Going the dHis-tance: Site-Directed Cu²⁺ Labeling of Proteins and Nucleic Acids

Abstract

Conspectus In this Account, we showcase site-directed Cu2+ labeling in proteins and DNA, which has opened new avenues for the measurement of the structure and dynamics of biomolecules using electron paramagnetic resonance (EPR) spectroscopy. In proteins, the spin label is assembled in situ from natural amino acid residues and a metal complex and requires no post-expression synthetic modification or purification procedures. The labeling scheme exploits a double histidine (dHis) motif, which utilizes endogenous or site-specifically mutated histidine residues to coordinate a Cu2+ complex. Pulsed EPR measurements on such Cu2+-labeled proteins potentially yield distance distributions that are up to 5 times narrower than the common protein spin labelthe approach, thus, overcomes the inherent limitation of the current technology, which relies on a spin label with a highly flexible side chain. This labeling scheme provides a straightforward method that elucidates biophysical information that is costly, complicated, or simply inaccessible by traditional EPR labels. Examples include the direct measurement of protein backbone dynamics at β-sheet sites, which are largely inaccessible through traditional spin labels, and rigid Cu2+–Cu2+ distance measurements that enable higher precision in the analysis of protein conformations, conformational changes, interactions with other biomolecules, and the relative orientations of two labeled protein subunits. Likewise, a Cu2+ label has been developed for use in DNA, which is small, is nucleotide independent, and is positioned within the DNA helix. The placement of the Cu2+ label directly reports on the biologically relevant backbone distance. Additionally, for both of these labeling techniques, we have developed models for interpretation of the EPR distance information, primarily utilizing molecular dynamics (MD) simulations. Initial results using force fields developed for both protein and DNA labels have agreed with experimental results, which has been a major bottleneck for traditional spin labels. Looking ahead, we anticipate new combinations of MD and EPR to further our understanding of protein and DNA conformational changes, as well as working synergistically to investigate protein–DNA interactions.

Cu(ii)-based DNA labeling identifies the structural link between transcriptional activation and termination in a metalloregulator

“…The DPA-Cu(II) motif is a straightforward spin labeling technique that orients the DPA into the helix and reports directly on DNA backbone distances. 10,11 Additionally, we have shown earlier that orientational selectivity 12,13 effects for the DPA-Cu(II) motif are negligible even at Q-band, which enables the measurement of distances even from data at a single magnetic eld. 11 The labeling of DNA with Cu(II) is described in ESI.…”

“…10,11 Additionally, we have shown earlier that orientational selectivity 12,13 effects for the DPA-Cu(II) motif are negligible even at Q-band, which enables the measurement of distances even from data at a single magnetic eld. 11 The labeling of DNA with Cu(II) is described in ESI. † 11,14 Electrophoretic mobility shi assays (EMSA) show that CueR binds to the DPA-DNA duplex (Fig.…”

“…11 The labeling of DNA with Cu(II) is described in ESI. † 11,14 Electrophoretic mobility shi assays (EMSA) show that CueR binds to the DPA-DNA duplex (Fig. S1 †).…”

The Advantages of EPR Spectroscopy in Exploring Diamagnetic Metal Ion Binding and Transfer Mechanisms in Biological Systems

“…In addition, the most used spin-label, MTSSL, is highly flexible, which leads to broad distance distributions. The use of other spin-labels, such as dHis-Cu(II), can lead to much narrower distance distribution [17,107], and can overcome the limitations associated with the use of MTSSL. dHis-Cu(II) spin-labeling has recently been shown to hold high affinity and stability against competitor metal ions (even in an acidic environment) to biomolecules.…”

Interpretation of EPR and optical spectra of Ni(II) ions in crystalline lattices at ambient temperature

“…[5][6][7] An enzymatically active diamagnetic ion in the biomolecules is replaced with a paramagnetic ion, or diamagnetic molecules are labeled with a paramagnetic ion to study them by electron paramagnetic resonance (EPR) spectroscopy. [8][9][10][11][12][13][14][15] This provides scope for structural characterization of biomolecules [16][17][18][19][20] and understanding the local environment around the paramagnetic center. [21][22][23][24] In view of this, many biologically important metal ions, such as copper(II), [25][26][27] cobalt (II), [28][29][30][31] iron(III), [32][33][34] manganese(II), [35,36] and other ions, [37][38][39] have been studied by EPR spectroscopy.…”