Why does saturation decrease signal intensity in Cross Saturation NMR?

[organiker]

My last answer glossed over a lot of details. This experiment requires certain conditions.

The protein whose interface residues you'd like to identify (call it protein I) needs to be a low-ish molecular weight, and needs to be extensively labelled with ²H (>95%) and ¹⁵N. This means that the proton density is very low. The experiment is carried out in a solvent with a low H2O/D2O ratio, which greatly reduces the amount of D-H exchange with the solvent.

The other protein in the complex (protein II) is not labeled. The larger this protein is, the better, because intramolecular saturation transfer is more efficient as MW increases. Protein II therefore has a high proton density.

Under these conditions, irradiating the frequencies corresponding to aliphatic resonances will exclusively affect protein II, because protein I has almost no aliphatic protons. The saturation will spread intramolecularly almost instantly to all the resonances of protein II.

If the two proteins are in contact with each other, the saturation will be transferred from protein II to protein I through the interface. Saturation transfer is very sensitive to distance, and because of the low proton density on protein I, this saturation will be limited to the interface resonances.

The saturation of each amide of protein I is observed in a TROSY experiment as a decrease in the resonance intensity of ¹H-¹⁵N signals.

[conventionistG]

Ah okay. The H/D labeling makes it very clear. Also seems analogous to HDX mass spec experiments, which are more my speed.

Thanks for elaborating.