The paramagnetic relaxation enhancement technique can be utilized to characterize and measure intermolecular distances and quantify transient and/or lowly populated biomolecular interactions. Here, we have applied this technique to capture interactions that precede protein condensation. The sensitivity of PREs enables atomic resolution identification, detection, and quantification of dynamic state that remain invisible and inaccessible to other NMR techniques.
Key steps in this protocol include removing the unconjugated nitroxide spin label and carefully analyzing the spectra to accurately measure peak height. Additionally, caution must be exercised during NMR experiment setup and pulse calibration. To begin, add 3-maleimido-PROXYL from a stock solution at 20 times molar excess of the 15N isotopically labeled protein of interest.
Incubate overnight at room temperature or four degrees Celsius protected from light and oxygen and with gentle rocking or nutation. The following day, remove the non-reacted free spin label to prevent non-specific solvent PRE using either extensive dialysis of the protein sample or using gel filtration. Then use Ellman's reagent to quantify free sulfhydryl groups in the solution.
Measure the absorbance using a microplate reader and construct the standard curve. To measure intramolecular PRE, prepare 15N isotopically enriched spin labeled protein to a concentration of at least 100 micromolar, but not more than 300 micromolar, in a buffer suitable for NMR. Then using a long stem glass pipette or micropipette, transfer the NMR sample to a five millimeter NMR tube appropriate for use in high-field magnets.
Place the sample in the magnet. Lock on the deuterium signal using the lock command and attune and match the proton channel according to facility protocols. Then adjust the shims using the top shim subroutine to optimize solvent signal suppression.
Next, calibrate the proton pulse using the P-OPT program. Then calibrate the 15N pulse against a standard sample. Determine the correct attenuation for shaped pulses using the shape tool subroutine.
Then open the appropriate pulse shape file by clicking the folder icon. The shaped pulses are found in the pulse parameters section of acquisition parameters. After loading the pulse definition file, click Analyze Waveform, followed by Integrate Shape.
Input the calibrated proton 90 degree hard pulse, desired shape pulse length, and rotation. Then calculate the power level of the shaped pulse by adding the change of power level to the attenuation for the calibrated 90 degree pulse. Record a standard proton 15N HSQC to optimize sweep width, carrier frequency, and check water suppression.
Finally, adjust the sweep width and the number of indirect dimension increments using the SW and TD commands or directly in the appropriate dialog boxes. Calibrate the shaped pulses as demonstrated previously. Then enter the calibrated pulse lengths in the pulse parameters section on the Acquisition Parameters tab.
To measure the amide proton T2 using the two time delay point approach, set the time delays by editing the VD list file. The first delay is set to 0.01 milliseconds. Using the relationship to the expected maximum PRE, choose the second delay.
Then determine a suitable value by comparing the first increments of the first and second delay spectra and adjusting the second delay such that the signal decays to between 40 to 50%of its initial value. Determine the number of complex points to record and the number of scans for sufficient signal averaging. Then use the command EXPT to calculate the experiment time and start the experiment with the command ZG.After dissolving sodium ascorbate in the NMR buffer, adjust the pH to match that of the original NMR buffer.
Then add the ascorbic acid to the NMR tube at a 10 times molar excess over the concentration of the spin label by placing a droplet below the rim of the tube. Cap the tube and carefully invert to mix. Spin at 200 to 400 G for 10 to 20 seconds in a hand cranked centrifuge to settle the sample at the bottom of the tube.
After wrapping the tube in foil, allow the reaction to proceed for at least three hours. Then record amide proton T2 on the diamagnetic sample using the same parameters used for the paramagnetic sample. Intramolecular amide proton gammas PREs were recorded on a self-associating intrinsically disordered fragment derived from the low complexity domain of the RNA binding protein EWSR1.
Residues in close sequential proximity to the spin label attachment point are expected to be significantly broadened and are not detectable in the spectrum. Residues that were sequentially spaced from the attachment point yet showed enhanced gamma were spatially close to the spin label. In the case of intrinsically disordered proteins, recording and analyzing purees will yield information about intramolecular contacts and transient interaction surfaces.
Combining PRE measurements with other biophysical methods such as dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, and computational methods can provide deeper insights. PRE measurement aids in characterizing transient sparsely populated state. This approach could be expanded to characterize interactions important for myriad biological processes such as molecular recognition, allosteric conformational selection, and assembly processes including assembling membraneless organelles via phase separation.
