13C NMR of a single molecule magnet: analysis of pseudocontact shifts and residual dipolar couplings

Paramagnetic triple decker complexes of lanthanides are promising Single Molecule Magnets (SMMs), with many potential uses. Some of them show preferable relaxation behavior, which enables the recording of well resolved NMR spectra. These axially symmetric complexes are also strongly magnetically anisotropic, and this property can be described with the axial component of the magnetic susceptibility tensor, χa. For triple decker complexes with phthalocyanine based ligands, the Fermi˗contact contribution is small. Hence, together with the axial symmetry, the experimental chemical shifts in 1H and 13C NMR spectra can be modeled easily by considering pseudocontact and orbital shifts alone. This results in the determination of the χa value, which is also responsible for molecular alignment and consequently for the observation of residual dipolar couplings (RDCs). A detailed analysis of the experimental 1H-13C and 1H-1H couplings revealed that contributions from RDCs (positive and negative) and from dynamic frequency shifts (negative for all observed couplings) have to be considered. Whilst the pseudocontact shifts depend on the average positions of 1H and 13C nuclei relative to the lanthanide ions, the RDCs are related to the mobility of nuclei they correspond to. This phenomenon allows for the measurement of the internal mobility of the various groups in the SMMs.

Development of a fluorescent-based single liposome assay for studying calcium transporter LMCA1

The morphological and functional unit of all the living organisms is the cell. The transmembrane proteins, localized in the plasma membrane of cells, play a key role in the survival of the cells themselves. These proteins perform a variety of different tasks, for example the control of the homeostasis. In order to control the homeostasis, these proteins have to regulate the concentration of chemical elements, like ions, inside and outside the cell. These regulations are fundamental for the survival of the cell and to understand them we need to understand how transmembrane proteins work. Two of the most important categories of transmembrane proteins are ion channels and transporter proteins. The ion channels have been depth studied at the single molecule level since late 1970s with the development of patch-clamp technique. It is not possible to apply this technique to study the transporter proteins so a new technique is under development in order to investigate the behavior of transporter proteins at the single molecule level. This thesis describes the development of a nanoscale single liposome assay for functional studies of transporter proteins based on quantitative fluorescence microscopy in a highly-parallel manner and in real time. The transporter of interest is the prokaryotic transporter Listeria Monocytogenes Ca2+-ATPase1 (LMCA1), a structural analogue of the eukaryotic calcium pumps SERCA and PMCA. This technique will allow the characterization of LMCA1 functionality at the single molecule level. Three systematically characterized fluorescent sensors were tested at the single liposome scale in order to investigate if their properties are suitable to study the function of the transporter of interest. Further studies will be needed in order to characterize the selected calcium sensor and pH sensor both implemented together in single liposomes and in presence of the reconstituted protein LMCA1.

La spettroscopia di forza basata sull'AFM nello studio dello spazio conformazionale e dei processi aggregativi di proteine prioniche

Le malattie neurodegenerative sono caratterizzate da aggregazione proteica, dipendente dalla perdita della usuale struttura fisiologica funzionale delle proteine coinvolte, a favore di conformazioni tossiche (patologiche). Il modello corrente descrive questi cambiamenti conformazionali come eventi rari e ritiene che non esista una sola conformazione patogena, ma che tali possibili conformazioni siano piuttosto eterogenee. La caratterizzazione di queste strutture è, di conseguenza, difficile con le tradizionali tecniche in bulk che permettono di studiare solo la conformazione media e non rendono possibile il riconoscimento delle caratteristiche dei conformeri individuali. Lo sviluppo delle tecniche di singola molecola ha permesso di studiare in modo approfondito le conformazioni possibili. In questo lavoro la spettroscopia di forza di singola molecola basata sull'AFM viene applicata a PrP (proteina responsabile delle encefalopatie spongiformi trasmissibili). Si studiano gli equilibri conformazionali del monomero e quelli di costrutti oligomerici, allo scopo di caratterizzare gli step iniziali dei processi aggregativi. Nel corso di questo lavoro di tesi è stato, in particolare, sviluppato un sistema di analisi dati, al fine di studiare in modo quantitativo le distribuzioni di eventi ottenute. Grazie a tale strumento è stato possibile riconoscere i segnali di unfolding della conformazione nativa del monomero e notare come essa sia presente anche in costrutti oligomerici, ad indicare come questo ripiegamento sia stabile anche in presenza di più monomeri ravvicinati. Si è osservato l'effetto del pH sulla stabilità di tale struttura, notando come pH acidi destabilizzino il ripiegamento nativo. Inoltre si è studiato il ruolo dell'orientazione dei monomeri nella formazione di strutture dimeriche. Monomeri e oligomeri di PrP sono stati descritti come proteine parzialmente strutturate il cui panorama energetico contiene molti minimi locali, dando origine a parecchie conformazioni transienti.

Analysis of the high-resolution ro-vibrational spectrum of DC3N in the far and mid infrared regions

Cyanoacetylene HC3N is a molecule of great astronomical importance and it has been observed in many interstellar environments. Its deuterated form DC3N has been detected in number of sources from external galaxies to Galactic interstellar clouds, star-forming regions and planetary atmospheres. All these detections relied on previous laboratory investigations, which however still lack some essential information concerning its infrared spectrum. In this project, high-resolution ro-vibrational spectra of DC3N have been recorded in two energy regions: 150 – 450 cm-1 and 1800 – 2800 cm-1. In the first window the ν7← GS, 2ν7 ← ν7, ν5 ← ν7, ν5+ν7 ← 2ν7, ν6+ν7 → 2v7, 4ν7 ← 2ν7 bands have been assigned, while in the second region the three stretching fundamental bands ν1, ν2, ν3 have been observed and analysed. The 150 – 450 cm-1 region spectra have been recorded at the AILES beamline at the SOLEIL synchrotron (France), the 1800 – 2800 cm-1 spectra at the Department of Industrial Chemistry “Toso Montanari” in Bologna. In total, 2299 transitions have been assigned. Such experimental transition, together with data previously recorded for DC3N, were included in a least-squares fitting procedure from which several spectroscopic parameters have been determined with high precision and accuracy. They include rotational, vibrational and resonance constants. The spectroscopic data of DC3N have been included in a line catalog for this molecule in order to assist future astronomical observations and data interpretation. A paper which includes this research work has been published (M. Melosso, L. Bizzocchi, A. Adamczyk, E. Cane, P. Caselli, L. Colzid, L. Dorea, B. M. Giulianob, J.-C. Guillemine, M-A. Martin-Drumel, O. Piralif, A. Pietropolli Charmet , D. Prudenzano, V. M. Rivillad, F. Tamassia, Extensive ro-vibrational analysis of deuterated-cyanoacetylene (DC3N) from millimeter wavelengths to the infrared domain, Jour. of Quant. Spectr. and Rad. Tran. 254, 107221, 2020).