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 Å 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.

Keywords: DEER; EPR spectroscopy; copper; proteins; spin labeling.

Figure 1

a) Overview of the DEER experiment. b) The mutations and resulting side chains of the most common spin label (R1) and the paramagnetic metal label reported herein (dHis–Cu²⁺–IDA). The widths of the resultant distance distributions are heavily influenced by the flexibility of the side chain.

Figure 2

a) Crystal structure of 6H/8H/28H/32H-GB1 (PDB: 4WH4). b) ESEEM and CW (inset) data for each of the individual dHis sites complexed with Cu²⁺–IDA. All data are indicative of coordination by two histidine residues and IDA for both the α-helix (28H/32H) and β-sheet (6H/8H) sites.

Figure 3

a) Baseline-subtracted time-domain DEER data for both the R1 (gray) and the Cu²⁺ samples (black) as well as the best fit (dotted black lines) from Tikhonov regularization; modulation depths have been scaled for comparison. b) The distance distributions for each sample. c,d) Structural models of 6R1/28R1-GB1 and 6H/8H/28H/32H-GB1 complexed with Cu²⁺–IDA with measured interspin distances.