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Biochemistry. 2003 Aug 5;42(30):9227-34.

Quantitative identification of the protonation state of histidines in vitro and in vivo.

Author information

1
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California 94143, USA.

Abstract

The C[bond]N coupling constants centered at the C(epsilon 1) and C(delta 2) carbons in histidine residues depend on the protonation state and tautomeric form of the imidazole ring, making them excellent indicators of pH or pK(a), and the ratio of the tautomeric states. In this paper, we demonstrate that the intensity ratios for the C(epsilon 1)-H and C(delta 2)-H cross-peaks measured with a constant time HSQC experiment without and with J(C[bond]N) amplitude modulation are determined by the ratios of the protonated and deprotonated forms and tautomeric states. This allows one to investigate the tautomeric state of histidines as well as their pK(a) in situations where changing the pH value by titration is difficult, for example, for in-cell NMR experiments. We apply this technique to the investigation of the bacterial protein NmerA and determine that the intracellular pH in the Escherichia coli cytoplasm is 7.1 +/- 0.1.

PMID:
12885258
DOI:
10.1021/bi0344679
[Indexed for MEDLINE]

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