Binding observing the Ligand

Theory: soon….

How to set an STD experiment?

1) Adjust everything at zgesgp (water suppression..);
2) wrpa 2 –> re 2;
3) rpar SCREEN_STD all (stddiffesgp.3);
4) Acqupars –> Lists à FQLIST –> edit à FQ2LIST –> E (edit); … (select);

Obs: 1) Reference (ex: -40, 40, 50, or 100 ppm –> avoid chemical shifts close to zero);

          2) Protein saturation – Be careful not to saturate any ligand peak.

The pulse sequence above contains important parameters that you can play with:

  • d20: saturation time     [Usually 0.5 – 5 sec] –> 2 sec is a good starting point

Important: The minimum value of d1 is d20, because DELTA1=d1-d31 and d31=d20. I like using d1 = d20.

  • d29: spinlock time    [10 – 50 msec] – 30 msec is a good starting point
  • sp9 : f2 channel – shaped pulse  for saturation     [40 – 60 dB] or cnst62: 20-200 Hz if ZGOPTNS (-DCALC_POWER)
  • NBL: NBL = number of irradiation frequencies (usually NBL = 2)

Processing

  • Type stdsplit – au program for processing (splits your data in 2: reference (STD off-resonance), and difference spectrum (reference – on-resonance spectrum). Process your reference and difference spectrum as a 1D.
–> 20 µM BSA + 400 µM ibuprofen (from a pill) + 400 µM tryptophan. The first experiment is a 1D 1H.
 
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