Monitoring Conformational Dynamics of Single Unmodified Proteins using Plasmonic Nanotweezers

This research aims to gain insight into label-free proteins at the single-molecule level in real time, with a particular focus on proteins that are challenging to investigate using current techniques or have relevance to disease. Plasmonic nanotweezers have recently demonstrated their ability to monitor conformational dynamics upon binding to small molecules, check disassembly kinetics, and elucidate free energy landscapes all at the single-molecule level. Currently, no established protein characterization technique is able to investigate label-free single-molecule protein conformational dynamics.

Plasmonic nanotweezers are believed to have the potential to fill this niche within the field. This unique advantage helps us to investigate the relationship between the conformational change of proteins and their biological functions and the implications in disease development. Future research will focus on intrinsically disordered and membrane proteins, as many of these are implicated in several diseases such as Alzheimer's, Parkinson's, and various cancers.

To begin, place the PEG-thiol coated sample into the 3D-printed flow cell using straight tweezers, ensuring the gold layer faces upwards. Peel off one side of the clear PET plastic double-sided tape cover. Carefully place the tape over the sample and flow cell, ensuring that the nano structures and intake/outtake holes in the flow cell remain uncovered.

Using rounded tweezers, gently press around the edges of the tape to secure its adhesion to the flow cell and sample. Peel off the other side of the tape and gently place a glass cover slip over the sample. Use rounded tweezers to press around the edges of the cover slip to secure its adhesion.

This process creates a liquid channel within the flow cell with a height of 50 micrometers and a volume of 3.5 microliters. Using a small pipette tip, mix equal parts of solution A and solution B of the duplicating silicone onto a microscope slide at a one-to-one ratio or is specified by the manufacturer. Fill the gaps between the cover slip and flow cell with a mixed duplicating silicone, gently pushing it under the cover slip.

Hold the flow cell upside down and carefully apply duplicating silicone around the inner wall of the hole. Gently move the silicone onto the visible edges of the fused silica underside of the sample, leaving the sample to dry with the gold layer facing upwards until the duplicating silicone has fully set. After verifying that all components are properly connected, load the microfluidic system UI on the computer.

Click on the play icon next to the MUX distributor and wire, which control the 12 by one rotary valve and the three by two way valve respectively to open their corresponding interfaces. To bypass the three by two way valve, turn on port one of the wire. To mount the flow cell into plasmonic nanotweezers, attach the inlet and outlet tubes to appropriate parts of the flow cell.

Place the flow cell on clean tissue with the gold layer facing upwards. Infuse buffer into the flow cell at a high flow rate of approximately 0.3 milliliters per minute. Check that the fluid moves across the sample inside the flow cell and that no fluid is visible on the exterior or underside.

Apply one to two drops of immersion oil onto the 100X objective. Place the flow cell into the plasmonic nanotweezers stage with the gold layer facing downwards. Secure the flow cell using metal clips over the magnets and lock the stage to keep it in place.

Turn on the white light source. Open the camera software and adjust the exposure time and gain until the nano structures become visible. Turn on the laser at a relatively high power and manually adjust the Z-axis until the laser spot is visible.

Turn on the piezoelectric controller and select the appropriate settings for the comm port and maximum voltage. Set the X, Y, and Z-axis values to half of the maximum voltage to allow for better stage alignment in all directions. Align the laser spot with one of the double nano hole or DNH structures using the main stage X, Y, and Z-axis control knobs.

Ensure the avalanche photo diode or APD is turned on. Gently close the enclosure to the plasmonic nanotweezers. Open the software associated with APD recording, such as a homemade LabVIEW UI.Edit the file name and set the cutoff frequency to one kilohertz.

Then specify the desired file path where the files will be saved. Turn off the white light source and turn the laser back on. After setting the laser power to an appropriate level, use the piezoelectric controls to adjust the X, Y, and Z-axes until the APD signal is as high as possible.

Avoiding APD saturation and with minimal standard deviation of the trace. Next, turn off the laser to preserve the lifespan of the nano structures. Run the syringe pump control unit and the distributor UI to set the valve and withdraw the desired amount of protein.

Using the microfluidic UI, infuse the protein solution at a flow rate similar to the withdrawal rate. Continue infusion at this rate until the protein solution reaches the flow cell. Then set the flow rate to an appropriate value for trapping and wait for the trace to be stabilized.

Verify that the volume and time on the syringe pump match the expected values. To record the APD signal data, adjust the X, Y, and Z axes as needed using the piezoelectric controller UI.Upon observing a large change in transmission and standard deviation, note down the time this occurs for future data sorting. If the protein needs to be released as part of the experiment, turn off the laser for approximately five seconds, then turn it back on.

The trace should have a large change in transmission and a significantly lower standard deviation, indicating a return to the baseline state. After completing the experiment, turn off the laser. Remove the flow cell from the three-axis stage and disconnect the microfluidic system tubing.

Place the flow cell on clean tissue with the gold layer facing upwards. Using a scalpel, carefully cut the glue beneath the glass cover slip before gently lifting it off and disposing of it. Hold the flow cell at an angle with the gold layer still facing upwards and use rounded tweezers to carefully remove the glue from the underside of the flow cell to free the sample.

Using straight tweezers, pick up the sample and rinse it thoroughly with isopropanol. Dry the sample using an air gun. A representative experiment carried out on in-situ iron loading to an apoferritin molecule demonstrates the use of plasmonic nanotweezers as a tool to investigate protein conformational dynamics.

The transmission trace of apoferritin remains stable when trapped in a PBS solution, indicating no significant conformational changes. Upon exposure to the ferrous solution, fluctuations in the standard deviations of the trace increased, indicating dynamic structural changes associated with iron loading. After 20 minutes of iron exposure, the transmission trace stabilized, indicating the transition of apoferritin to its holoform.

The probability density function plots demonstrated a shift in voltage distribution upon iron loading, further supporting the conformational transition from apoferritin to holoferritin.