A general model to optimise CuII labelling efficiency of double-histidine motifs for pulse dipolar EPR applications.

Joshua L Wort,Katrin Ackermann,David G Norman,Bela E Bode

doi:10.1039/d0cp06196d

Abstract

Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to studies of biomolecules underpinning health and disease by providing highly accurate and precise geometric constraints. Combining double-histidine (dH) motifs with CuII spin labels shows promise for further increasing the precision of distance measurements, and for investigating subtle conformational changes. However, non-covalent coordination-based spin labelling is vulnerable to low binding affinity. Dissociation constants of dH motifs for CuII-nitrilotriacetic acid were previously investigated via relaxation induced dipolar modulation enhancement (RIDME), and demonstrated the feasibility of exploiting the dH motif for EPR applications at sub-μM protein concentrations. Herein, the feasibility of using modulation depth quantitation in CuII-CuII RIDME to simultaneously estimate a pair of non-identical independent KD values in such a tetra-histidine model protein is addressed. Furthermore, we develop a general speciation model to optimise CuII labelling efficiency, depending upon pairs of identical or disparate KD values and total CuII label concentration. We find the dissociation constant estimates are in excellent agreement with previously determined values, and empirical modulation depths support the proposed model.

Highlights

This covalent attachment of nitroxide radicals through sulfhydryl moieties is convenient and robust,[35,37,38,39] it results in the labelling of all accessible cysteine residues and so is often intractable in systems which contain essential structural or functional cysteines
Dissociation constants of dH motifs for CuII–nitrilotriacetic acid were previously investigated via relaxation induced dipolar modulation enhancement (RIDME), and demonstrated the feasibility of exploiting the dH motif for Electron paramagnetic resonance (EPR) applications at sub-mM protein concentrations
RIDME, isotherms can be measured at plateau because modulation depth increases asymptotically, and CuII–chelate can be added to saturation

Summary

Introduction

This covalent attachment of nitroxide radicals through sulfhydryl moieties is convenient and robust,[35,37,38,39] it results in the labelling of all accessible cysteine residues and so is often intractable in systems which contain essential structural or functional cysteines. Considering the simple case of a tetra-histidine (double dH) protein labelled with CuII spin-label, optimisation of labelling efficiency for PDEPR applications can become nontrivial.[58] Under conditions of partial loading, all CuII species whether dH bound or not can be detected, but not all species will contribute to dipolar modulation of the detected echo This has obvious implications for measurement sensitivity, being further exacerbated if each site coordinates CuII spin-label with differential affinity and must be treated as nonidentical and independent. For s identical sites with microscopic association constant, K1, and t identical sites with microscopic association constant, K2, it is known from multinomial theorem that Z can be expressed as a double sum over all i permutations of s, and j permutations This is equivalent to the form given for 2 non-identical independent binding sites.[59] in the case of s identical sites, t is 0, so this binomial term reduces to unity, and yields the familiar expression given above in (eqn 7). Under the approximation of monoexponential T1 behaviour, the asymptotic modulation depth (DTmix) for a given ratio of Tmix and T1, is given as:

D À1 Tmix

Experimental procedures

Results and discussion

Conclusions