Novel spin labels for EPR investigation of protein–peptide interactions

Site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a well-established method that can provide specific information about the structure, dynamics, and conformational changes of biomacromolecules. SDSL involves introducing a cysteine residue at selected site (either via mutation, for proteins, or via direct synthesis for peptides) and then covalently labeling it with a spin label, which is used as the EPR-detectable probe. In particular, pulse dipolar EPR (PDS-EPR) experiments on doubly spin-labelled protein/substrate systems allow the measurement of distances between spin centers, providing insights about the molecular interaction. The research presented in this thesis aims to evaluate new spin probes using Calmodulin (CaM) as a model protein. Since CaM has already been widely studied, the availability of structural and functional data makes it an excellent model protein for EPR-based structural analysis. CaM is a highly dynamic Ca(2+)-binding protein that undergoes significant conformational changes upon binding Ca(2+) and subsequently one of its protein substrates .  We investigated the intermolecular distances between an analog of the M13 peptide (the archetypical CaM substrate) and CaM through site-directed spin labeling, in which both components were selectively labeled. The peptide, M13C, was synthesized with an additional cysteine residue at the N-terminus, serving as the labeling site. CaM was engineered to include cysteine mutations at three distinct positions, each representative of different structural environments. Both the peptide and the protein were labeled with a nitroxide-based paramagnetic probe, in order to perform pulsed dipolar EPR spectroscopy. In addition, one of the two labels was changed to a photoexcitable triplet-state label with the goal of performing PDS-EPR in combination with photoexcitation. The thesis, at first, involved the fine tuning of the purification and labeling protocols for both the peptide and the protein. Then the PDS-EPR experiments coupled to the modelling of the system in silico. Overall, the optimized protocols for both the peptide and the protein enabled the effective application of the new spin probes in PDS-EPR experiments. The thesis highlighted both the beneficts and drawbacks associated with the spin labels utilized. A future direction will be to further evaluate the performance of these spin labels in cell-mimetic environments, with the long-term goal of employing them to investigate protein–protein interactions in-cell.