A comparison of cysteine-conjugated nitroxide spin labels for pulse dipolar EPR spectroscopy
Date
13/12/2021Grant ID
BB/R013780/1
BB/T017740/1
RPG-2018-397
099149/Z/12/Z
Keywords
Metadata
Show full item recordAbstract
The structure-function and materials paradigms drive research on the understanding of structures and structural heterogeneity of molecules and solids from materials science to structural biology. Functional insights into complex architectures are often gained from a suite of complementary physicochemical methods. In the context of biomacromolecular structures, the use of pulse dipolar electron paramagnetic resonance spectroscopy (PDS) has become increasingly popular. The main interest in PDS is providing long-range nanometre distance distributions that allow for identifying macromolecular topologies, validating structural models and conformational transitions as well as docking of quaternary complexes. Most commonly, cysteines are introduced into protein structures by site-directed mutagenesis and modified site-specifically to a spin-labelled side-chain such as a stable nitroxide radical. In this contribution, we investigate labelling by four different commercial labelling agents that react through different sulfur-specific reactions. Further, the distance distributions obtained are between spin-bearing moieties and need to be related to the protein structure via modelling approaches. Here, we compare two different approaches to modelling these distributions for all four side-chains. The results indicate that there are significant differences in the optimum labelling procedure. All four spin-labels show differences in the ease of labelling and purification. Further challenges arise from the different tether lengths and rotamers of spin-labelled side-chains; both influence the modelling and translation into structures. Our comparison indicates that the spin-label with the shortest tether in the spin-labelled side-group, (bis-(2,2,5,5-Tetramethyl-3-imidazoline-1-oxyl-4-yl) disulfide, may be underappreciated and could increase the resolution of structural studies by PDS if labelling conditions are optimised accordingly.
Citation
Ackermann , K , Chapman , A & Bode , B E 2021 , ' A comparison of cysteine-conjugated nitroxide spin labels for pulse dipolar EPR spectroscopy ' , Molecules , vol. 26 , no. 24 , 7534 . https://doi.org/10.3390/molecules26247534
Publication
Molecules
Status
Peer reviewed
ISSN
1420-3049Type
Journal article
Rights
Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/4.0/).
Description
This research was funded, in whole or in part, by the Wellcome Trust (099149/Z/12/Z and 204821/Z/16/Z). B.E.B. and K.A. acknowledge support from the Leverhulme Trust (RPG-2018–397). B.E.B. acknowledges equipment funding from BBSRC (BB/R013780/1 and BB/T017740/1).Collections
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