Nuclear Magnetic Resonance

       Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy, is a research technique that exploits the magnetic properties of certain atomic nuclei. This type of spectroscopy determines the physical and chemical properties of atoms or the molecules in which they are contained. It relies on the phenomenon of nuclear magnetic resonance and can provide detailed information about the structure, dynamics, reaction state, and chemical environment of molecules. The intramolecular magnetic field around an atom in a molecule changes the resonance frequency, thus giving access to details of the electronic structure of a molecule and its individual functional groups. 

      This protocol describe procedures for the preparation of in-cell NMR samples, as well as for the setup of NMR experiments and their application to in-cell studies, using human α-synuclein overexpressed in Escherichia coli as an example. The expressed protein is labelled with 13C and 15N stable isotopes to enable the direct recording of 13C-detected NMR experiments. The entire procedure covers 24 h, including cell transformation, cell growth overnight, setup of the spectrometer and NMR experiment recording.


     A. Reagents and Equipment

         Reagents

  •  LB broth base (powder)
  •  Isopropyl β-D-1-thiogalactopyranoside (IPTG)
  •  Carbenicillin disodium salt
  • Agar for microbiology
  • Sodium Chloride (NaCl)
  • Calcium chloride hydrate (CaCl2.H2O)
  • Disodium hydrogen phosphate dihydrate (Na2HPO4.2H2O)
  • Monopotassium phosphate (KH2PO4)
  • Deuterium oxide (D2O)
  • Magnesium Sulfate (MgSO4)
  • Biotin
  • Thiamin-HCl
  • BL21(DE3)gold E. coli
  •  LB broth (20 mg/ml LB broth base)
  • M9-labelled medium composition: 47 mM Na2HPO4·2H2O, 22 mM KH2PO4, 8 mM NaCl, 2 mM MgSO4, 0.1 mM CaCl2, 1 μg/ml biotin, 1 μg/ml thiamin-HCl, 4 mg/ml 13C6 glucose, 1 mg/ml (15NH4)2SO4 and 50 ng/ml carbenicillin
  • Expression plasmid: pT7-7 asyn WT
  • Standard sample A, pulse calibration: 0.1 M 15N-labelled urea and 0.1 M 13C-labelled methanol in 2H-labelled DMSO
  • Standard sample B, 13C sensitivity: 40% (v/v) dioxane in 2H-labelled benzene
  • Standard sample C, setup of protein experiments: 1.0 mM 13C, 15N-labelled ubiquitin in 10 mM phosphate buffer, pH 6.5, 0.02% NaN3, 10% (v/v) D2O

         Equipment

  • All the equipment necessary for expression, characterization and purification of recombinant proteins
  • NMR spectrometer fully equipped for triple-resonance experiments of biological macromolecules
  • NMR data-processing software (i.e., TOPSPIN for Bruker machines, VNMR for Agilent spectrometers and so on)
  • Visualization and analysis software for NMR spectra (e.g., TOPSPIN, CARA, Sparky, NMRView, CCPN and so on)
  • Cryogenically cooled probe head with 13C-direct detection capabilities (either inverse-detection triple-resonance probe head with 13C cooled preamplifier or 13C direct detection triple resonance probe head) with z-field gradients. Sensitivity should allow for recording 2D HN and 2D CON correlation experiments on a purified α-synuclein sample of ~0.1 mM concentration on the order of tens of minutes
  • High-throughput 5-mm NMR tubes
  • Sonicator Sonix Vibracell VCX-750 equipped with microtip

