Combining Orientation Selective Light-Induced Pulsed Dipolar Spectroscopy with Molecular Dynamics

This thesis is developed on Light-Induced Pulsed Dipolar Spectroscopy (LiPDS) methods that aim to measure structural information by recording the dipolar interaction between pairs of labels attached to the biological structure containing unpaired electrons, which can be interpreted to provide a distribution of inter-label distances. LiPDS represents an attractive tool to study biological systems containing spin active moieties, where at least one spin centre is a photoexcited triplet state. The use of light induced techniques relies on the characteristic spin polarisation mechanism that can facilitate and increase in sensitivity due to the non-Boltzmann population of the triplet, when this spin is used for detection. Moreover, the Zero Field Splitting (ZFS) interaction in triplet states gives rise to a broad and distinctive shape of the triplet spectrum, enabling to selectively excite only a small portion of it. As a result, only limited molecular orientations with respect to the applied magnetic field are excited allowing to gain insights on spin labels orientations as well. Previous results on two model peptide systems have been performed by the EPR group in collaboration with the University of Oxford and Manchester. This project accounts for the complete structure using Molecular Dynamic (MD) simulations in order to better understand the peptide structure and corroborate our dipolar data. Simulating the LiPDS traces based on conformations extracted from the MD trajectories yields a library of time traces, which are then fitted to the experimental data. Furthermore, analysis of orientation selection effects in the LiPDS data allows information on the relative orientations of the two spin centres with respect one another to be determined alongside the inter-spin distances. The first model system examined is a bis-labelled peptide containing a free base tetraphenylporphyrin and a permanent spin centre in the form of a conformationally rigid TOAC nitroxide radical, the second peptide is made of two free base tetraphenylporphyrin chromophores, both systems are separated by alpha-helical bridges chosen as highly rigid structures with a defined conformational preference. This research project was carried out both at the Department of Chemistry in Padova and at the National Research Facility for EPR of the University of Manchester.