This protocol can be used for detection and characterization of protein conformational dynamics, which are essential for understanding a large variety of cellular processes. Compared to other methods, this method does not require specialized sample preparation steps and provides in comprehensive characterization of the kinetics, thermodynamics, and structural aspects of confirmational equilibrium. CPMG relaxation dispersion can be applied more widely for the characterization of confirmational dynamics in nucleic acids and other biomolecules, as well as for the characterization of ligand nanoparticle interactions.
Our protocol is aimed at first-time CPMG users. So it is a good starting point. However, we do expect users to know the basic steps for running conventional NMR experiments.
To set up an NMR experiment for the first time, first download and unzip these supplemental files. Copy the Bits. vv and trosy_15N_CPMG.
vv files in the pulse program folder to the pulse program directory. Open the acquisition software and use the EDC command to copy a previously run hydrogen nitrogen 15 HSQC experiment into a new experiment. Use the pulse program command to load the trosy_15N_CPMG.
vv pulse program file into the newly created experiment. Then use the instructions from the end of the pulse program file to set up the CPMG experiment. To set up a routine NMR experiment, introduce the sample to the magnet and perform all of the basic NMR acquisition steps.
Set P1 to the duration of the hydrogen hard 90 degree pulses and P7 to the duration nitrogen 15 hard 90 degree pulses. In the acquisition window, set the center and spectral width for the hydrogen and nitrogen 15 dimensions. Set the relaxation delay to 0.7 T2 and use the VC list command to create a list of integer numbers corresponding to N.After confirming that each entry in the list corresponds to a different CPMG field according to CPMG filed equals times 4N divided by D30, check that the first number in the list is zero.
Set L8 to the number of entries in the VC list, L3 to the number of real points for the indirect dimension, and 1TD to L8 times L3 times two. To optimize the water suppression, set the receiver gain to one and type EDC pull to open the pulse program file. On line 91, remove the semi-colon preceding goto 999, and save the file.
Using the GS command, adjust the SPDB0 parameters to minimize the intensity of the FID signal. When the signal intensity has been modified, reintroduce a semi-colon at line 91 of the pulse program file and save the file. To optimize SPDB11, set the receiver gain to one and the type EDC pull to open the pulse program file.
On line 168, remove the semi-colon preceding goto 999, and save the file. Using the GS command, adjust the SPDB11 parameters to minimize the intensity of the FID signal. When the signal intensity has been modified, reintroduce the semi-colon at line 168 and save the file.
To optimize SPDB2, set the receiver gain to one and enter EDC pull to open the pulse program file. On line 179, remove the semi-colon preceding goto 999, and save the file. Using the GS command, adjust the SPDB2 parameters to minimize the intensity of the FID signal.
When the signal intensity has been modified, re-introduce the semi-colon at line 179 of the pulse program file and save the file. To optimize PLDB2, set the receiver gain to one and enter EDC pull to open the pulse program file. On line 184, remove the semi-colon preceding goto 999, and save the file.
Using the GS command, adjust the PLDB2 parameters to minimize the intensity of the FID signal. When the signal intensity has been modified, reintroduce the semi-colon at line 184 of the pulse program file and save the file. Run the RGA command to optimize the receiver gain.
Then run the ZG command to start the experiment. In this figure, the results of the relaxation dispersion profiles acquired for each peak in the hydrogen nitrogen 15 Trosy spectrum can be observed. From the acquired relaxation dispersion profiles, it's possible to estimate the exchange contribution to the nitrogen 15 transverse relaxation of each backbone AMI group.
By plotting the transverse relaxation on the 3D structure of the protein under investigation it's possible to identify the structural regions undergoing confirmational exchange on the microsecond millisecond timescale. Modeling of the relaxation dispersion curves using the Carver-Richards equations returns the thermodynamic and kinetic parameters on the exchange process, such as the fractional populations of the states in equilibrium and the rate of exchange among these states. The temperature dependence of these thermodynamic and kinetic parameters can then be modeled using the van't Hoff and I-ring equations, respectively, to obtain detailed information on the energetics of the confirmational exchange.
It is crucial to carefully optimize all of the acquisition parameters, in particular P7.It is also important to produce highly pure and homogenous samples to avoid spurious dispersions. The kinetic, thermodynamic, and chemical chip parameters obtained using this protocol can be used to derive energetic and structural information about the species undergoing the confirmational exchange. Characterization of the protein confirmational dynamics obtained by CPMG methods provides crucial information towards understanding signaling and enzymatic activity, as well as new perspectives for drug design.
