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.
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.
- Fersht A (1998) Structure and mechanism in protein science: a guide to enzyme catalysis and protein folding. W. H. Freeman and Company, New YorkGoogle Scholar
- Grey MJ, Tang YF, Alexov E, McKnight CJ, Raleigh DP, Palmer AG (2006) Characterizing a partially folded intermediate of the villin headpiece domain under non-denaruring conditions: contribution of His41 to the pH-dependent stability of the N-terminal subdomain. J Mol Biol 355:1078–1094CrossRefGoogle Scholar
- Mandel M (1965) Proton magnetic resonance spectra of some proteins. I. Ribonuclease oxidized ribonuclease lysozyme and cytochrome c. J Biol Chem 240:1586–1592Google Scholar
- Schmidt L, Christensen HEM, Harris P (2006) Structure of plastocyanin from the cyanobacterium Anabaena variabilis. Acta Crystallogr D 62:1022–1029Google Scholar
- Shimahara H, Yoshida T, Shibata Y, Shimizu M, Kyogoku Y, Sakiyama F, Nakazawa T, Tate S, Ohki S, Kato T, Moriyama H, Kishida K, Tano Y, Ohkubo T, Kobayashi Y, Ha (2007) Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase. J Biol Chem 282:9646–9656CrossRefGoogle Scholar
- Sudmeier JL, Evelhoch JL, Jonsson NBH (1980) Dependence of NMR lineshape analysis upon chemical rates and mechanisms - implications for enzyme histidine titrations. J Magn Reson 40:377–390Google Scholar
- Vuister GW, Bax A (1992) Resolution enhancement and spectral editing of uniformly C-13-enriched proteins by homonuclear broad-band C-13 decoupling. J Magn Reson 98:428–435Google Scholar