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Abstract Pulsed dipolar EPR spectroscopy (PDS) in combination with site‐directed spin labeling is a powerful tool in structural biology. However, the commonly used spin labels are conjugated to biomolecules via rather long and flexible linkers, which hampers the translation of distance distributions into biomolecular conformations. In contrast, the spin label copper(II)‐nitrilotriacetic acid [Cu2+(NTA)] bound to two histidines (dHis) is rigid and yields narrow distance distributions, which can be more easily translated into biomolecular conformations. Here, we use this label on the 71 kDaYersiniaouter protein O (YopO) to decipher whether a previously experimentally observed bimodal distance distribution is due to two conformations of the biomolecule or of the flexible spin labels. Two different PDS experiments, that is, pulsed electron–electron double resonance (PELDOR aka DEER) and relaxation‐induced dipolar modulation enhancement (RIDME), yield unimodal distance distribution with the dHis‐Cu2+(NTA) motif; this result suggests that the α‐helical backbone of YopO adopts a single conformation in frozen solution. In addition, we show that the Cu2+(NTA) label preferentially binds to the target double histidine (dHis) sites even in the presence of 22 competing native histidine residues. Our results therefore suggest that the generation of a His‐null background is not required for this spin labeling methodology. Together these results highlight the value of the dHis‐Cu2+(NTA) motif in PDS experiments.
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Endogenous Cu(II) Labeling for Distance Measurements on Proteins by EPR
Abstract In‐cell measurements of the relationship between structure and dynamics to protein function is at the forefront of biophysics. Recently, developments in EPR methodology have demonstrated the sensitivity and power of this method to measure structural constraints in‐cell. However, the need to spin label proteins ex‐situ or use noncanonical amino acids to achieve endogenous labeling remains a bottleneck. In this work we expand the methodology to endogenously spin label proteins with Cu(II) spin labels and describe how to assess in‐cell spin labeling. We quantify the amount of Cu(II)‐NTA in cells, assess spin labeling, and account for orientational effects during distance measurements. We compare the efficacy of using heat‐shock and hypotonic swelling to deliver spin label, showing that hypotonic swelling is a facile and reproducible method to efficiently deliver Cu(II)‐NTA intoE. coli. Notably, over six repeats we accomplish a bulk average of 57 μM spin labeled sites, surpassing existing endogenous labeling methods. The results of this work open the door for endogenous spin labeling that is easily accessible to the broader biophysical community.
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- Award ID(s):
- 2407706
- PAR ID:
- 10642346
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Chemistry – A European Journal
- Volume:
- 30
- Issue:
- 72
- ISSN:
- 0947-6539
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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