     B. Reagent and Equipment Set-up

         Reagent Set-up

  • 5X M9 Medium: Dissolve 42 g of Na2HPO4·2H2O, 15 g of KH2PO4 and 2.5 g of NaCl in 100 ml of H2O. Autoclave the solution and store it at room temperature (~25 °C). Use the solution within 3 months.
  • 1X M9 Medium: Dilute 5× M9 in a 5-fold dilution with ddH2O.
  • 1M MgSO4: Dissolve 12.4 g of MgSO4 in 100 ml of H2O. Filter-sterilise the solution and store it at room temperature.  Use the solution within 3 months.
  • 0.1M CaCl2: Dissolve 14.7 g of CaCl2·H2O in 100 ml of H2O. Autoclave the solution and store it at ROOM TEMPERATURE. Use the solution within 3 months.
  • 1M IPTG: Dissolve 238.3 mg of IPTG in 1 ml of H2O. Filter-sterilise the solution and store it at −20 °C. Use the solution within 6 months.
  • 1000X Biotin: Dissolve 15 mg of biotin in 15 ml of H2O. Filter-sterilise the solution and store it at −20 °C. Use the solution within 3 months.
  • 1000X Thiamine-HCl: Dissolve 15 mg of thiamine in 15 ml of H2O. Filter-sterilise the solution and store it at −20 °C. Use the solution within 3 months.
  • 1000X Carbenicillin: Dissolve 0.5 g of carbenicillin in 10 ml of H2O. Filter-sterilise the solution and store it at −20 °C. Use the solution within 3 months.
  • 13C6-Glucose/15N-ammonium sulfate mix: Dissolve 1 g of 13C6-glucose and 250 mg of 15N-ammonium sulfate in 5 ml of water (warm if necessary). Filter-sterilise the solution. Use it immediately or keep it at 4 °C for no longer than 2-3 days.
  • 50 ml labelled medium: Autoclave a 250-ml flask containing 38.7 ml of H2O. Add sterile stock solutions to the flask as follows: 10 ml of 5× M9, 100 μl of 1M MgSO4, 50 μl of CaCl2, 50 μl of biotin, 50 μl of thiamine, 1 ml of 13C6-glucose/15N-ammonium sulfate mix and 50 μl of carbenicillin. Use the medium immediately or keep it at 4 °C for no longer than 2-3 days.

         Equipment Set-up

  • NMR spectrometer: The following setup is designed for measurements at a 16.4 T Bruker AVANCE 700 spectrometer equipped with a 13C direct-detection cryogenically cooled probe head. For other magnetic field strengths, pulses should be scaled accordingly.
  • Determine the sensitivity of 13C of the NMR instrument. A standard sample is generally provided by the spectrometer manufacturer to test the 13C sensitivity (such as standard sample A). In addition, it is very useful to test the performance of the instrument on a 13C, 15N protein sample of known concentration (such as standard sample C).
  • Determine the pulse lengths of hard 90° pulses for 1H, 13C and 15N on the spectrometer standard sample (such as standard sample B). For 1H and 13C, do this in both direct and indirect acquisition mode setups.
  • Check the performance of the amplifiers by measuring a couple of additional 90° pulses of longer duration with respect to the hard pulse to verify that the experimental pulse lengths correspond to those calculated assuming that the amplifiers are linear. This is important to avoid errors in setting the power levels for the shaped pulses necessary for the experiments below, which are calculated under this assumption.

     C. Assay procedure

          Sample preparation: growth of cells

  1. Freshly transform BL21(DE3)gold E. coli cells with the expression plasmid pT7-7 asyn WT.
  2. Inoculate a single colony in 70 ml of LB broth supplemented with 100 μg/ml carbenicillin in a 250-ml flask.
  3. Grow the cells overnight in a shaker at 27 °C at 380 rpm (20 mm oscillation amplitude). The next day, the OD600 value should be between 4 and 6.
  4. Centrifuge 35 ml of the culture (2 000 g) gently at 4 °C for 20 min.
  5. Re-suspend the cells in 50 ml of 13C, 15N-labelled M9 medium in a 250-ml flask.
  6. Let the flask shake at 37 °C and 380 rpm for ~10–15 min.
  7. Induce the culture by adding IPTG to a final concentration of 1 mM.
  8. Grow the cells for 4 h after induction. OD600 value typically ranges between 5 and 6. While the cells are growing for 4 h, you can prepare the NMR data sets (Steps 12–16).

          Sample preparation for in-cell NMR

  1. Centrifuge 25 ml of the culture (2 000 g) at 4 °C for 20 min.
  2. Re-suspend the cells using 300 μl of their culture medium and 60 μl of D2O by pipetting gently to dissolve the cell pellet completely. The final volume should be adjusted at 600 μl; the amount of cells should be ~50% of the entire sample volume.
  3. Transfer the sample into the NMR tube and measure it immediately. Rapidity of execution is important to limit sample degradation.

