Histidine side-chain dynamics and protonation monitored by 13C CPMG NMR relaxation dispersion
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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.
KeywordsPlastocyanin Histidine side-chain dynamics 13C CPMG NMR relaxation dispersion
We thank Lise-Lotte Jespersen for technical assistance. This study was supported by the Danish Agency for Science, Technology and Innovation, grants, 21-04-0519 and 272-07-0466, Carlsbergfondet grant 1624/40, Novo Nordisk Fonden grant 2003-11-28, and Villum Kann Rasmussen Fonden grant 8.12.2003.
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