG concentrations of ca.…”

“…In addition to this, a second factor was incorporated into the calculation to allow for the differences in mean free path (λ) between Si (λ Si ) and O (λ O ). The final equation used for calculating the SiO 2 free oxygen concentration (atom %) was (1) where λ O /λ Si = 3.39 nm/4.09 nm = 0.83. Table 2 shows the corrected values without the underlying silicon substrate contributions.…”