Single-Molecule FRET Analyses of NMDA Receptors

Abstract

Single-molecule fluorescence resonance energy transfer (smFRET) enables the real-time observation of conformational changes in a single protein molecule of interest. These observations are achieved by attaching fluorophores to proteins of interest in a site-specific manner and investigating the FRET between the fluorophores. Here we describe the method wherein the FRET is studied by adhering the protein molecules to a slide using affinity-based interactions and measuring the fluorophores' fluorescence intensity from a single molecule over time. The resulting information can be used to derive distance values for a point-to-point measurement within a protein or to calculate kinetic transition rates between various conformational states of a protein. Comparing these parameters between different conditions such as the presence of protein binding partners, application of ligands, or changes in the primary sequence of the protein can provide insights into protein structural changes as well as kinetics of these changes (if in the millisecond to second timescale) that underlie functional effects. Here we describe the procedure for conducting analyses of NMDA receptor conformational changes using the above methodology and provide a discussion of various considerations that affect the design, execution, and interpretation of similar smFRET studies.

Keywords: Fluorescence resonance energy transfer; Glutamate receptors; NMDA receptors; Protein dynamics; Single-molecule methods.

Figure 1.. Slide Preparation and Protein Surface Attachment

A) A silicone template is applied to the cleaned smFRET slide to create an oval-shaped area on the slide. The ends of the oval area are marked on the other side of the slide. B) PEG solutions can be applied to the oval area by pipette to coat the surface of the slide with biotin molecules. C) The PEG solution and silicone template are removed and the slide is washed and dried. D) A chamber is attached to the slide aligned with the PEG-treated area. E) Press-fit tubing connectors are attached to each port of the chamber to create a liquid-tight seal. Solutions (such as streptavidin solution, antibody solutions, and protein sample) can be injected into one port of the chamber by pipette, and the solution in the chamber will flow out of the exit port. F) Different strategies can be employed to attach the NMDA receptors to the slide surface. The simplest method for attaching the receptors to the slide surface involves attaching a Twin-Strep-tag (yellow) to the C-terminus of the NMDA receptor (cyan). This tag acts like a biotin molecule (yellow) and will attach to streptavidin molecules (red) on the surface of the slide. This allows for recording of the donor (blue star) and acceptor (green star) fluorescence intensities over time. If adding a Twin-Strep-tag to the protein is not feasible, antibodies can instead be used. A biotinylated secondary antibody and an anti-NMDA receptor primary antibody (brown) are instead used to bridge the gap between the receptor and the streptavidin-coated surface.

Figure 2.. Diagram of smFRET Confocal Microscope

Excitation light (green) from the laser sources is reflected by a dichroic beamsplitter into the microscope body, where it excites the sample fluorophores. After the fluorophores undergo FRET, fluorescence emission light is emitted by the donor and acceptor fluorophores and collected by the objective. The collected emission light from the donor and acceptor (orange and red, respectively) passes through the dichroic beamsplitter and the confocal pinhole. A longpass beamsplitter reflects the donor emission towards the donor detector while a separate mirror directs the acceptor emission to the acceptor detector. Bandpass emission filters in front of the donor and acceptor detectors allow only donor or acceptor emitted wavelengths to pass into the detectors, respectively.

Figure 3.. Single-Molecule FRET Example Data

A) An image of a 20 μm × 20 μm area of the smFRET slide. The image shows the detected acceptor intensity during the donor excitation window, which represents the FRET signal. Disparate spots that each represent an individual NMDA receptor that has been labeled with fluorophores can be clearly seen spread across the slide surface. Individual molecules, such as the one within the yellow circle, can be selected for fluorescence intensity recording. B) Donor (Alexa 555, blue) and acceptor (Alexa 647, green) fluorescence intensity traces over time for an individual NMDA receptor molecule. Note the anticorrelation during Section 1 of the trace before the acceptor photobleaches where an increase in donor is matched by a decrease in acceptor and vice-versa. This anticorrelation indicates that FRET is occurring. Also note the single photobleaching step in the acceptor channel that marks the end of Section 1 of the trace and the single photobleaching step in the donor that marks the end of Section 2 of the trace. These single steps in each channel indicate that only one fluorophore of each type is present during this recording. Section 1 of the trace is the section where FRET is occurring and is the only section of the trace that is used to calculate the FRET efficiency. C) FRET efficiency values over time calculated from the donor and acceptor intensity traces. Note the presence of two distinct FRET levels, a low one around 0.40 FRET and a high one around 0.80 FRET. Efficiency traces can be analyzed to provide information about rates of transitions between states in a given condition. D) A histogram of the FRET efficiency values taken from the FRET efficiency time trace. The histogram visually shows the low and high FRET populations at around 0.40 and 0.80 FRET, respectively. Many efficiency traces from many molecules can be pooled into a single histogram showing the overall distribution of FRET efficiencies sampled by the protein under a given condition.