Terthiophene as a novel Spin Label for the EPR investigation of Peptide-Protein interactions

Electron paramagnetic resonance (EPR) spectroscopy is widely employed in structural biology for the determination of intra- and intermolecular distances in biomolecular complexes. Such measurements rely on the introduction of paramagnetic spin labels through site-directed spin labeling (SDSL), often combined with site-directed mutagenesis (SDM) to selectively control the labeling position within the protein sequence. Among pulsed EPR techniques, double electron–electron resonance (DEER) allows for distance measurements between pairs of stable radicals, whereas Laser-Induced Magnetic Dipole spectroscopy (Laser-IMD) enables distance measurements between a stable radical and a photo excitable triplet-state label. This thesis reports the study of terthiophene (TT) as a novel photo excitable spin label for pulsed EPR spectroscopy. The acetyl-imidazole-functionalized TT was selected because it can be linked to cysteine residues through a one-atom covalent linker, which was expected to limit conformational flexibility and produce narrow distance distributions. Upon excitation with 355 nm laser pulses, TT populates the triplet state. At the same wavelength, it also forms the radical anion, raising the question whether TT is more suitable for Laser-IMD or DEER measurements. This study was carried out on the CaM-M13C system, which mimics the interaction between calmodulin and the myosin light chain kinase (MLCK) that occurs during smooth muscle contraction. Previously prepared single-cysteine CaM mutants (positions 6, 100 and 114) and M13C peptide were employed as model systems for TT labeling and EPR characterization. CaM mutants were already available with the SPIRO nitroxide spin-label. TT labeling tests were conducted both on M13C and unlabeled CaM mutants. The labeled peptide (M13TT) was purified by reverse phase chromatography (RPC). UV-Vis spectroscopy was used both to characterize the RPC fractions and to determine the labeling efficiency. Echo detected EPR was employed to characterize the triplet-state spectra of free TT, M13TT, CaM6S-M13TT, CaM100S-M13TT and CaM114S-M13TT. DEER and Laser-IMD measurements were performed on the peptide-protein complexes to determine inter-spin distance distribution between the SPIRO nitroxide on CaM and the TT on the peptide. The acetyl-imidazole-functionalized TT derivative exhibited limited aqueous solubility, slow labeling kinetics, and marked pH sensitivity, requiring mixed DMSO/water conditions for efficient conjugation. While these conditions enabled peptide labeling, they proved incompatible with protein substrates, representing a significant limitation for broader biomolecular applications of TT-based spin labels. Photoinduced radical anion formation significantly complicated Laser-IMD measurements, limiting the overall suitability of TT for this technique. The radical generation may originate from charge-transfer processes involving a neighboring tryptophan residue within the peptide sequence. Furthermore, the measured distance distributions were broader than initially expected, suggesting that the conformational flexibility of TT may still be significant. These findings indicate that shorter thiophene-based labels, such as dithiophene derivatives, may represent more suitable candidates for future pulsed EPR distance measurements.