No abstract

In this work, we explore the potential of a rigid Cu 2+ spin-labeling technique, the double histidine (dHis) motif, along with Q-band electron paramagnetic resonance to report on the relative orientations of the spin labels. We show that the precision of the dHis motif, coupled with the sensitivity and resolution of Q-band frequencies, may allow for the straightforward determination of the relative orientation of the dHis-Cu 2+ labels using double electron− electron resonance (DEER). We performed Q-band DEER measurements at different magnetic fields on a protein containing two dHis Cu 2+ sites. These measurements exhibited orientational selectivity such that each discrete magnetic field yielded a unique DEER signal. We determined the relative orientation of the two metal centers by simulating the orientationally selective DEER data. These relative orientations were validated by visual analysis of the protein crystal structure modified with dHis sites. The simple visual analysis was shown to agree well with the angular values determined via simulation of the experimental data. The combination of the dHis-Cu 2+ motif along with the advantages of the Q-band can aid in the accurate measurement of protein structural and conformational dynamics.

Cu based distance measurements using the double-histidine (dHis) motif by pulsed ESR present an attractive strategy to obtain precise, narrow distance distributions that can be easily related to protein backbone structure (Cunningham et al., Angew. Chem., Int. Ed., 2015, 54, 633). The Cu-ion is introduced as a complex with the iminodiacetic acid (IDA) chelating agent, which enhances binding selectivity to the two histidine residues that are site-selectively placed on the protein through mutagenesis. However, initial results of this method produced weak dipolar modulations. To enhance applicability of the double histidine motif using IDA, we perform a systematic examination of the possible causes of these weak dipolar modulations. We examine the efficiency of the Cu-ion to form the Cu-IDA complex in solution. In addition, we analyze the selectivity of Cu-IDA binding to dHis sites at both α-helical and β-strand environments. Our results indicate that the dHis motif on the β-sheet sites have high affinity towards Cu-IDA while the dHis sites on α-helices show poor affinity for the metal-ion complex. We are able to use our new findings to optimize conditions to maximize dHis loading while minimizing both free Cu and unbound Cu-IDA complex in solution, allowing us to double the sensitivity of the Double Electron-Electron Resonance (DEER) experiment. Finally, we illustrate how Cu-based CW-ESR and DEER can be combined to obtain information on populations of different Cu-complexes in solution.

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.