Enzymatic Spin-Labeling of Protein N- and C-Termini for Electron Paramagnetic Resonance Spectroscopy

Abstract

Electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for investigating the structure and dynamics of proteins. The introduction of paramagnetic moieties at specific positions in a protein enables precise measurement of local structure and dynamics. This technique, termed site-directed spin-labeling, has traditionally been performed using cysteine-reactive radical-containing probes. However, large proteins are more likely to contain multiple cysteine residues and cysteine labeling at specific sites may be infeasible or impede function. To address this concern, we applied three peptide-ligating enzymes (sortase, asparaginyl endopeptidase, and inteins) for nitroxide labeling of N- and C-termini of select monomeric and dimeric proteins. Continuous wave and pulsed EPR (double electron electron resonance) experiments reveal specific attachment of nitroxide probes to ether N-termini (OaAEP1) or C-termini (sortase and intein) across three test proteins (CheY, CheA, and iLOV), thereby enabling a straightforward, highly specific, and general method for protein labeling. Importantly, the linker length (3, 5, and 9 residues for OaAEP1, intein, and sortase reactions, respectively) between the probe and the target protein has a large impact on the utility of distance measurements by pulsed EPR, with longer linkers leading to broader distributions. As these methods are only dependent on accessible N- and C-termini, we anticipate application to a wide range of protein targets for biomolecular EPR spectroscopy.

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

“…Nevertheless, due to the challenges of spin-labeling appreciable concentrations of p-FRQ, the modulation depths of the DEER data, which reflect the number of spins in range to produce specific dipolar interactions, are small (∼2-4%). These values compare to ∼10-12% for a well-ordered, highly labeled pair of sites under these instrument conditions (Dunleavy, et al, 2023 ). The single Cys spin-labeled variant 490SL produced log-linear time domain traces indicative of a uniform background with little evidence of dimerization, consistent with the MALS data showing p-FRQ to be monomeric (Fig.…”