Combining rigid Cu( ii ) labels and pulsed-EPR techniques enables distance constraint measurements that are incisive probes of protein structure and dynamics. However, the labels can lead to a dipolar signal that is biased by the relative orientation of the two spins, which is typically unknown a priori in a bilabeled protein. This effect, dubbed orientational selectivity, becomes a bottleneck in measuring distances. This phenomenon also applies to other pulsed-EPR techniques that probe electron–nucleus interactions. In this work, we dissect orientational selectivity by generating an in silico sample of Cu( ii )-labeled proteins to evaluate pulse excitation in the context of double electron–electron resonance (DEER) at Q-band frequencies. This approach enables the observation of the contribution of each protein orientation to the dipolar signal, which provides direct insights into optimizing acquisition schemes to mitigate orientational effects. Furthermore, we incorporate the excitation profile of realistic pulses to identify the excited spins. With this method, we show that rectangular pulses, despite their imperfect inversion capability, can sample similar spin orientations as other sophisticated pulses with the same bandwidth. Additionally, we reveal that the efficiency of exciting spin-pairs in DEER depends on the frequency offset of two pulses used in the experiment and the relative orientation of the two spins. Therefore, we systematically examine the frequency offset of the two pulses used in this double resonance experiment to determine the optimal frequency offset for optimal distance measurements. This procedure leads to a protocol where two measurements are sufficient to acquire orientational-independent DEER at Q-band. Notably, this procedure is feasible with any commercial pulsed-EPR spectrometer. Furthermore, we experimentally validate the computational results using DEER experiments on two different proteins. Finally, we show that increasing the amplitude of the rectangular pulse can increase the efficiency of DEER experiments by almost threefold. Overall, this work provides an attractive new approach for analyzing pulsed-EPR spectroscopy to obtain microscopic nuances that cannot be easily discerned from analytical or numerical calculations.
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Cu(II) EPR Reveals Two Distinct Binding Sites and Oligomerization of Innate Immune Protein Calgranulin C
S100A12 or Calgranulin C is a homodimeric antimicrobial protein of the S100 family of EF-hand calcium-modulated proteins. S100A12 is involved in many diseases like inflammation, tumor invasion, cancer and neurological disorders like Alzheimer’s disease. The binding of transition metal ions to the protein is important as the sequestering of the metal ion induces conformational changes in the protein, inhibiting the growth of various pathogenic microorganisms. In this work, we probe the Cu(II) binding properties of Calgranulin C. We demonstrate that the two Cu(II) binding sites in Calgranulin C show different coordination environments in solution. Electron spin resonance (ESR) spectra of Cu(II)-bound protein clearly show two distinct components at higher Cu(II):protein ratios, which is indicative of the two different binding environments for the Cu(II) ions. The g|| and A|| values are also different for the two components, indicating that the number of directly coordinated nitrogens in each site differs. Furthermore, we perform Continuous Wave (CW)-titrations to obtain the binding affinity of the Ca(II)-loaded protein to Cu2+ ions. We observe a positive cooperativity in binding of the two Cu(II) ions. In order to further probe the Cu2+ coordination, we also perform Electron Spin Echo Envelope Modulation (ESEEM) experiment. We perform ESEEM at two different fields where one Cu(II) binding site dominates over the other. At both sites we see distinct signatures of Cu(II)-histidine coordination. However, we clearly see that the ESEEM spectra corresponding to the two Cu2+ binding sites are significantly different. There is clear change in the intensity of the double quantum (DQ) peak with respect to the nuclear quadrupole interaction (NQI) peak at the two different fields. Furthermore, ESEEM along with Hyperfine Sublevel Correlation (HYSCORE) show that only one of the two Cu(II) binding sites has backbone coordination, confirming our previous observation. Finally, we perform Double Electron Electron Resonance (DEER) spectroscopy to probe if the difference in binding environment is due to the Cu(II) binding to different sites in the protein. We obtain a distance distribution with a sharp peak at ~ 3 nm and a broad peak at ~ 4 nm. The shorter distance agrees with the Cu(II)-Cu(II) distance expected for a dimer from the crystal structure. The longer distance is consistent with the Cu(II)-Cu(II) distance when oligomerization occurs.
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- NSF-PAR ID:
- 10098379
- Date Published:
- Journal Name:
- Applied magnetic resonance
- Volume:
- 49
- ISSN:
- 0937-9347
- Page Range / eLocation ID:
- 1299-1311
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/https://doi.org/10.1007/s00723-018-1053-7
