We investigate the structural dynamics of proteins by NMR spectroscopy to understand the molecular function better. NMR stands for nuclear magnetic resonance spectroscopy. It uses effect that the nuclear spins of hydrogen atoms can transition between two energetically different states in an electromagnetic field.

We can use NMR spectroscopy to study the structural dynamics of intrinsically disordered proteins. These proteins are highly flexible, constantly changing shape, and frequently only fold into a stable structure when they interact with other molecules. We use NMR relaxation experiments to study how the protein backbone moves over very short time scales, ranging from picoseconds to nanoseconds, while focusing on each specific residue along the protein chain.

This protocol will guide you step-by-step through setting up NMR relaxation experiments on an NMR spectrometer. Aimed at scientists with basic NMR knowledge, it ensures a fast, accurate setup and introduces key concepts in protein backbone dynamics in an accessible way. This protocol makes NMR relaxation pulse sequences easily accessible to scientists studying protein dynamics.

NMR relaxation experiments provide residue-specific dynamic insights. Using this experimental approach, you can better understand backbone dynamics in proteins, the impact of protein-protein interaction on structured dynamics, and structural changes in response to environmental changes. To begin, download the NMR pulse programs to the directory on the computer operating the NMR spectrometer and adjust the TopSpin version.

Download the NMR-shaped pulses to the directory and verify that the TopSpin version is updated accordingly. After determining the spectral widths and appropriate acquisition times, type EDC in the TopSpin software to a new directory to copy the HSQC experiment. To open the pulse sequence parameters, type ASED.

Click on the three dots next to the pool prog parameter to change the pulse program to nitrogen 15R1 row experiment. Double-click on the pulse program, click on set PULPROG to the dataset, and confirm with OK.Type ASED to open the pulse sequence parameters. Enter all missing gradient strengths and gradient lengths as given in the pulse sequence.

Next, enter for the loop counter L3 half of the nitrogen 15TD entry in the acquisition parameter window. Enter for the loop counter L6 the number of recorded relaxation data points for fitting the relaxation curve. Set the adiabatic tanh/tan half NL pulse as the shape of the nitrogen 15-SP8 pulse.

Then set the adiabatic tanh/tan half second NL pulse as the shape of the nitrogen 15 SP9 pulse. Ensure the pulse lengths are sufficiently long for adiabaticity with P8 set to 3000 microseconds. Then set the interscan recovery delay D1 to at least two seconds or longer.

Set the dummy scans to at least 64. As a starting point, set the number of scans to four and use multiples of four if the signal to noise ratio is too low. Set the O1 to the calibrated carrier frequency, O2P to 176 parts per million, and copy the O3P from the proton nitrogen 15 HSQC experiment.

Now set the pulse length P7 to the 90 degree pulse length calibrated earlier. Then copy the pulse power level of the 90 degree pulse to PLW3 and PLW7. Afterward, set the pulse lengths P1 and P19 to the 90 degree proton pulse length.

Set the number of increments in the indirect dimension TD equals L6 by two by L6.Next, set the shape pulse SP5 to an I-BURP2 shape and the pulse length P15 to 2000 microseconds. Then open the shape tool display by clicking the E next to the I-BURP2 shaped pulse in the TopSpin pulse sequence parameter window. To simulate the shape pulse, click the start NMR simulation button.

Check the shaped pulse length and the rotation angle in the simulation window and click on start NMR SIM to proceed. Check the excitation range in the simulation and select the appropriate I-BURP2 pulse length to cover the proton spectral dispersion while avoiding excitation of the water. Set P15 to the shaped pulse length from the simulation window with the best I-BURP2 pulse.

Now set to adjust the carrier frequency of the I-BURP2 pulse, shifting the excitation range left or right to avoid water magnetization disturbance. Then open the Bruker shape tool and click start NMR simulation to determine the appropriate power level of the shaped pulse. Set the I-BURP2 pulse length to the shaped pulse length, and note the soft rectangular 90 degree proton pulse length shown in the simulation window.

Type CALPOWLEV to calculate the power level difference in decibels between the hard 90 degree proton pulse and the soft rectangular 90 degree proton pulse. Copy the power level of the hard 90 degree proton pulse to SPW5 and adjust by adding the memorized difference in decibels. To determine the power level of the spin-lock, calculate the corresponding 90 degree nitrogen 15 pulse length.

Use CALPOWLEV to calculate the power difference in decibels between the spin-lock power and the hard 90 degree nitrogen 15 pulse. Copy the power level of the hard 90 degree nitrogen 15 pulse to PLW7 and adjust the spin-lock power level PLW8 by adding the calculated power difference. Copy the power level of the spin-lock PLW8 to the power levels SPW8 and SPW9.

To determine the power level of the nitrogen 15 decoupling, use CALPOWLEV to calculate the power difference between the power of the 90 degree nitrogen 15 decoupling pulse and the 90 degree nitrogen 15 hard pulse. Copy the power level of the 90 degree nitrogen 15 hard pulse PLW7 to the decoupling power level PLW31 and adjust by adding the calculated power difference in decibels. For temperature compensation, include line define TEMP_ in the pulse program.

Set P18 to the maximum duration of the spin-lock used in the nitrogen 15 R1 row experiment. In the case of nitrogen 15 carbon 13 labeled samples, include the line define LABEL_CN in the pulse program. Set P4 to the calculated pulse length.

Use CALPALLEV to calculate the power difference in decibels between P4 and the carbon 13 hard pulse. Copy the power level of the carbon 13 hard pulse to PLW4 and add the calculated power difference in decibels. Copy PLW4 to PLW2.

To determine the appropriate relaxation delays for sampling, run the first eight free induction decays or FIDs and process them. Choose VP list entries where the peak intensity of the longest delay experiment decreases to at least one over E, but not lower than 25%compared to the shortest delay experiment. Type RGA in the command line to determine the receiver gain.

Start a test run of the nitrogen 15R1 row experiment by typing ZG in the command line. Check that the water signal is suppressed for all delays. Also, check increment nine, the second increment of the quadrature detection scheme.