The biological function of macromolecules such as protein, DNA, and RNA depends on their folding and on the relative movements of domains with dimensions of a few nanometers. This length scale can be accessed by distance measurements between paramagnetic spin centers employing Electron Paramagnetic Resonance (EPR) pulsed dipolar spectroscopy (PDS) techniques.
In order to use this spectroscopic methods, the biomolecule has to contain either stable or transient paramagnetic centers, which can be metal ions or clusters, amino acid radicals, or organic cofactor radicals. If the biomolecule is diamagnetic, it can be spin-labeled with nitroxides or a diamagnetic metal may be substituted with a paramagnetic one. Nitroxides are the most employed spin probes in PDS, especially for structural studies in proteins were they can be attached to specific sites following a protocol of mutagenesis and site-directed spin labeling (SDSL) on cysteine residues. However, the introduction of a spin label can modify the structure around the labeling site and in some regions it may even interfere with the correct folding. For this reason, exploiting endogenous probes i.e. paramagnetic centers which are naturally present in the protein, represents an primary task in PDS. Indeed, PSD has been entatively performed on various classes of proteins naturally containing metal base-prosthetic groups such as and low-spin ferric heme centers, iron-sulfur cluster, Mn clusters for which mainly the ¢mS Æ §1/2 transition can be selected. Utilizing endogenous probes for EPR detection only causes minimal functional perturbation to the macromolecules. Another advantage is that they are firmly anchored in the protein and, therefore, are not fraught with the problem of flexible linkers as the commonly used spin labels.
In recent years photoexcited triplet state of porphyrin has been introduced in the selection of spin labels for PDS applications. In their ground state, these chromophores are diamagnetic and thus EPR-silent, but, upon laser photoexcitation, their triplet state can be populated via intersystem crossing from the lowest excited singlet state, generating in this way the paramagnetic center. The inter-system crossing mechanism makes the population of the triplet sublevels different from the Boltzmann distribution, significantly enhancing the intensity of their EPR signals. Moreover porphyrin-derivative groups are suitable to be exploited as endogenous probes because are present in numerous systems such as heme-protein and photosynthetic proteins. The orthogonal labeling method, based on the use of spectroscopically nonidentical labels which can be addressed selectively in the EPR experiment, is attracting increasing interest in the spectroscopic community. Triplet states work very efficiently as orthogonal labels, adding to the spectroscopic selectivity the advantage of behaving as photoinduced spin probes. This feature allows to perform PDS in the presence of light excitation to measure intramolecular triplet-nitroxide distances or in the absence of light excitation revealing intermolecular nitroxide-nitroxide interactions.
While the feasibility of the PDS experiment had already been demonstrated for a photoexcited porphyrin moiety interacting with a nitroxide radical [Di Valentin, M.; Albertini, M.; Zurlo, E.; Gobbo, M. and Carbonera, D. J. Am. Chem. Soc., 2014, 136, 6582 -6585], the accuracy of the new labeling approach for distance determination, and the theoretical frame describing the behavior of polarized high-spin systems for application in dipolar techniques were still laking. In this thesis work a complete spectroscopic and theoretical characterization of photoexcited triplet state probes has been carried out. The reliability and versatility of such spin labels has been tested employing different dipolar pulse schemes and exploiting diverse chromophores for the photogeneration of the paramagnetic center, both in peptide-based model systems and in protein belonging to different classes. The study has been completed with an exhaustive theoretical description.
The reliability and the accuracy of the new labeling approach has been demonstrated by measuring the dipolar traces of a spectroscopic ruler composed by a-helix peptides of increasing length, labeled with a porphyrin chromophore, that upon photoexcitation gives the EPR-active species, and a nitroxide artificial amino acid. The good correlation between the distances obtained by experimental PDS data and calculation, is used to asses the accuracy of the new labeling approach.
In PDS, there are different pulse sequences that exploits diverse mechanisms to induce the dipolar oscillations. Such pulse schemes have been tested on the triplet state in order to classify the performances of the various PDS techniques with the novel labeling approach. The availability of different light-induced PDS sequences increases the versatility of triplet state probes allowing to select case by case the pulse scheme that guarantees the best signal-to-noise ratio.
The new labeling approach has been extended to two paradigmatic proteins: the light-harvesting complex Peridinin-Chlorophyll a-Protein fromAmphidinium Cartarae and the human Neuroglobin belonging to the globins family where the endogenous prosthetic groups have been exploited to photo-generated the triplet state. In the photosynthetic protein the dipolar trace arising from the interaction between the triplet state of one of the carotenoids in the photoactive site and a nitroxide, introduced via site-directed spin labeling, have been measured. This allowed to identify the pigment involved in the photoprotective mechanism and demonstrated that, not only porphyrin-derivatives, but also other chromophores can be used as spin probes. In human neuroglobin the Zn-substitution of the heme has allowed to populate the triplet state of the Zn protoporphyrin IX and successfully measure the dipolar trace proving the applicability of this labeling procedure on the class of hemeproteins.
The full characterization of triplet state probes has been completed with a theoretical study based on the density matrix formalism. First, the analytic formula describing the modulation of the dipolar trace for a simplified radical-triplet state system has been obtained, highlighting a time an analogous dependence to the radical-radical case. Subsequently, a program for time-domain numerical calculation of radical-triplet state dipolar traces has been implemented and employed for a quantitative characterization of triplet state probes in PDS.