Nuclear Magnetic Resonance to Study Atomic Level Protein-Protein Interactions

Nuclear magnetic resonance, or NMR, spectroscopy enables atomic-level protein-protein interaction detection.

To study interactions between globular and helical proteins, label purified wild-type or mutant globular proteins with ¹⁵N — a nitrogen isotope. In an NMR tube, mix the labeled globular and unlabeled helical proteins in a solvent containing an NMR standard. Insert the tube into the NMR spectrometer.

The spectrometer magnets generate a strong magnetic field, aligning the ¹⁵N-labeled globular protein nuclei parallel to the field. A radiofrequency, or RF, pulse excites the ¹⁵N-nuclei to align antiparallel to the field.

The pulse frequency required for the transition of ¹⁵N nuclei between the two states defines the resonance frequency. Normalizing the ¹⁵N-resonance frequency to the NMR standard measures the ¹⁵N-chemical shift.

The magnetic field is adjusted to align the ¹H-nuclei — bonded to ¹⁵N — parallel to the field. The RF pulses are adjusted, and the ¹H-chemical shift is recorded.

During protein interactions, the chemical environment surrounding the interacting amino acid residues changes, altering the ¹⁵N-¹H chemical bonds and corresponding chemical shifts. Plot the ¹⁵N-¹H NMR spectra.

Without helical proteins, wild-type globular proteins exhibit well-resolved spectral peaks. When helical and wild-type globular proteins interact, the spectral peak is reduced. In contrast, incubating helical proteins with mutant globular proteins results in the spectral peaks reappearing, indicating no interaction between them.

Turn on the airflow with the eject command "ej". This will bring the sample up from the magnet. Now, place the sample within a spinner on top of the magnet in the opening. Insert with the command "ij". Wait until the sample settles inside the magnet before proceeding.

Create a new data set using the "edc" command, and load Standard Proton NMR parameters by selecting the experiment "ZGPR". Fill in the name, experiment number, and processed data folder number fields.

Select the solvent in the "Set Solvent" field and click on "Execute 'getprosol'" to read standard probehead head and solvent-dependent parameters. Lock the sample to the deuterated solvent using the "Lock" command, and wait until it is finished sweeping and achieves lock.

Correct the resonance frequency of the magnet by tuning the sample using the automatic tuning command "atma". Monitor the wobble curve until the automatic tuning is complete.

Shim the magnetic field using "TOPSHIM". Shimming makes adjustment to the magnetic field to achieve uniformity around the sample. It is good practice to store the same values with the command "wsh" and read them using "rsh" before topshim if using the same or similar samples.

Now, adjust the receiver gain with the "rga" command to achieve the maximum signal-to-noise ratio. Place the center of the spectrum on the water resonance offset and set the 90-degree proton pulse at high power using "calibo1p1".

Collect the proton spectrum using the Zero Go "zg" command and process with "efp". This includes exponential multiplication, the free induction decay incorporating line broadening, Fourier transformation of FID, and "pk" to apply phase correction.

Apply the automatic phase correction "apk" and the automatic baseline correction "absn" using the polynomial without integration option. Create a new data set for the SOFAST HMBC experiment by selecting "SFHMQC3GPPH" in experiment.

Copy the optimized P1 and O1 from the proton spectrum and populate P1-dependent pulses by using the command "getprosol 1H p1 plw1" where P1 is the optimized P1 value and plw1 is the power level for P1. Now, optimize the CNST54 constant to set the offset for amide chemical shift. Also, optimize CNST55 to define the bandwidth in order to encompass the spectral regions of interest, allowing the receiver gain to be optimized.

To select these parameters, extract the first FID from the two-dimensional spectrum, and look for the observed signal to define them. In addition, vary the relaxation delay, number of scans, and dummy scans to obtain acceptable signal sensitivity with the command "gs", which enables go and scan to monitor data quality in real-time. Finally, record the spectra using Zero Go "zg".