          Preparation of the NMR data sets on a standard protein sample

  1. Prepare a data set by recording a 1D 1H NMR spectrum over a spectral width of 20 ppm, centred on the water signal (~4.7 ppm). An NMR pulse sequence that has very good performance in terms of water suppression is the excitation sculpting sequence.
  2. Prepare a data set by recording a 1D 13C NMR spectrum. Record the spectrum over a spectral width of 250 ppm, centred at 100 ppm In general, 1H is irradiated during the recycle delay to take advantage of the 1H-13C NOE effect, which might be significant in IDPs; 1H and 15N are decoupled during acquisition of the 13C free induction decay (FID).
  3. Prepare a data set by recording a 2D 1H-15N correlation experiment (2D HN SOFAST)40 (Steps 3–9). Set the spectral width to 15 ppm for 1H and 34 ppm for 15N, 2,048 points and 256 increments (acquisition times of 96 ms and 53 ms), respectively.
  4. Set the 1H carrier at 4.7 ppm and the 15N carrier at 120 ppm
  5. Band-selective 1H-shaped pulses (90° and 180° pulses) should be centred at ~9 ppm or even higher to avoid as much perturbation of the water resonance as possible.
  6. Calculate the power level for the 1H 90° PC9-shaped pulses; typical duration at 16.4 T is 2,200 μs.
  7. Calculate the power level for the 1H 180° Reburp-shaped pulses; typical duration at 16.4 T is 1,600 μs.
  8. Set the recycle delay (acquisition time plus recovery delay) to ~350 ms as a good compromise between fast recycling and safe operation of the instrument. This can be further reduced if necessary, by checking the instrument specifications carefully.
  9. Acquire the 2D 1H-15N correlation experiment and process it with standard parameters to verify spectral quality. Typically raw data are multiplied by an apodization function, zero-filled and Fourier-transformed in both dimensions. Proper zero- and first-order phase correction and baseline correction in both dimensions are applied after the Fourier transformation.
  10. Prepare a data set by recording a 2D CON correlation experiment (Steps 10–16). Set the spectral width to 30 ppm for C′ and 40 ppm for 15N, 1,024 points and 376 increments (acquisition times of 96 ms and 33 ms), respectively. All the 13C NMR experiments use the IPAP approach to suppress the Cα-C′ coupling in the direct acquisition dimension where the in-phase (IP) and antiphase (AP) components are acquired and stored separately, doubling the number of FIDs recorded in the indirect dimensions. Set the 13C carrier at 173 ppm and the 15N carrier at 122.5 ppm to also include signals of prolines. For 13C adiabatic pulses, set the carrier at 112 ppm Cα pulses are given at 52 ppm, whereas Cα/Cβ are given at 39 ppm Proton-selective pulses are applied at 9 ppm.
  11. Calculate the power level of the Q5 (or time-reversed Q5)- and Q3-shaped pulses; typical duration's at 16.4 T are 300 μs (Q5) and 220 μs (Q3) for C′ or for Cα/Cβ and 860 μs (Q3) for Cα.
  12. Calibrate the adiabatic Chirp π pulse used to invert both C′ and Cα/Cβ with typical duration of 500 μs, 25% smoothing, 80 kHz sweep and 11.3 kHz strength centred at 112 ppm.
  13. Select the variant of the experiment to be acquired (Box 1). For the Hα-flip/HN-flip versions, only hard 1H pulses are necessary, whereas for the HN-BEST variant the same pulses described above for the 2D HN SOFAST experiment should be used, although in the second channel of the spectrometer rather than in the first.
  14. Set the recycle delay to ~700 ms for the Hα-flip variant and to ~250 ms for the HN-flip/HN-BEST variants.
  15. Acquire the 2D CON correlation experiment and pre-process the data to combine the acquired FIDs properly to achieve homonuclear virtual decoupling.
  16. Process the data with standard parameters to verify spectral quality. Typically, raw data are multiplied by an apodization function, zero filled and Fourier transformed in both dimensions. Proper zero- and first-order phase correction and baseline correction in both dimensions are applied after the Fourier transformation. In all cases, decoupling of 1H and 15N (when appropriate) is achieved with Waltz-16 (1.7 kHz) and Garp-4 (1.0 kHz) sequences, respectively. When cycling fast (<300 ms), the power for 15N decoupling should be reduced using adiabatic decoupling as in the 2D HN SOFAST experiment (p5m4sp180 sequence using a Chirp pulse of 2 ms, 25% smoothing, 4 kHz sweep, 0.7 kHz strength). To allow for long 13C acquisition time and even faster pulsing, 15N virtual decoupling can be implemented.
  17. Prepare a data set by recording a 2D CACO correlation experiment (Steps 17–21). Set the spectral width to 30 ppm for C′ and 50 ppm for Cα, 1,024 points and 800 increments (acquisition times of 96 ms and 22 ms), respectively. All the 13C NMR experiments use the IPAP43–45 approach to suppress the Cα-C′ coupling in the direct acquisition dimension where the IP and AP components are acquired and stored separately, doubling the number of FIDs recorded in the indirect dimensions. For 2D CBCACO experiments, set the spectral width in the indirect dimension to 76 ppm with the carrier at 39 ppm; all the other parameters are the same.
  18. Set the 13C carrier at 173 ppm for C′ and at 48 for Cα in the indirect dimension, the 15N carrier at 122.5 ppm and 1H at 4.7 ppm All the shaped and decoupling pulses are as for the CON experiments.
  19. Set the recycle delay to ~700 ms.
  20. Acquire the 2D CACO correlation experiment and pre-process the data to combine the acquired FIDs properly to achieve homonuclear virtual decoupling, as described for the 2D CON experiments.
  21. Process the data with standard parameters to verify spectral quality. Typically, raw data are multiplied by an apodization function, zero filled and Fourier transformed in both dimensions. Proper zero- and first-order phase correction and baseline correction in both dimensions are applied after the Fourier transformation.

