Electron paramagnetic resonance (EPR) in combination with the recently developed double-histidine (dHis)-based Cu2+ spin labeling has provided valuable insights into protein structure and conformational dynamics. To relate sparse distance constraints measured by EPR to protein fluctuations in solution, modeling techniques are needed. In this work, we have developed force field parameters for Cu2+–nitrilotriacetic and Cu2+–iminodiacetic acid spin labels. We employed molecular dynamics (MD) simulations to capture the atomic-level details of dHis-labeled protein fluctuations. The interspin distances extracted from 200 ns MD trajectories show good agreement with the experimental results. The MD simulations also illustrate the dramatic rigidity of the Cu2+ labels compared to the standard nitroxide spin label. Further, the relative orientations between spin-labeled sites were measured to provide insight into the use of double electron–electron resonance (DEER) methods for such labels. The relative mean angles, as well as the standard deviations of the relative angles, agree well in general with the spectral simulations published previously. The fluctuations of relative orientations help rationalize why orientation selectivity effects are minimal at X-band frequencies, but observable at the Q-band for such labels. In summary, the results show that by combining the experimental results with MD simulations precise information about protein conformations as well as flexibility can be obtained.

Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis-Cu II NTA label to obtain site-specific information on dynamics for both an a-helix and b-sheet site of GB1, the immunoglobulin binding domain of protein G. Spectral features found in our CW-EPR measurements were consistent with the overall rigid nature of GB1 and with predictions from molecular dynamics simulations. Using this information, we show the potential of this approach to elucidate the role of dynamics in substrate binding of a functionally necessary a-helix in human glutathione transferase A1-1 (hGSTA1-1). We observe two dynamical modes for the helix. The addition of the inhibitor GS-Met and GS-Hex resulted in hGSTA1-1 to favor the more rigid active state conformation, while the faster mode potentially aids the search for substrates. Together the results illustrate the remarkable potential of the dHis-based labelling approach to measure sitespecific dynamics using room temperature lineshape analysis.

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.

Recent advances in site-directed Cu2+ labeling of proteins and nucleic acids have added an attractive new methodology to measure structure-function relationship in biomolecules. Despite the promise, accessing the higher sensitivity...

Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis-Cu II NTA label to obtain site-specific information on dynamics for both an a-helix and b-sheet site of GB1, the immunoglobulin binding domain of protein G. Spectral features found in our CW-EPR measurements were consistent with the overall rigid nature of GB1 and with predictions from molecular dynamics simulations. Using this information, we show the potential of this approach to elucidate the role of dynamics in substrate binding of a functionally necessary a-helix in human glutathione transferase A1-1 (hGSTA1-1). We observe two dynamical modes for the helix. The addition of the inhibitor GS-Met and GS-Hex resulted in hGSTA1-1 to favor the more rigid active state conformation, while the faster mode potentially aids the search for substrates. Together the results illustrate the remarkable potential of the dHis-based labelling approach to measure sitespecific dynamics using room temperature lineshape analysis.