Dec 20, 2024

Public workspacetrNOE.nan

DOI
dx.doi.org/10.17504/protocols.io.8epv5rr9ng1b/v1
  • NAN KB1,
  • Alex Eletsky2,
  • John Glushka2
  • 1Network for Advanced NMR ( NAN);
  • 2University of Georgia
NAN-KB UGA
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.8epv5rr9ng1b/v1
Protocol Citation: NAN KB, Alex Eletsky, John Glushka 2024. trNOE.nan. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rr9ng1b/v1
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: Working
We use this protocol and it's working
Created: May 30, 2024
Last Modified: December 20, 2024
Protocol Integer ID: 100905
Keywords: protein nmr, assignment, backbone, amides, 15N, hsqc
Funders Acknowledgements:
NSF
Grant ID: 1946970
Disclaimer
This protocol is part of the knowledge base content of NAN: The Network for Advanced NMR ( https://usnan.nmrhub.org/ )
Specific filenames, paths, and parameter values apply to spectrometers in the NMR facility of the Complex Carbohydrate Research Center (CCRC) at the University of Georgia.
Abstract
This protocol describes running a 2D 1H-1H trNOE (NOESY) pulse sequence with ZQ coherence suppression and presaturation for water suppression. This produces a 2D phase-sensitive dataset with 1H-1H correlations due to NOEs.

This pulse sequence used is a standard 2D NOESY experiment but is applied to a mixture of a low MW ligand and a macromolecule to which it weakly binds. Since the sign of NOEs are dependent on the tumbling (correlation) time of the molecule, a low MW compound (fast tumbling) has generally weak 'positive' NOE signals whereas a high MW macromolecule has stronger 'negative' signals.

Generally two datasets are collected. The NOESY spectrum of the ligand alone is acquired with a suitably long NOE mixing time in order to see significant crosspeaks. Then a NOESY spectrum is acquired of the mixture of ligand and macromolecule in a molar ratio of 10-100: 1, generally with a shorter NOE mixing time. Under certain binding kinetics, the NOEs of the ligand while bound to the macromolecule will dominate and the crosspeaks will be of opposite sign from that of the ligand alone.
In some cases, additional control spectra of the macromolecule alone and of the ligand alone with the shorter mixing time are collected to resolve ambiguities.

Main applications
  • measuring 1H-1H distances in small molecule ligands when bound to large molecules
  • structure determination of bound conformations of small molecule ligands

Required isotope labeling: natural abundance.

Optimal MW: ligand < 2000 Da; macromolecule > 5 kDa.
Field strength and sample temperature are often chosen to achieve zero NOE transfer for free ligand (ωτc = 1.1), though this may not always be possible.

Pulse sequence used is noesygpphzspr.
Attachments
Icon for biotop.pdf
biotop.pdf
2.5MB
Guidelines
Generally two datasets are collected. The NOESY spectrum of the ligand alone is acquired with a suitably long NOE mixing time in order to see significant crosspeaks. Then a NOESY spectrum is acquired of the mixture of ligand and macromolecule in a molar ratio of 10-100: 1, generally with a much shorter NOE mixing time. Under certain binding kinetics, the NOEs of the ligand while bound to the macromolecule will dominate and the crosspeaks will be of opposite sign from that of the ligand alone.
In some cases, additional control NOESY spectra of the macromolecule alone and of the ligand alone with the shorter mixing time are collected to resolve ambiguities.

Before start
A sample must be inserted in the magnet either locally by the user after training or by facility staff if running remotely.
This protocol requires a sample is locked, tuned/matched on the 1H channel, and shimmed. At a minimum, 1H 90° pulse width and offset O1 should be calibrated and a 1D proton spectrum with water suppression has been collected according to the protocol PRESAT_bio.nan.


Create trNOE experiment
Create trNOE experiment
Start with existing the Dataset containing 1D PRESAT data in EXPNO 1 acquired with protocol PRESAT_bio.nan
Click on Acquire -> 'Create Dataset' button to open dataset entry box or type edc command.

The EXPNO is automatically incremented by +1 by default.
Directory should be the same as preliminary 1D.

The Title text box will copied from the previous experiment. Edit appropriately.
Load the starting parameter set: Check 'Read parameterset' box, and click Select.

For standard NAN parameter sets, change the Source directory at upper right corner of the window:
Source = /opt/NAN_SB/par
Click 'Select' to bring up list of parameter sets.
Select trNOE_xxx.par, where xxx=900,800 or 600*.




