Using the amide 15N CEST NMR experiment to study slow exchange between ‘visible’ protein states

Slow exchange between ‘visible’ protein states is often studied using the two-dimensional ZZ exchange class of magnetisation transfer experiments. However, the cross-peaks that arise due to magnetisation transfer between different states can lead to additional overlap in the two-dimensional ZZ exchange NMR spectrum. To overcome this overlap problem, here we have explored the utility of the ¹⁵N CEST experiment as an alternative to the ¹H^N–¹⁵N ZZ exchange experiment to study exchange between ‘visible’ protein states. In the case of two-state exchange, the ¹H^N–¹⁵N correlation map contains two correlations for each exchanging site, one arising from each state. Thus, two ¹⁵N CEST profiles can be recorded for each of these sites using a single ¹⁵N CEST experiment. We find that site-specific exchange parameters can then be obtained by simultaneously analysing both these ¹⁵N CEST profiles recorded at a single ‘high’ B₁ field supplemented with experimentally derived information regarding the initial magnetisation or as in the case of the ZZ exchange experiment, the minor state population. The utility of the ¹⁵N CEST based approach to characterise exchange between visible protein states is demonstrated by studying the interconversion of the ∼18 kDa T34A mutant of T4 lysozyme between its native state and a minor state populated to ∼21 % (exchange rate ∼5 s⁻¹) at 40 °C.