Bioelectronic Measurement of Target Engagement to a Membrane-Bound Transporter.

The ability to detect and characterize drug binding to a target protein is of high priority in drug discovery research. However, there are inherent challenges when the target of interest is an integral membrane protein (IMP). Assuming successful purification of the IMP, traditional approaches for measuring binding such as surface plasmon resonance (SPR) and fluorescence resonance energy transfer (FRET) have been proven valuable. However, the mass dependence of SPR signals may preclude the detection of binding events when the ligand has a significantly smaller mass than the target protein. In FRET-based experiments, protein labeling through modification may inadvertently alter protein dynamics. Graphene Bio-Electronic Sensing Technology (GBEST) aims to overcome these challenges. Label-free characterization takes place in a microfluidic chamber wherein a fluid lipid membrane is reconstituted directly above the GBEST sensor surface. By leveraging the high conductivity, sensitivity, and electrical properties of monolayer graphene, minute changes in electrostatic charges arising from the binding and unbinding of a ligand to a native IMP target can be detected in real time and in a mass-independent manner. Using crude membrane fractions prepared from cells overexpressing monocarboxylate transporter 1 (MCT1), we demonstrate the ability to (1) form a fluid lipid bilayer enriched with MCT1 directly on top of the GBEST sensor and (2) obtain kinetic binding data for an anti-MCT1 antibody. Further development of this novel technology will enable characterization of target engagement by both low- and high-molecular-weight drug candidates to native IMP targets in a physiologically relevant membrane environment.

The lipid phase preference of the adenosine A2A receptor depends on its ligand binding state.

Giant unilamellar protein vesicles (GUPs) were formed with the adenosine A2A receptor (A2AR) incorporated in the lipid bilayer and protein partitioning into the liquid ordered and liquid disordered phases was observed. When no ligand is bound, A2AR partitions preferentially into the liquid disordered phase of GUPs, while ligand-bound A2AR partitions into the liquid ordered phase.

Evaluation of dextran(ethylene glycol) hydrogel films for giant unilamellar lipid vesicle production and their application for the encapsulation of polymersomes.

Giant Unilamellar Vesicles (GUVs) prepared from phospholipids are becoming popular membrane model systems for use in biophysical studies. The quality, size and yield of GUVs depend on the preparation method used to obtain them. In this study, hydrogels consisting of dextran polymers crosslinked by poly(ethylene glycol) (DexPEG) were used as hydrophilic frameworks for the preparation of vesicle suspensions under physiological ionic strength conditions. A comparative study was conducted using hydrogels with varied physicochemical properties to evaluate their performance for GUV production. The prepared GUVs were quantified by flow cytometry using the Coulter Principle to determine the yield and size distribution. We find that hydrogels of lower mechanical strength, increased swellability and decreased lipid interaction favour GUV production, while their resulting size is determined by the surface roughness of the hydrogel film. Moreover, we embedded polymersomes into the crosslinked hydrogel network, creating a DexPEG - polymersome hybrid film. The re-hydration of lipids on those hybrid substrates led to the production of GUVs and the efficient encapsulation of polymersomes in the lumen of GUVs.

Dynamics of Hydrogel-Assisted Giant Unilamellar Vesicle Formation from Unsaturated Lipid Systems.

While current research is centered on observing biophysical properties and phenomena in giant unilamellar vesicles (GUVs), little is known about fabrication parameters that control GUV formation. Using different lipids and rehydration buffers, we directly observe varying dynamics of hydrogel-assisted GUV formation via fluorescence microscopy. We observe the effects of buffer ionic strength, osmolarity, agarose density, and pH on the formation of GUVs using neutral and charged lipids. We find that increasing rehydration buffer ionic strength correlates with increased vesicle size and rate of GUV formation. Increasing buffer acidity increased the rate of GUV formation, while more basic environments slowed the rate. For buffers containing 500 mM sucrose, GUV formation was overall inhibited and only tubules formed. Observations of GUV formation dynamics elucidate parametric effects of charge, ionic strength, pH, and osmolarity, demonstrating the versatility of this biomimetic platform.

G Protein-Coupled Receptors Incorporated into Rehydrated Diblock Copolymer Vesicles Retain Functionality.

G protein-coupled receptor (GPCR) is incorporated into polymeric vesicles made up of diblock copolymer bilayers. Successfully incorporated GPCRs exhibit correct biased physiological orientation and respond to various ligands. After extended dehydrated storage via lyophilization and subsequent rehydration, diblock copolymer polymersomes retain their shape and incorporated GPCR retains its function.

The Functional Activity of the Human Serotonin 5-HT1A Receptor Is Controlled by Lipid Bilayer Composition.

Although the properties of the cell plasma membrane lipid bilayer are broadly understood to affect integral membrane proteins, details of these interactions are poorly understood. This is particularly the case for the large family of G protein-coupled receptors (GPCRs). Here, we examine the lipid dependence of the human serotonin 5-HT1A receptor, a GPCR that is central to neuronal function. We incorporate the protein in synthetic bilayers of controlled composition together with a fluorescent reporting system that detects GPCR-catalyzed activation of G protein to measure receptor-catalyzed oligonucleotide exchange. Our results show that increased membrane order induced by sterols and sphingomyelin increases receptor-catalyzed oligonucleotide exchange. Increasing membrane elastic curvature stress also increases this exchange. These results reveal the broad dependence that the 5-HT1A receptor has on plasma membrane properties, demonstrating that membrane lipid composition is a biochemical control parameter and highlighting the possibility that compositional changes related to aging, diet, or disease could impact cell signaling functions.

Human serotonin receptor 5-HT(1A) preferentially segregates to the liquid disordered phase in synthetic lipid bilayers.

We demonstrate successful incorporation of the G protein coupled receptor 5-HT1A into giant unilamellar vesicles using an agarose rehydration method. With direct observation using fluorescence techniques, we report preferential segregation of 5-HT1A into the cholesterol-poor liquid disordered phase of the membrane, contradicting previous reports of lipid raft segregation. Furthermore, altering the concentration of cholesterol and sphingomyelin in ternary mixtures does not alter 5-HT1A segregation into the liquid disordered phase.

[12]Annulene radical anions revisited: evaluation of structure assignments based on computed energetic and electron spin resonance data.

We report density functional and coupled cluster calculations on numerous monocyclic and bicyclic (CH)12(*-) isomers. At the RCCSD(T)/cc-pVDZ//UB3LYP/6-31+G* level, a nearly planar, bond-equalized radical anion of 1,7-di-trans-[12]annulene (4a(*-)) is lowest in energy; several other isomers and conformations lie within 3 kcal/mol of 4a(*-). RCCSD(T)/AUG-cc-pVDZ//UB3LYP/6-31+G* results place the all-cis isomer 3(*-) slightly below 4a(*-) in energy. Validation studies on the heptalene radical anion, [16]annulene radical anion, and tri-trans-[12]annulene radical anion indicate that electron spin resonance (ESR) hyperfine coupling constants (aH values) computed at the BLYP/EPR-III level on DFT geometries give much better agreement with experimental values than those computed using B3LYP/6-31G*. We were unable to locate any C12H12(*-) isomer that could account for the ESR spectrum previously attributed to a highly twisted structure for the 1,7-di-trans-[12]annulene radical anion. Our computed energetic and ESR data for [12]annulene radical anions and their valence isomers suggest that 4a(*-) may have been made, yet its ESR spectrum was incorrectly assigned to the bicyclic isomer 6b(*-). Finally, the computed (1)H NMR shift values of the dianion of 4 reveal a distinct diatropic ring current that should aid in its characterization.