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) Cu 2+ -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. Cu 2+ 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.

Rotameric preferences of a protein spin label at edge‐strand β‐sheet sites

“…Use of alternative labeling methods that feature greatly reduced ambiguity would be an ideal means for overcoming these issues. In particular, recently developed rigid spin labels such as the bifunctional spin label RX, the double histidine Cu 2+ chelating motif, and rigid Gd 3+ tags would offer advantages in the β‐sheet environment.…”

“…The mutant plasmids coding for E15C, E15C/K28C, T44C, and K28C/T44C GB1 were generated using previously described methods . The resulting plasmids were then transformed into BL21(DE3) E. coli cells and subsequently utilized for overexpression, purification, and eventual spin labeling via methods described previously …”

“…This process, known as site‐directed spin labeling (SDSL), involves the site‐specific placement of either stable organic radicals or paramagnetic metals within a protein. While metals such as Mn 2+ , Cu 2+ , and Gd 3+ show great promise in recent work, the most commonly utilized spin probe is the nitroxide‐functionalized side‐chain R1. R1 is generated by reacting the thiol‐specific methanothiosulfonate spin label (MTSSL) with cysteine residues.…”

A triarylmethyl spin label for long-range distance measurement at physiological temperatures using T 1 relaxation enhancement

“…Recent advances make this possible with the facile introduction of high-affinity Cu 2+ binding motifs consisting either of a genetically engineered pentapeptide in loops [16] or histidine pairs introduced into regular secondary structure [17]. A problem encountered upon the introduction of TAM1 is a reduction of the protein solubility [9] at least for some small soluble proteins, a problem that is solved by tethering the protein to a solid support prior to labeling.…”

“…[28] has an affinity too low for the present method. Various high affinity Cu 2+ peptide binding sites have been reported in the literature; for example, studies described in References [16], [17], and [29] all involve Cu 2+ binding sites with nanomolar K d values.…”