Nuclear Magnetic Resonance to Study Atomic Level Protein-Protein Interactions

Overview

This video describes the nuclear magnetic resonance spectroscopy technique to study protein-protein interactions between ¹⁵N-labeled wild-type and mutant envoplakin proteins and the unlabeled vimentin protein. The successful interaction between wild-type envoplakin and vimentin leads to extensive line broadening and peak disappearance in the NMR spectra, whereas the absence of an interaction between the mutated envoplakin and vimentin results in well-resolved peaks in the NMR spectra.

Protocol

1. NMR Methods

1. NMR Sample Preparation
   1. Purify ¹⁵N-labeled wild-type or R1914E E-PRD protein using 20 mM Tris-HCl, 1 mM DTT, pH 7. Protein stock solutions usually range from 0.3 to 1 mM with volumes of about 1 mL.
      NOTE: Protein can be concentrated to >100 µM using an MWCO 3 kDa, 5 mL centrifugal ultrafiltration device to bring the concentration into a suitable range for sample preparation.
   2. Purify a sample of unlabeled VimRod protein using 20 mM Tris-HCl, 1 mM DTT, pH 7.
   3. In a final volume of 500 µL, add wild-type or mutant E-PRD protein to a final concentration of 100µM, deuterium oxide (D₂O) to a final concentration of 10% (v/v), and DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) to a final concentration of 20 µM. Bring the sample volume up to 500 µL using 20 mM Tris-HCl, 1 mM DTT, and pH 7. Representative sample preparation is described in Table 1.
      NOTE: The 0 ppm resonance of DSS is used to calibrate the ¹H chemical shifts as well as for indirect referencing of the ¹⁵N chemical shifts of the protein. D₂O is used for the deuterium lock signal to keep the spectrometer operating at a constant net magnetic field.
   4. Make up a second sample of E-PRD, D₂O, and DSS as in the previous step and add VimRod to a final concentration of 50 µM before bringing the volume up to 500 µL.
   5. Transfer the 500 µL samples to 5 mm wide NMR tubes for the experiment.

2. NMR Experimental Setup

1. 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 by the opening and insert it with the command “ij”. Wait until the sample settles inside the magnet before proceeding.
2. Create a new dataset using the “edc” command and load standard ¹H NMR parameters by selecting experiment “ZGPR” (Figure 1). Fill in the NAME, EXPNO (experiment number), and PROCNO (processed data folder number) fields. Select the solvent in the “Set solvent” field and click on “Execute ‘getprosol’” to read the standard probe head and solvent-dependent (prosol) parameters.
3. Lock the sample to the deuterated solvent, i.e., D₂O, using the command “lock” and wait until it is finished sweeping and achieves lock.
4. 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.
5. Shim the magnetic field using TOPSHIM (command “topshim”). Shimming is the process of adjustments to a magnetic field to achieve uniformity around the sample. It is good practice to store the shim values with the command “wsh” and read them using “rsh” before topshim, if using the same or similar samples.
6. Adjust the receiver gain with “rga” command to achieve maximum signal-to-noise ratio.
7. Place the center of the spectrum on the water resonance offset (o1) and set the 90-degree proton pulse (p1) at high power using “calibo1p1”.
8. Collect the proton spectrum using the zero go “zg” command and process with “efp” which includes exponential multiplication (“em”), the free induction decay (FID) incorporating line broadening, “ft” Fourier transformation of FID and “pk” to apply phase correction.
9. Apply the automatic phase correction “apk” and the automatic baseline correction “absn” using the polynomial without integration option.
10. Create a new dataset (as in 1.2.2) for the SOFAST HMQC experiment by selecting “SFHMQC3GPPH” in the experiment.
11. Copy optimized P1 and O1 from proton spectrum and populate P1 dependent pulses by using command “getprosol 1H p1 plw1”, where p1 is the optimized P1 value and plw1 is the power level for P1.
12. Optimize the CNST54 constant to set the offset for amide chemical shift and CNST55 to define the bandwidth in order to encompass the spectral regions of interest which allows the receiver gain to be optimized (Figure 2). To select these parameters, extract the first FID (free induction decay) from the two-dimensional spectrum and look for the observed signal to define them. In addition, vary the relaxation delay (D1), number of scans (NS), and dummy scans (DS) to obtain acceptable signal sensitivity with the command “gs”, which enables go and scan to monitor data quality in real-time.
13. Record the spectra using Zero Go “zg”.

Table 1. NMR Sample Preparation.

Sample	1 mM E-PRD in buffer A (µL)	1mM VimRod in buffer A1 (µL)	Buffer A (µL)	200 µM DSS in D2O (µL)	Buffer B2 (µL)	Total Volume (µL)
E-PRD alone	50	0	50	50	350	500
E-PRD + VimRod	50	50	0	50	350	500
1Buffer A: 20 mM Tris-HCl, 1 mM DTT, pH 7
2Buffer B: 23 mM Tris-HCl, 1.14 mM DTT, pH 7

Representative Results

Figure 1: Screen Capture of the Setup of the NMR Experiment. The window shown is used to set up a standard experiment to collect an HSQC dataset. Experiment parameters are read adjacent to Experiment. The ZGPR experiment shown is chosen as an initial experiment to load the standard and solvent-dependent proton parameters. The Title window is used to input experimental details for record-keeping purposes. To collect the HSQC spectrum the ZGPR experiment is replaced with SFHMQC3GPPH.

Figure 2: Adjusting of NMR Experimental Parameters. The window shown is used for entering the basic parameters for the NMR pulse sequence in order to optimize the signal.

Materials

Name	Company	Catalog Number	Comments
Ammonium chloride (15N, 99%)	Cambridge Isotope Laboratories	NLM-467	Isotope labelling for NMR
D2O, Deuterium Oxide (D, 99.8%)	Cambridge Isotope Laboratories	DLM-2259	NMR sample preparation
DSS, Sodium 2,2-dimethyl-2- silapentane-5-sulfonate-D6 (D,98%)	Cambridge Isotope Laboratories	DLM-8206	Reference for NMR
Precision 5 mm NMR Tubes, 7” long	SJM/Deuterotubes	BOROECO-5-7	NMR tubes
NMR spectrometer (14.1 Tesla)	Bruker		Acquisition of NMR data
TCI 5mm z-PFG cryogenic probe	Bruker		Acquisition of NMR data
Software
Bruker TopSpin 4.0.1	Bruker		Processing of NMR data