Abstract
Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) in combination with site-directed spin labeling (SDSL) of proteins and oligonucleotides is a powerful tool in structural biology. Instead of using the commonly employed gem-dimethyl-nitroxide labels, triarylmethyl (trityl) spin labels enable such studies at room temperature, within the cells and with single-frequency electron paramagnetic resonance (EPR) experiments. However, it has been repeatedly reported that labeling of proteins with trityl radicals led to low labeling efficiencies, unspecific labeling and label aggregation. Therefore, this work introduces the synthesis and characterization of a maleimide-functionalized trityl spin label and its corresponding labeling protocol for cysteine residues in proteins. The label is highly cysteine-selective, provides high labeling efficiencies and outperforms the previously employed methanethiosulfonate-functionalized trityl label. Finally, the new label is successfully tested in PDS measurements on a set of doubly labeled Yersinia outer protein O (YopO) mutants.
Highlights
The combination of site-directed spin labeling (SDSL) with electron paramagnetic resonance (EPR)spectroscopy has proven to be a valuable tool in structural biology [1,2,3]
Spectroscopy has proven to be a valuable tool in structural biology [1,2,3]
The most established spin label is the methanethiosulfonate-functionalized nitroxide MTSSL 1 (Figure 1), which reacts with cysteine residues to form the disulfide bonded side chain R1 [26,27]
Summary
The combination of site-directed spin labeling (SDSL) with electron paramagnetic resonance (EPR)spectroscopy has proven to be a valuable tool in structural biology [1,2,3]. Have been very successful in providing information on the structure, conformational changes and dynamics of proteins [12,13,14,15,16,17,18], oligonucleotides [19,20,21,22,23] and their complexes [24,25]. Most of these studies rely on spin labeling with nitroxides. MTSSL provides high labeling yields and site selectivity through a combination with site-directed mutagenesis, which places the cysteines and the R1 side chain at the desired positions in the protein.
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