Exchangeable deuterons introduce artifacts in amide 15N CEST experiments used to study protein conformational exchange

Protein molecules sample different conformations in solution and characterizing these conformations is crucial to understanding protein function. N-15 CEST experiments are now routinely used to study slow conformational exchange of protein molecules between a visible' major state and invisible' minor states. These experiments have also been adapted to measure the solvent exchange rates of amide protons by exploiting the one bond deuterium isotope effect on the amide N-15 chemical shifts. However at moderately high temperatures (similar to 50 degrees C) that are sometimes required to populate protein minor conformers to levels (similar to 1%) that can be detected by CEST experiments solvent H/D exchange can lead to dips' in low (B1N)-N-15 CEST profiles that can be wrongly assigned to the conformational exchange process being characterized. This is demonstrated in the case of similar to 18kDa T4 Lysozyme (T4L) at 50 degrees C and the similar to 11kDa E. coli hibernation promoting factor (HPF) at 52 degrees C. This problem is trivially solved by eliminating the exchangeable deuterons in the solvent by using either an external D2O lock or by using a small amount (similar to 1-3%) of a molecule like d6-DMSO that does not contain exchangeable deuterons to lock the spectrometer.