Probing orientations of single fluorescent labels on a peptide reversibly binding to the human delta-opioid receptor.

We report the first in-depth study of single-molecule polarization behavior of a species that is undergoing reversible binding with its biological receptor. We examine the utility of the information in single-molecule polarization measurements for investigations of binding. The human delta-opioid receptor, which is a G protein-coupled receptor, was incorporated into a supported lipid bilayer. A Cy3 label was covalently attached by a hydrophilic linker to a peptide agonist, Deltorphin II (5,6 Ile-Ile). The fluorescence excitation was alternated between s- and p-polarization using a microscope having the capability of total internal reflectance fluorescence (TIRF) excitation. The polarization behavior reveals that nonspecific binding events for this system give emission that is mostly s-polarized, while binding to the receptor gives emission that has a strong component of p-polarization. The results show that a high signal-to-noise ratio is achievable with single-molecule polarization measurements. The experiment detected 37 binding events of short duration (<30 s) and 35 binding events of long duration (from 30 s to 500 s). The polarization studies indicate that the receptors in the bilayer do not freely rotationally diffuse in the plane of the bilayer when the peptide is bound. The system exhibits two types of polarization behavior. One type has the dye label with fixed orientation, which sometimes abruptly switches. The other type has the dye orientation continuously fluctuating over time, typically exhibiting occasional periods of fixed orientation. For a long binding event of fixed orientation, it is established through analysis of the variance that the orientation actually is fluctuating through a range of angles on the order of 6 degrees. It is shown that precise measurements of reorientation are achievable, with a detection limit of 1.3 degrees for a typical single-molecule signal.

Peptide-labeled quantum dots for imaging GPCRs in whole cells and as single molecules.

We report a robust and practical method for the preparation of water-soluble luminescent quantum dots (QDs) selectively coupled through an amine or thiol linkage to peptide ligands targeted to G-protein coupling receptors (GPCRs) and demonstrate their utility in whole-cell and single-molecule imaging. We utilized a low molecular weight ( approximately 1200 Da) diblock copolymer with acrylic acids as hydrophilic segments and amido-octyl side chains as hydrophobic segments for facile encapsulation of QDs (QD 595 and QD 514) in aqueous solutions. As proof of principle, these QDs were targeted to the human melanocortin receptor (hMCR) by chemoselectively coupling the polymer-coated QDs to either a hexapeptide analog of alpha-melanocyte stimulating hormone or to the highly potent MT-II ligand containing a unique amine. To label QDs with ligands lacking orthogonal amines, the diblock copolymers were readily modified with water-soluble trioxa-tridecanediamine to incorporate freely available amine functionalities. The amine-functionalized QDs underwent facile reaction with the bifunctional linker NHS-maleimide, allowing for covalent coupling to GPCR-targeted ligands modified with unique cysteines. We demonstrate the utility of these maleimide-functionalized QDs by covalent conjugation to a highly potent Deltorphin-II analog that allowed for selective cell-surface and single-molecule imaging of the human delta-opioid receptor (hDOR).

Lipid bilayers on polyacrylamide brushes for inclusion of membrane proteins.

The ability of neutral polymer cushions to support neutral lipid bilayers for the incorporation of mobile transmembrane proteins was investigated. Polyacrylamide brush layers were grown on fused silica using atom-transfer radical polymerization to provide polymer layers of 2.5-, 5- and 10-nm thickness. Lipid bilayers composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) were formed by vesicle fusion onto bare fused silica and onto each of the polyacrylamide layers. Bilayer fluidity was assessed by the diffusion of a probe, NBD-labeled phosphatidylcholine, using fluorescence recovery after photobleaching. A transmembrane protein, the human delta-opioid receptor, was inserted into each lipid bilayer, and its ability to bind a synthetic ligand, DPDPE, cyclic[2-d-penicillamine, 5-d-penicillamine]enkephalin, was detected using single-molecule fluorescence spectroscopy by labeling this ligand with a rhodamine dye. The transmembrane protein was observed to bind the ligand for all bilayers tested. The protein's electrophoretic mobility was probed by monitoring the fluorescence from the bound ligand. The 5-nm polyacrylamide thickness gave the fastest diffusion for the fluorescent lipid probe (D(1) = 2.0(+/-1.2) x 10(-7) and D(2) = 1.2(+/-0.5) x 10(-6) cm(2)/s) and also the largest electrophoretic mobility for the transmembrane protein (3 x 10(-8) cm(2)/V.s). The optimum in polymer thickness is suggested to be a tradeoff between decoupling from the substrate and increasing roughness of the polymer surface.

Lactone cleavage reaction kinetics of rhodamine dye at liquid/liquid interfaces studied by micro-two-phase sheath flow/two-photon excitation fluorescence microscopy.

Two-photon excitation fluorescence microscopy was combined with the two-phase microflow system in order to measure the fast interfacial reaction rate at liquid/liquid interfaces. The lactone cleavage kinetics of octadecylrhodamine B (C(18)RB) at the toluene/water and heptane/water interfaces was studied by this new method. The organic solution containing the nonfluorescent lactone of C(18)RB was made to flow as an inner flow with an aqueous outer sheath flow. The diameter of the inner flow was <20 microm. A focused fundamental beam of a Ti:sapphire pulse laser of 780 nm was irradiated to the interface, and emitted fluorescence from the fluorescent product was detected by a charge-coupled device (CCD) camera or a streakscope. The increase in the concentration of the fluorescent form of C(18)RB was measured along the interface of the inner flow of the toluene/water and heptane/water systems for 80 micros just after the contact of two phases. The analysis made by the time-dependent Langmuir adsorption model with the aid of the digital simulation method gave the cleavage reaction rate constants of the lactone form of C(18)RB at the liquid/liquid interfaces.