Abstract
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.
Highlights
Research into the functional characteristics of molecules and materials is underpinned by the fundamental dogma that the molecular structure determines properties
The ever-increasing complexity of biological systems under study has been accompanied by a rise in awareness that structural context is of crucial relevance, and integrative structural biology is becoming increasingly important for consolidating information from a variety of methods into a holistic model
The sharp component in MPSL-labelled GB1 spectra suggests that some free label might be present in the samples despite the PD10 column used to remove the free label
Summary
Research into the functional characteristics of molecules and materials is underpinned by the fundamental dogma that the molecular structure determines properties. Structure determination lies at the core of chemistry, and virtually every undergraduate will learn Bragg’s Law describing X-ray diffraction [1]. The prevalence of structure as a determinant of all properties persists when studying the molecules of life. The fundamental hypothesis that all biomolecular functions are encoded in the structure [2] remains the central dogma of structural biology. The ever-increasing complexity of biological systems under study has been accompanied by a rise in awareness that structural context is of crucial relevance, and integrative structural biology is becoming increasingly important for consolidating information from a variety of methods into a holistic model. Similar approaches allow integrating results from multiple methods for materials characterisation
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