A general model to optimise Cu^IIlabelling efficiency of double-histidine motifs for pulse dipolar EPR applications

Abstract

A multi-site speciation model facilitates double-histidine motif labelling efficiency optimisation for pulse dipolar EPR measurements. Results suggest affinities differing by a factor of 10 between an α-helical and a β-sheet double-histidine motif.

Pulse dipolar EPR spectroscopy reveals buffer modulated cooperativity of metal templated protein dimerization

“…First, measurements were performed as a nine-point pseudo-titration series (where each datapoint was a discrete sample) for Cu II , in the presence of phosphate buffer (150 mM NaCl, 42.4 mM Na2HPO4 and 7.6 mM KH2PO4, pH 7.4), having been used extensively for previous EPR methodological work involving GB1 and Cu II -NTA. 28,32,33 In Figure 2, the ∆ 𝑀−𝑅1 behaviour was consistent with a reduced apparent binding affinity of Cu II for the dHis motif in phosphate buffer, with <80% occupancy at a metal:protein ratio of 1:1. Indeed, ∆ 𝑀−𝑅1 was systematically lower for the phosphate buffer series than the Tris-HCl buffer series (ESI and vide infra for details).…”

Pulse dipolar EPR for determining nanomolar binding affinities

“…42 In recent years, the use of genetically encoded doublehistidine (dHis) motifs to bind paramagnetic metal ions, in particular Cu II , has been established for PDS. 7,27,[43][44][45] Here, orthogonal labelling, that is, the combination of two spectroscopically distinct spin labels (in this case methanethiosulfonate (MTSL) 6,46 and Cu II -nitrilotriacetic acid (Cu II -NTA), 43 Fig. 1B) with the RIDME method, has enabled PDS measurements in the sub-mM concentration regime.…”

Nanomolar Pulse Dipolar EPR Spectroscopy in Proteins: CuII–CuII and Nitroxide–Nitroxide Cases

“…group G protein G, B1 domain (GB1) constructs (I6R1/K28R1 and I6H/N8H/K28H/Q32H) were used as biological model systems (Figure ). GB1 has been used extensively in previous EPR methodology studies. − ,− We have shown previously that nitroxide-detected Cu II –nitroxide and Cu II –Cu II RIDME are similar in sensitivity and roughly two orders of magnitude more sensitive than Cu II –Cu II PELDOR when limited to rectangular pulses . Here, we endeavored to test the sensitivity of the most widespread pulse dipolar EPR methodology, nitroxide–nitroxide PELDOR .…”