Evaluation of different spin labels for the study of protein-peptide interaction in cellular extract using Electron Paramagnetic Resonance spectroscopy techniques

Calmodulin (CaM) is a protein involved in cellular signaling and undergoes specific conformational changes in the presence of Ca 2+ ions and target peptides like M13. 1 In this study, some CaM mutants were labeled with two different nitroxide probes.Then, M13 was synthesized via SPPS and an aliquot was labeled with a porphyrin.Using EPR spectroscopy, CaM's M13 TPP CaM's target peptide, M13, has a sequence rich in hydrophobic amino acids, allowing good interaction with CaM, which literally wraps the peptide creating a hydrophobic tunnel around it. 3,4M13 (residues 1-19 of the peptide) was obtained through solid-phase peptide synthesis (SPPS), with a 63% final yield and a 92% purity.Porphyrin TPP was added to the N-terminal unprotected amine as TPP-COOH, by a standard coupling reaction. Lock-in Amplifier CaM114-MTSSL in ascorbate CaM114-SPIRO in ascorbate CaM114-MTSSL in cellular extract CaM114-SPIRO in cellular extractEvaluation of different spin labels for the study of protein-peptide interaction in cellular extract using Electron Paramagnetic Resonance spectroscopy techniquesThe next steps include bringing EPR measurements directly into the cell, as well as the possibility of exploring the protein-ligand interaction by means of pulsed EPR measurements coupled to photoexcitation that could provide distance measurements between the protein and the porphyrin-labeled peptide.