          NMR Measurements

  1. Prepare the NMR spectrometer for the measurements, as described in the previous section.
  2. Set the experimental temperature, shim the magnet with an NMR sample containing 600 μl of cell growth culture medium and tune the probe head.
  3. Insert the in-cell NMR sample into the magnet, check the tuning of the probe head and quickly verify 1H and 13C pulse lengths.
  4. Measure and process a 1D 1H and 13C NMR spectra to check sample conditions and its reproducibility.
  5. On each sample, perform one of the following 2D experiments: the 2D 1H-15N correlation experiment (2D HN SOFAST), the 2D 13C′-15N correlation experiment , or the 2D 13C′-13Cα correlation experiment (2D CACO) or 2D 13C′-13Cα/β (2D CBCACO).
  6. Process the spectra and analyse them, for example, by comparing them with analogous ones acquired on cell lysates and on purified protein samples.

          Quality control

  1. To assess the absence of protein leakage, remove the measured sample from the NMR tube and centrifuge it at 1,500 g for 10 min at 25 °C. Collect the supernatant and centrifuge it further at 10,000 g for an additional 10 min at 25 °C. Adjust the volume to 600 μl by adding fresh culture medium containing 5% (v/v) D2O, transfer it in an NMR tube and measure a 2D HN SOFAST experiment. The resulting spectrum should be almost empty.
  2. To check cell viability, before and after each NMR experiment prepare a sample by taking 5 μl of the cell suspension from the NMR tube and diluting it in a 1-ml solution of LB. Spread 100 μl of this diluted sample per 60-mm-diameter LB agar plate containing 50 ng/ml carbenicillin. The plates should present a comparable number of cells.

          Cell extract sample preparation

  1. Remove the sample from the NMR tube and sonicate the cell suspension in a 1.5-ml microtube using a 750-W sonicator equipped with a 3-mm-diameter microtip (the amplitude of the vibration is set to 40%).
  2. Perform 5–7 cycles of 10 s of pulsed sonication; each cycle should be followed by 1 min of cooling time in an ice/water bath.
  3. Remove cell debris by two steps of centrifugation at 16,000 g for 15 min each at 4 °C.
  4. Collect the supernatant, transfer it into an NMR tube and measure it immediately.