Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

This method can help answer key questions in the protein interaction field such as whether protein binds to a small or a large molecule directly. The main advantage of this technique is that it is simple and fast way to detect direct interactions at the atomic level. Turn on the air flow 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 dataset 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 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.

Achieve uniformity around the sample. It is good practice to store this in 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 dataset 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. Set the processing parameters to the size of the direct F2 and indirect F1 dimensions of the spectrum, with optional linear prediction in the indirect dimension. Select QSINE as the Window function and enter a Sine bell shift of two to process the two-dimensional spectrum.

Enter the command xfb to process the data in both directions with window function and Fourier transformation. Use the command apk2d to carry out automatic phase correction in both directions. Correct the baseline with the automatic baseline correction function abs2 for 2D data.

This applies a polynomial function between the ppm values defined in the processing parameters, and will produce a 2D spectrum for further analysis. If planning to perform serial processing for comparison of interaction data with another molecule, store the processing parameters with the command wpar and recall them with rpar. Enter the command pp to begin the process of peak picking.

Define the ppm range, minimum intensity, and maximum number of peaks based on expected peaks. Then click OK.Verify the results by visual inspection. If needed, re-run the process until results are satisfactory based on spectra quality.

Generate a peak list with the pp command. This peak list contains data height and peak intensity information by default. The peak list can be exported to subsequent spectrums and can be read by other programs.

Now observe the protein HSQC spectra for changes in peak intensities or movement in chemical shifts that indicate interaction with another molecule. If the interacting molecule is large, expect reductions in peak intensities along with disappearance of some peaks. Import the peak list to the next data set by clicking on the peaks tab and selecting import with a right click in the peaks window.

Visualize the peaks over the spectrum and if needed, shift them to new positions. Click on reset intensities for complete table to generate a peak list for the spectrum that includes intensities. This peak list will carry over the position information from the stored peak list.

Export the peak lists from different datasets to a spreadsheet or other mathematical program for analysis by selecting the Export function. Calculate the change in peak intensities with the function peak intensity in complex spectrum, peak intensity in protein spectrum for each peak. Values can be converted to percentage change by multiplication by 100.

Note that peak volumes are also useful, although peak intensities are easier to measure for peaks that are positioned close to each other, as is usually the case for proteins with a high density of relatively broad peaks. The N15HSQCs were acquired for the wild type E-PRD, as well as the R1914E mutant, in the presence or absence of VimRod. The spectrum of wild type E-PRD shows the expected number of well resolved peaks, indicative of a properly folded protein.

In the presence of VimRod, the spectrum shows extensive line broadening and peak disappearance, corresponding to binding between E-PRD and VimRod. Little change is observed between the spectrum of the R1914E mutant on its own and upon addition of VimRod, indicating a lack of binding to this mutant E-PRD. Here the E-PRD peak intensities in the presence and absence of VimRod were compared and plotted as the relative peak intensities to indicate the range of peak broadening in the E-PRD complex.

To validate and quantitate the binding of VimRod and E-PRD, MST analysis using His6-VimRod labeled with fluorescent RED-tris-NTA dye as the target, was performed using decreasing concentrations of the E-PRD ligand. The data were fit with a standard model of one-site ligand binding and gave a KD of 25.7 plus or minus 2.1 micromolar. While attempting this procedure, it is important to remember to use identical acquisition and processing conditions.

will struggle because of access to isotope labeled protein samples for collection of NMR spectra. The implications of this technique extend toward therapy of cancer, infection, or neurodegenerative disease because they involve formation of protein interactions that are compromised in the disease.