Histidine side-chain dynamics and protonation monitored by ¹³C CPMG NMR relaxation dispersion

The use of ¹³C NMR relaxation dispersion experiments to monitor micro-millisecond fluctuations in the protonation states of histidine residues in proteins is investigated. To illustrate the approach, measurements on three specifically ¹³C labeled histidine residues in plastocyanin (PCu) from Anabaena variabilis (A.v.) are presented. Significant Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion is observed for ¹³Cε¹ nuclei in the histidine imidazole rings of A.v. PCu. The chemical shift changes obtained from the CPMG dispersion data are in good agreement with those obtained from the chemical shift titration experiments, and the CPMG derived exchange rates agree with those obtained previously from ¹⁵N backbone relaxation measurements. Compared to measurements of backbone nuclei, ¹³Cε¹ dispersion provides a more direct method to monitor interchanging protonation states or other kinds of conformational changes of histidine side chains or their environment. Advantages and shortcomings of using the ¹³Cε¹ dispersion experiments in combination with chemical shift titration experiments to obtain information on exchange dynamics of the histidine side chains are discussed.