Spectroscopie IR et spectrométrie de mobilité ionique appliquées aux structures de systèmes chargés isolés d'intérêt pharmaceutique

Structural properties and noncovalent interactions within biologically relevant molecular systems are crucial for their activity. They are especially important for the specific binding of drugs on receptors in living organisms. Besides, studying molecules in the gas phase allows measuring their intrinsic properties because of the absence of solvent effects. Precise information can then be extracted from the comparison between experimental data and state-of-the-art quantum chemistry calculations. Our research group aims to describe the structural changes occurring within peptides, oligonucleotides and pharmaceutically relevant complexes: we compare results from simulations and two complementary experimental techniques, IRMPD spectroscopy and ion mobility spectrometry. In this PhD thesis, I have tested for the first time the performances of a QM/MM method for the simulation of the IR spectra of isolated biomolecular systems. It allowed me to simulate the IR spectrum of amyloid β-protein strands containing more than 250 atoms. We found that the solvent in which these strands are diluted has a big influence on their gas-phase secondary structure: in the case of polar solvents, globular ones dominate, whereas a weak dielectric constant tends to favor helical structures. The structural changes upon receptor binding on vancomycin, a glycopeptide antibiotic, have also been studied. The receptor binding site in the protonated complexes is not the pocket which can be seen in X-ray scattering data, but the specific interactions responsible for the high binding constant of the complex in solution remains intact in the deprotonated species.Les caractéristiques structurales et les interactions intra et/ou intermoléculaires non-covalentes jouent un rôle primordial dans l'activité des molécules d'intérêt biologique, notamment lors de la reconnaissance moléculaire entre un médicament et son récepteur. Par ailleurs, on peut connaître par les études en phase gazeuse (sans les effets du solvant) les propriétés intrinsèques des systèmes moléculaires. Des calculs de chimie quantique élaborés peuvent ainsi être comparés aux résultats des expériences pour en tirer des informations précises. L'équipe AMIBES tente ainsi de décrire les structures de peptides, d'oligonucléotides ou de complexes d'intérêt pharmaceutiques. Pour cela, nous prenons appui sur deux techniques expérimentales complémentaires, la spectroscopie IRMPD et la spectrométrie de mobilité ionique, dont les résultats sont comparés avec des simulations. Dans ce travail de thèse, j'ai d'abord testé les prédictions d'une méthode QM/MM en ce qui concerne le calcul des spectres IR d'espèces d'intérêt biologique isolées. J'ai ensuite utilisé cette méthode pour simuler les spectres IR de brins de la protéine amyloïde β en phase gazeuse. Il ressort de ces études que leur structure secondaire varie selon l'état de protonation et le solvant dans lequel ils sont dissous: un solvant polaire favorise les structures globulaires, alors qu'une constante diélectrique plus basse permet de conserver une structure en hélice α. Je me suis également intéressé aux changements de structure induits par la complexation d'un antibiotique, la vancomycine, avec un modèle de son récepteur. En mode positif, le site de fixation de celui-ci est différent de celui révélé par la cristallographie, mais les interactions spécifiques responsables de la grande constante d'affinité en solution sont conservées dans le complexe déprotoné

Proton irradiation of DNA nucleosides in the gas phase

International audienceThe four DNA nucleosides guanosine, adenosine, cytidine and thymidine have been produced in the gas phase by a laser thermal desorption source, and irradiated by a beam of protons with 5 keV kinetic energy. The molecular ions as well as energetic neutrals formed have been analyzed by mass spectrometry in order to shed light on the ionization and fragmentation processes triggered by proton collision. A range of 8-20 eV has been estimated for the binding energy of the electron captured by the proton. Glycosidic bond cleavage between the base and sugar has been observed with a high probability for all nucleosides, resulting in predominantly intact base ions for guanosine, adenosine, and cytidine but not for thymidine where intact sugar ions are dominant. This behavior is influenced by the ionization energies of the nucleobases (G < A < C < T), which seems to determine the localization of the charge following the initial ionization. This charge transfer process can also be inferred from the production of protonated base ions, which have a similar dependence on the base ionization potential. Other dissociation pathways have also been identified, including further fragmentation of the base and sugar moieties for thymidine and guanosine, respectively, and partial breakup of the sugar ring without glycosidic bond cleavage mainly for adenosine and cytidine. These results show that charge localization following ionization by proton irradiation is important in determining dissociation pathways of isolated nucleosides, which could in turn influence direct radiation damage in DNA

