SMD:  Single-Molecule Detection Fluroescence Spectroscopy

Summary. 
The Sakmar Laboratory is committed to developing new approaches to probe GPCR dynamics and intermolecular interactions, such as the binding of a specific ligand to a specific receptor, in real time.  Typical ensemble assays of receptor-ligand interactions rely on decades-old formulations of receptor theory and assume equilibrium or pseudo-equilibrium conditions.  In studies of surface phenomena that occur on cell membranes, it would be preferable to measure individual binding events and then to use statistical analysis to derive kinetic rate constants and thermodynamic parameters where possible.  The Sakmar Laboratory has implemented an arsenal of tools to enable single-molecule detection (SMD) fluorescence experiments of GPCRs reconstituted in solid-supported bilayer membranes with defined lipid composition.  For example, members of the Sakmar Laboratory have established a method for site-specific introduction of unnatural amino acids (see section on UAA mutagenesis) in GPCRs and other proteins expressed in mammalian cells in culture.  They have also demonstrated bio-orthogonal chemical modification of these UAAs in the prototypical GPCR, rhodopsin.  One particularly useful method for SMD studies of GPCRs is called TIRF (total internal reflection fluorescence) microscopy.  The Sakmar Laboratory recently assembled a TIRF microscopy workstation capable of SMD. 

SMD Using TIRF. 
In TIRF microscopy, a light source is focused on the surface of a thin glass slide at a critical angle of incidence that creates an evanescent field that emanates a short distance (typically about 200 microns) above the opposite surface of the glass.  A fluorescent probe with the correct photochemical properties that lies within the evanescent field becomes excited and emits a photon, which can be detected by the optical collection system of a suitable epifluorescence microscope.  The TIRF method can be used for SMD experiments, Förster resonance energy transfer (FRET) applications, or imaging.  A brief description of TIRF microscopy can be found by clicking here.

Dr. Thomas Huber designed a novel strategy suitable for SMD-TIRF experiments of GPCRs.  In preliminary experiments, expressed recombinant epitope-tagged chemokine receptors were immuno-captured in a self-assembling, oriented, tethered membrane bilayer on the surface of a TIRF chip.  The custom chip surfaces can be prepared by sequential perfusion of different protein solutions in a simple semi-microfluidics chamber.  Dr. Huber recorded SMD images of single fluorescently labeled chemokines (MIP-1α-Alexa-647) bound to recombinant expressed CCR5 chemokine receptor embedded in the tethered bilayer membrane.  Each fluorescence spot represents the fluorescent chemokines that bind to CCR5.  The membrane-receptor assembly is stable for hours and can be interrogated repeatedly using a continuous flow cell chamber.  For example, the MIP-1α-Alexa-647 can be washed out of the chip flow cell and a different concentration of MIP-1α-Alexa-647, or a different labeled chemokine, can be added in turn.  Competition experiments with small molecules or with chemokine mixtures can be carried out as well. 

Novel SMD-TIRF-FRET Methods. 
SMD-TIRF-FRET (fluorescence resonance energy transfer) experiments can be carried out using the general approach and microscope already in place, provided that an appropriate fluorescent donor-acceptor pair can be adapted.  In FRET, excitation of the donor fluorophores will cause emission from the acceptor fluorophores provided that they are situated together within their characteristic Förster distance (~2-8nm distance range).  To study receptor-ligand interactions, or protein-protein interactions, if two molecules of interest, one harboring the donor and the other the acceptor, form a complex that allows the fluorophores to come together within their Förster distance, then FRET will occur. If no FRET signal is observed, it suggests that the target molecules have no direct interaction.  The key to making clear-cut interpretations of FRET signals relies on establishing a relevant positive control.  In one particular formulation under development, a GPCR is labeled with Alexa-494 at a Cys residue through maleimide chemistry.  The Sakmar Laboratory is also developing Europium(III) dye conjugates to label other signaling components.  Europium(III) dyes exhibit superb photochemical properties, which will allow fluorescent imaging at the ultimate SMD level.




























































































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