The Double‐Histidine Cu²⁺‐Binding Motif: A Highly Rigid, Site‐Specific Spin Probe for Electron Spin Resonance Distance Measurements

Abstract

The development of ESR methods that measure long-range distance distributions has advanced biophysical research. However, the spin labels commonly employed are highly flexible, which leads to ambiguity in relating ESR measurements to protein-backbone structure. Herein we present the double-histidine (dHis) Cu2+-binding motif as a rigid spin probe for double electron–electron resonance (DEER) distance measurements. The spin label is assembled in situ from natural amino acid residues and a metal salt, requires no postexpression synthetic modification, and provides distance distributions that are dramatically narrower than those found with the commonly used protein spin label. Simple molecular modeling based on an X-ray crystal structure of an unlabeled protein led to a predicted most probable distance within 0.5 of the experimental value. Cu2+ DEER with the dHis motif shows great promise for the resolution of precise, unambiguous distance constraints that relate directly to protein-backbone structure and flexibility.

RIDME distance measurements using Gd(iii) tags with a narrow central transition

“…2-Step phase cycles were applied to the first two observe pulses and to the pump pulse resulting in an overall 8-step phase cycle ( 4 ; receiver = (x, Àx) 4 ). A 2-step phase cycle was applied to the first MW pulse and a 4-step phase cycle was applied to the third and fourth MW pulses, resulting in an overall 8-step phase cycle (f 1 = (x) 4 , (Àx) 4 ; f 3 = f 4 = (x, Àx, y, Ày) 2 ; receiver = (x) 4 , (Àx) 4 ). The frequencies were set in a symmetric way with respect to the resonant mode of the cavity (centered at 94.9 GHz), with n 1 = 94.95 GHz and n 2 = 94.85 GHz.…”

Nucleotide‐Independent Copper(II)‐Based Distance Measurements in DNA by Pulsed ESR Spectroscopy

“…[13] Electron spin-echo envelope modulation results further suggest the preferential binding of Cu 2+ to the DPAb inding site over non-specific DNAcoordination. Thed ata was acquired at am agnetic field of 3360 G. Thes pectrum of control DNAs hows peaks below 4MHz, which is consistent with coordination to the imidazole [14] of guanine.T he spectrum of DPA-DNAi sm arkedly different. Thed ata was acquired at am agnetic field of 3360 G. Thes pectrum of control DNAs hows peaks below 4MHz, which is consistent with coordination to the imidazole [14] of guanine.T he spectrum of DPA-DNAi sm arkedly different.…”

Inhibitor‐Directed Spin Labelling—A High Precision and Minimally Invasive Technique to Study the Conformation of Proteins in Solution

“…C) The introduction of metal ions as spin centers can be achieved by engineering two closely spaced His residues. Together with Cu 2+ ions and iminodiacetic acid (IDA), a stable spin center is formed …”