Single-photon absorption of isolated collagen mimetic peptides and triple-helix models in the VUV-X energy range

Cartilage and tendons owe their special mechanical properties to the fibrous collagen structure. These strong fibrils are aggregates of a sub-unit consisting of three collagen proteins wound around each other in a triple helix. Even though collagen is the most abundant protein in the human body, the response of this protein complex to ionizing radiation has never been studied. In this work, we probe the direct effects of VUV and soft X-ray photons on isolated models of the collagen triple helix, by coupling a tandem mass spectrometer to a synchrotron beamline. Single-photon absorption is found to induce electronic excitation, ionization and conversion into internal energy leading to inter- and intra-molecular fragmentation, mainly due to Gly-Pro peptide bond cleavages. Our results indicate that increasing the photon energy from 14 to 22 eV reduces fragmentation. We explain this surprising behavior by a smooth transition from excitation to ionization occurring with increasing photon energy. Moreover, our data support the assumption of a stabilization of the triple helix models by proline hydroxylation via intra-complex stereoelectronic effects, instead of the influence of solvent

Mass Spectral Signatures of Complex Post-Translational Modifications in Proteins: A Proof-of-Principle Based on X-ray Irradiated Vancomycin

Characterizing post-translational modifications (PTM) of proteins is of key relevance for the understanding of many biological processes, as these covalent modifications strongly influence or even determine protein function. Among the different analytical techniques available, mass spectrometry is attracting growing attention because recent instrumental and computational improvements have led to a massive rise of the number of PTM sites that can be identified and quantified. However, multiple PTM occurring at adjacent amino acid residues can lead to complex and dense chemical patterns that are a challenge to characterize. By means of X-ray synchrotron radiation coupled to mass spectrometry, and through the test-case of the glycopeptide antibiotic vancomycin, we show that such a pattern has a unique and robust signature in terms of photon energy and molecular environment. This highlights the potential of this technique in proteomics and its value as a tool to understand the biological roles of PTM

Stabilities of nanohydrated thymine radical cations: insights from multiphoton ionization experiments and ab initio calculations

Multi-photon ionization experiments have been carried out on thymine-water clusters in the gas phase. Metastable H2O loss from T+(H2O)n was observed at n ≥ 3 only. Ab initio quantum-chemical calculations of a large range of optimized T+(H2O)n conformers have been performed up to n = 4, enabling binding energies of water to be derived. These decrease smoothly with n, consistent with the general trend of increasing metastable H2O loss in the experimental data. The lowest-energy conformers of T+(H2O)3 and T+(H2O)4 feature intermolecular bonding via charge-dipole interactions, in contrast with the purely hydrogen-bonded neutrals. We found no evidence for a closed hydration shell at n = 4, also contrasting with studies of neutral clusters

Fragmentation processes of ionized 5-fluorouracil in the gas phase and within clusters

We have measured mass spectra for positive ions produced from neutral 5-fluorouracil by electron impact at energies from 0 to 100 eV. Fragment ion appearance energies of this (radio-)chemotherapy agent have been determined for the first time and we have identified several new fragment ions of low abundance. The main fragmentations are similar to uracil, involving HNCO loss and subsequent HCN loss, CO loss, or FCCO loss. The features adjacent to these prominent peaks in the mass spectra are attributed to tautomerization preceding the fragmentation and/or the loss of one or two additional hydrogen atoms. A few fragmentions are distinct for 5-fluorouracil compared to uracil, most notably the production of the reactive moiety CF+. Finally, multiphoton ionization mass spectra are compared for 5-fluorouracil from a laser thermal desorption source and from a supersonic expansion source. The detection of a new fragment ion at 114 u in the supersonic expansion experiments provides the first evidence for a clustering effect on the radiation response of 5-fluorouracil. By analogy with previous experiments and calculations on protonated uracil, this is assigned to NH3 loss from protonated 5-fluorouracil