On the use of the Cu2+-iminodiacetic acid complex for double histidine based distance measurements by pulsed ESR.
Phys Chem Chem Phys
; 19(31): 20959-20967, 2017 Aug 09.
Article
em En
| MEDLINE
| ID: mdl-28745737
ABSTRACT
Cu2+ based distance measurements using the double-histidine (dHis) motif by pulsed ESR present an attractive strategy to obtain precise, narrow distance distributions that can be easily related to protein backbone structure (Cunningham et al., Angew. Chem., Int. Ed., 2015, 54, 633). The Cu2+-ion is introduced as a complex with the iminodiacetic acid (IDA) chelating agent, which enhances binding selectivity to the two histidine residues that are site-selectively placed on the protein through mutagenesis. However, initial results of this method produced weak dipolar modulations. To enhance applicability of the double histidine motif using IDA, we perform a systematic examination of the possible causes of these weak dipolar modulations. We examine the efficiency of the Cu2+-ion to form the Cu2+-IDA complex in solution. In addition, we analyze the selectivity of Cu2+-IDA binding to dHis sites at both α-helical and ß-strand environments. Our results indicate that the dHis motif on the ß-sheet sites have high affinity towards Cu2+-IDA while the dHis sites on α-helices show poor affinity for the metal-ion complex. We are able to use our new findings to optimize conditions to maximize dHis loading while minimizing both free Cu2+ and unbound Cu2+-IDA complex in solution, allowing us to double the sensitivity of the Double Electron-Electron Resonance (DEER) experiment. Finally, we illustrate how Cu2+-based CW-ESR and DEER can be combined to obtain information on populations of different Cu2+-complexes in solution.
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2017
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Article