Click OK at bottom of window to create the new EXPNO directory.
It will be the active experiment in the acquisition window and should now be listed on your data browser.
Load pulse calibrations
Load pulse calibrations
Use getprosol to load correct pulse widths and power levels.

Loading pulse widths and power levels with getprosol:

Examine the PRESAT dataset acquisition parameters in Exp 1 (type 'ased'), previously collected according to the protocol PRESAT_bio.nan (see step 1). The P1 pulse width value should be calibrated - note its value (inspect table or type 'p1').
If necessary, it can be recalibrated by typing pulsecal.

Then execute the getprosol command:
getprosol 1H [ calibrated P1 value] [power level attenuation for P1 (PLdB1)]
e.g. getprosol 1H 9.9 -13.14.
Where for example, the calibrated P1=9.9 at power level -13.14 dB attenuation
Inspect and adjust parameters
Inspect and adjust parameters
Other than the 1H pulsewidth, the default parameters from trNOE_xxx.par will provide suitable starting values. Typically the only parameters to change will be NS = number of scans in order to increase the signal to noise.
Select the 'Acqpars' tab to display acquisition parameters. Two display modes can be selected, the full display mode (click on the 'A' icon or type eda), or pulse program-specific mode (click on the 'pulse' icon, or type ased). The former gives you access to all parameters and provides an overview of all spectral dimensions at once, while the latter is useful because it only displays acquisition parameters used in the pulse sequence and can be parsed sequentially as a checklist.

First examine the specific dimension parameters in Acqpars 'eda' mode (click 'A' icon):



Parameters to check:
  • SW[ppm]: 1H(F2 and F1) ~9-15 ppm
  • O1P: 1H H2O offset, typically ~4.7.
  • 2 TD: Number of 1H time domain real points (2048-8192, depending on resolution desired)
  • 1 TD: 2 X number of 1H time domain real points (256-1024, depending on resolution desired and time available)
  • NS: multiple of 4-16; increase for for higher signal to noise ( S/N increases as square root of NS )
  • DS: 8-32 'dummy' scans that are not recorded; allows system to reach steady state equilibration.
  • DIGMOD - 'baseopt' (zero 1st order phase correction)


Then examine the parameters in the pulse program-specific 'ased' mode (click on the 'pulse' icon).





Most of the default parameters should be appropriate, however it's useful to compare values in the fields against suggestions in the pulseprogram comments. In general, only a few may need to be changed.

  • D1: recycle delay (~1-2 s)
  • D8: NOE mixing time
  • NS: multiple of 4-16; increase for for higher signal to noise ( S/N increases as square root of NS )
  • P1 - calibrated 1H 90º high power pulse (see step 1)

Acquire and Process Data
Acquire and Process Data
Type 'rga' or click on 'Gain' in Topspin Acquire menu to execute automatic gain adjustment.
Type 'zg' or click on 'Run' in Topspin Acquire menu to begin acquisition.
You can always check the first FID by typing 'efp' to execute an exponential multiplied Fourier transform. It will ask for a FID #, choose the default #1. You can evaluate the 1D spectrum for amide proton signal to noise and water suppression.

To take a look at the 2D, wait for >= 16 FIDs and then click on 'Proc.Spectrum' on the Topspin Process menu. This will execute an automated processing macro. Although the resolution will be poor, you can evaluate the signal to noise (S/N) and whether the 15N offset and spectral width are appropriate.
This can be repeated at any time as additional FIDs are acquired.
The example spectrum region below shows both 'positive' crosspeaks for the tyrosine (blue, ~7.1 ppm), which does NOT bind to BSA, so behaves as a small molecule; and 'negative' crosspeaks for the tryptophan (yellow ~7.23 and 7.64 ppm) which does bind and behaves the same as the large BSA protein.



Protocol references
J.Cavanaugh, W.Fairbrother, A.Palmer, N.Skelton: Protein NMR Spectroscopy: Principles and Practice.
Academic Press 2006 ; Hardback ISBN: 97801216449189, eBook ISBN: 9780080471037

Dr. V. Higman: Protein NMR, a practical guide. https://protein-nmr.org.uk

J. Schleucher, M. Schwendinger, M. Sattler, P. Schmidt, O. Schedletzky, S.J. Glaser, O.W. Sorensen & C. Griesinger, J. Biomol. NMR 4, 301-306 (1994)

S. Grzesiek & A. Bax, J. Am. Chem. Soc. 115, 12593-12594 (1993)