Conservation of the oligomeric state of native VDAC1 in detergent micelles.

The voltage-dependent anion-selective channel (VDAC) is an intrinsic β-barrel membrane protein located within the mitochondrial outer membrane where it serves as a pore, connecting the mitochondria to the cytosol. The high-resolution structures of both the human and murine VDACs have been resolved by X-ray diffraction and nuclear magnetic resonance spectroscopy (NMR) in 2008. However, the structural data are not completely in line with the findings that were obtained after decades of research on biochemical and functional analysis of VDAC. This discrepancy may be related to the fact that structural biology studies of membrane proteins reveal specific static conformations that may not necessarily represent the physiological state. For example, overexpression of membrane proteins in bacterial inclusion bodies or simply the extraction from the native lipid environment using harsh purification methods (i.e. chaotropic agents) can disturb the physiological conformations and the supramolecular assemblies. To address these potential issues, we have developed a method, allowing rapid one step purification of endogenous VDAC expressed in the native mitochondrial membrane without overexpression of recombinant protein or usage of harsh chaotropic extraction procedures. Using the Saccharomyces cerevisiae isoform 1 of VDAC as a model, this method yields efficient purification, preserving VDAC in a more physiological, native state following extraction from mitochondria. Single particle analysis using transmission electron microscopy (TEM) demonstrated conservation of oligomeric assembly after purification. Maintenance of the native state was evaluated using functional assessment that involves an ATP-binding assay by micro-scale thermophoresis (MST). Using this approach, we were able to determine for the first time the apparent KD for ATP of 1.2 mM.

Novel octreotide dicarba-analogues with high affinity and different selectivity for somatostatin receptors

A limited set of novel octreotide dicarba-analogues with non-native aromatic side chains in positions 7 and/or 10 were synthesized. Their affinity toward the ssts1-5 was determined. Derivative 4 exhibited a pan-somatostatin activity, except sst4, and derivative 8 exhibited high affinity and selectivity toward sst5. Actually, compound 8 has similar sst5 affinity (IC50 4.9 nM) to SRIF-28 and octreotide. Structure-activity relationships suggest that the Z geometry of the double-bond bridge is that preferred by the receptors. The NMR study on the conformations of these compounds in SDS(-d25) micelles solution shows that all these analogues have the pharmacophore beta-turn spanning Xaa7-D-Trp8-Lys9-Yaa10 residues. Notably, the correlation between conformation families and affinity data strongly indicates that the sst5 selectivity is favored by a helical conformation involving the C-terminus triad, while a pan-SRIF mimic activity is based mainly on a conformational equilibrium between extended and folded conformational states.

Insight into the mechanism of transient trapping in micellar electrokinetic chromatography

Transient trapping is a new mechanism of on-line sample concentration and separation that has recently been presented. It involves the injection of a short length of micellar solution in front of the sample, making it similar to sweeping in partial-filling MEKC. Here, we examine the mechanism of transient trapping by the use of computer simulations and compare it to sweeping in MEKC for the two analytes, sulforhodamine B and 101. The simulation results confirm the mechanism for concentration and separation originally proposed. The mechanism for concentration is similar to sweeping since the analytes are picked and accumulated by the micelles that penetrate the sample zone. The mechanism for separation is however quite unique since the concentrated analytes are trapped for a few seconds on the sample/micelle boundary before they are released as the concentration of micelle is reduced as it undergoes electromigration dispersion and the analytes separate down a micelle gradient. Simulation results suggested that a significant contribution of band broadening arises from the micelle gradient, with shallower gradients resulting in broader peaks. However, this is offset by an increase in selectivity, such that resolution was enhanced even though the peaks are broader. Transient trapping analysis with similar resolution to those obtained by sweeping MEKC could be achieved in 1/10 of the time and 1/4 of the capillary length, which results in a 2-3 times increase in sensitivity.

Efficient coupling of transducin to monomeric rhodopsin in a phospholipid bilayer

G protein-coupled receptors (GPCRs) are seven transmembrane domain proteins that transduce extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization, the function of which has only been established for a few different receptor systems. One challenge in understanding the function of oligomers relates to the inability to separate monomeric and oligomeric receptor complexes in membrane environments. Here we report the reconstitution of bovine rhodopsin, a GPCR expressed in the retina, into an apolipoprotein A-I phospholipid particle, derived from high density lipoprotein (HDL). We demonstrate that rhodopsin, when incorporated into these 10 nm reconstituted HDL (rHDL) particles, is monomeric and functional. Rhodopsin.rHDL maintains the appropriate spectral properties with respect to photoactivation and formation of the active form, metarhodopsin II. Additionally, the kinetics of metarhodopsin II decay is similar between rhodopsin in native membranes and rhodopsin in rHDL particles. Photoactivation of monomeric rhodopsin.rHDL also results in the rapid activation of transducin, at a rate that is comparable with that found in native rod outer segments and 20-fold faster than rhodopsin in detergent micelles. These data suggest that monomeric rhodopsin is the minimal functional unit in G protein activation and that oligomerization is not absolutely required for this process.

Self-assembling cannabinomimetics: supramolecular structures of N-alkyl amides

Certain fatty acid N-alkyl amides from the medicinal plant Echinacea activate cannabinoid type-2 (CB2) receptors. In this study we show that the CB2-binding Echinacea constituents dodeca-2E,4E-dienoic acid isobutylamide (1) and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (2) form micelles in aqueous medium. In contrast, micelle formation is not observed for undeca-2E-ene-8,10-diynoic acid isobutylamide (3), which does not bind to CB2, or structurally related endogenous cannabinoids, such as arachidonoyl ethanolamine (anandamide). The critical micelle concentration (CMC) range of 1 and 2 was determined by fluorescence spectroscopy as 200-300 and 7400-10000 nM, respectively. The size of premicelle aggregates, micelles, and supermicelles was studied by dynamic light scattering. Microscopy images show that compound 1, but not 2, forms globular and rod-like supermicelles with radii of approximately 75 nm. The self-assembling N-alkyl amides partition between themselves and the CB2 receptor, and aggregation of N-alkyl amides thus determines their in vitro pharmacological effects. Molecular mechanics by Monte Carlo simulations of the aggregation process support the experimental data, suggesting that both 1 and 2 can readily aggregate into premicelles, but only 1 spontaneously assembles into larger aggregates. These findings have important implications for biological studies with this class of compounds.

Self-assembly into nanoparticles is essential for receptor mediated uptake of therapeutic antisense oligonucleotides

Antisense oligonucleotides (ASOs) have the potential of revolutionizing medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated with nanoparticles to enhance their stability and cellular uptake; however, one of the biggest challenges is the poor understanding of their uptake mechanism, which is needed for designing better ASOs with high activity and low toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (P-PMO), 2?Omethyl phosphorothioate (2?OMe) and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Deuchenne muscular dystrophy (DMD). We show that P-PMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. P-PMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations P-PMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in-vitro. In-vivo, the activity of P-PMO was significantly decreased in SCARA1 knock-out mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2?OMe as shown by competitive inhibition and co-localization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that P-PMO and tcDNA have higher binding profiles to the receptor compared to 2?OMe. These results demonstrate receptor-mediated uptake for a range of ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles.