Feb 20, 2025

Public workspaceBio - 2H/13C/13C Respiration CP 3D

This protocol is a draft, published without a DOI.
  • Songlin Wang1
  • 1NMRFAM, University of Wisconsin-Madison
NMRFAM Facility
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Protocol Citation: Songlin Wang 2025. Bio - 2H/13C/13C Respiration CP 3D . protocols.io https://protocols.io/view/bio-2h-13c-13c-respiration-cp-3d-dd7h29j6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: May 23, 2024
Last Modified: February 20, 2025
Protocol Integer ID: 100297
Keywords: 2H/13C/13C Respiration CP 3D , Protein dynamic
Funders Acknowledgements:
National Science Foundation
Grant ID: 1946970
Abstract
Purpose 
2H/13C/13C 3D correlation experiment using Respiration CP for 2H/13C transfer and RFDR for 13C/13C transfer. This experiment is used to mapping the site-specific dynamics for proteins.  

Scope 
Site-specific dynamic measurement of protein.
Materials
Definitions:
TermDefinition
CPCross Polarization
MASMagic Angle Spinning
RFDRRadio-Frequency Driven Recoupling
Instrument: The 2H/13C/13C Respiration CP dataset in this SOP was acquired using a 900MHz NMR spectrometer with a Bruker Avance III console. The probe is a Phoenix 1.6 mm HCN probe. For the best result, this experiment should be executed after properly adjusting the shimming and magic angle.

Sample:
  1. Type: Protein
  2. Labeling: perdeuterated U-13C labeling samples.
  3. Minimum amount: ~50 nmol for 1.6 mm rotor.

Schematic pulse sequence of 2H/13C/13C Respiration-CP 3D:




Example Parameter set for a Phoenix 1.6mm probe using 20 kHz MAS on 900 MHz Bruker spectrometer (pulse sequence: RespirationCP3d):

Pulse Parameters:
1H 90o: (p14, plw14, cnst14) = (2.5 µs, 101 W, 100 kHz)
1C 90o:(p13, plw13, cnst13) = (5 µs, 90 W, 50 kHz)
2N 90o: (p15, plw15, cnst15) = (8.8 µs, 100 W, 28.4 kHz)

Respiration CP: [p35, cnst37, cnst 51, cnst 31, L1]
=[2 µs, 40 kHz, 40 kHz, 55 kHz, 16]

RFDR: [p36, L14]
=[15 µs, 48]

1H-decoupling for acquisition- (CPDPRG4, p24, cnst24) = (SPINAL64_p24, 184 µs, 2.5 kHz)

1H-decoupling for RFDR- (CPDPRG7, cnst27) = (cw_p27, 0 kHz)

Acquisition Parameters:
MAS rate (wr) in Hz: cnst20 = 20000
Rotor period (wr): d20 = (1/cnst20) = 50 µs
Number of scans: ns = 192
Recycle delay: d1 = 0.1 sec

13C Direct dimension: t3 or F3:
Carrier frequency: O1p = 40 ppm
Acquisition time: aq(F3) = 20.5 ms
Spectral width: sw(F3) = 441.986 ppm

13C indirect dimension: t2 or F2:
FnMode: States-TPPI
Carrier frequency: O1p = 40 ppm
Maximum evolution time: aq (F2) = 2.4 ms
Spectral width: sw (F2) = 88.397 ppm
Dwell time: IN_F (F2) = 50 µs
Total t2 points: TD (F2) = 96

2H indirect dimension: t1 or F1:
FnMode: States-TPPI
Carrier frequency: O3p = 0 ppm
Maximum evolution time: aq(F1) = 0.23 ms
Spectral width: sw(F1) = 200 kHz
Dwell time: IN_F (F1) = 5 µs Total t1 points: TD (F1) = 92

Safety warnings
Operator: User should be knowledgeable to operate MAS probes and use Topspin
Before start
MAS rate: 10-20 kHz for 1.6 mm rotor

Temperature: Note that the temperature increases by 5 -10 °C due to frictional heating from spinning at 10-20 kHz for 1.6 mm rotors. Adjust the variable temperature (VT) control set point temperature accordingly to achieve desired sample temperature. For microcrystals and fibrils, typical sample temperature range is 0 to 10 °C, requiring a VT set point temperature of -10 to 0 °C. For membrane proteins and/or liposomes, the desired sample temperature depends on the phase transition of interest and can vary over a wide range depending on the sample.

Below optimizations are required before setting up the hNH2d experiment. Example SOP for each optimization is attached at "References" section.
  1. Calibrate 1H, 2H, and 13C solid pulses (p14, plw14, p13, plw13, p15, and plw15)
  2. Optimize 2H-13C Respiration CP (p37, cnst37, p35, cnst31, cnst51, L1)
  3. Optimize low-power 1H decoupling (cpdprg4, p24, and cnst24)
  4. Optimize 13C/13C RFDR mixing (p36, L14, cpdprg7, p27, and cnst27)

Setup time: ~0.5 hours, presuming initial calibrations have already been completed.
Procedure
Procedure
Load “RespirationCP3d-NAN” pulse program and parameter set “RespirationCP3d-NAN_par”.
Use Topspin command “edc” to open a new experiment.
Input “RespirationCP3d-NAN” as PULPROG
Type “rpar”, then load file “RespirationCP3d-NAN_par”.
Note that the parameters will be off if using different spectrometers/probes. Please consult to your facility manager/staff to get proper starting parameter set.
Set the optimized pulse parameter values. The optimizations that need to be done and parameters need to be updated are listed in the "Before start" section under "Guidelines & Warnings".
Set the 2H carrier frequency (O3p) at ~0ppm, 1H carrier frequency (O2p) at ~4ppm, and 13C carrier frequency (O1p) at 40 ppm. For example, type “O1p” and enter, then input 40 to the opened window.
Set acquisition parameters.
Direct dimension (t3 or F3)
  1. Acquisition time (AQ, s): depending on 13C T2, default is ~20ms.
  2. Spectral width (SW, ppm): ~400 ppm.
  3. Carrier frequency (O3p, ppm): 40 ppm.
Indirect 2H dimension (t1 or F1)
  1. Spectral width (SW, Hz): the 2H dimension has spin sidebands at n*ωR (Hz, n≥1). So the spectral width should be able to cover most of the sidebands. The default n is 5, so the default spectral width is ~5ωR in Hz.
  2. Increment of delay (IN_F, s): coupled with spectral with. Increment of delay=1/SW.
  3. Carrier frequency (O1p, ppm): 0 ppm.
  4. Hypercomplex scheme (FnMODE): States-TPPI
  5. Maximum evolution time (AQ, s): The evolution time equals to (Increment of delay) * (TD). Once increment of delay is fixed as described above, change number of TD to adjust the total evolution time. The default value is ~0.25ms.
Indirect 13C dimension (t2 or F2)
  1. Spectral width (SW. ppm): For perdeuterated proteins, the region of interest is the aliphatic region, so the spectral width should cover from ~0 ppm to ~80ppm. The default spectral width is ~90 ppm.
  2. Increment of delay (IN_F, s): coupled with spectral with and must be rotor synchronized. For example, 50 µs for 20 kHz MAS spinning rate.
  3. Carrier frequency (O2p, ppm): 40 ppm for proteins.
  4. Hypercomplex scheme (FnMODE): States-TPPI
  5. Maximum evolution time (AQ, s): depending on 13C T2, the default is ~10 ms. The evolution time equals to (Increment of delay)*(TD). Once increment of delay is fixed as described above, change number of TD to adjust the total evolution time.
Set the recycle delay
Use recycle delay of 1.3*T1 (2H) for maximum sensitivity per unit time. Note that the 2H T1 is very short. The default value is 0.1s.
Due to the short recycle delay, only low-power 1H decoupling is allowed, which should be sufficient for perdeuterated proteins.
Adjust measurement time as required by increasing number of scans in multiples of 4.
Validation
Start the experiment and monitor the first ~20-30 rows
Process first dimension FT to check for adequate signal correctly arraying indirect dimension.
Protocol references