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Theses

First principles and proceedings of action-FRET : probing the molecular conformation of gas phase proteins and macro-ions

Abstract : In this thesis, I discuss the application and development of mass spectrometry (MS) - LASER coupled techniques for the characterization and measurement of trapped biomolecules in the gas phase. In broad terms, this thesis demonstrates the potential and perspectives of action-FRET a novel structural biology tool amenable to the gas phase. The fundamentals rely on a well attested resonance quantic process known as Förster resonance energy transfer (FRET). As of yet it has been a widely utilized method to scrutinize molecular structure in solution. The mo-tivation has been to transpose this occurrence to the instrumental settings of a mass spectrom-eter, its gas confinement and, in doing so, overcome the earlier limitations of the technique and stride into the theoretical and experimental study of well determined systems as well as those whose structure were presently undetermined - all without the influence of the environment of a solvated medium. The first chapter of this thesis offers a general overview on peptides and proteins plus how they can be studied. Subsequent chapters include how the work carried out herein ads towards their study, moving the technique towards a gold standard of native mass spectrometry (native MS). In the second chapter, a treatment of the synthetic steps and preparations is given detailing the mechanistics of the reactions at play and above all outlining the experimental procedures and providing any information on any observations made. The third chapter describes, and is devoted to an introduction of the instrumental setup outlaid as it stands giving an account on the LASER, optical pieces and the mass spectrometer employed throughout the course of this thesis, effectively setting the premises of thought and understand-ing for subsequent chapters and methodology. Chapter four presents energy transfer, in particular to the Foster Resonance Energy Transfer and furthermore outlines the developed technique central to the mass spectrometry method to look at donor-acceptor chromophores espoused to biomolecular systems and their photofrag-ments — what is nicknamed as action-FRET. Chapter five reviews and discusses the study of a macromolecule: Ubiquitin, by action-FRET. The first gas phase experiment on the protein to have ever been realised. The information and content gathered from this adapted experiment is compared to work found elsewhere giving an appraisal on the potential of action-FRET and providing an idea to what future insights the technique could bring. In chapter six the reader is introduced to the project of establishing action-FRET in the negative mode of the mass spectrometer’s ionization source as opposed to its positive mode. Suitable pairs of donor-acceptor chromophores to validate the energy transfer under a negative regime were explored. These where profiled and characterized before being adapted to a biomolecular system. The results provide a different flavor of complimentary structural and conformation information, the first of its kind for negative mode action-FRET. The seventh and final chapter is devoted to future developments. The conclusive work tends to grant a further understanding of neurodegenerative diseases that afflict our societies. Chiefly that of the likes of Alzheimer’s: it’s the mechanism of action pertinent to other neurodegenera-tive pathologies; Parkinson’s, Huntington’s but also prion diseases or amyloid neuropathy. In doing so a contribution is presented on a way to trace a strategy in how to tackle and treat such diseases
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Geoffrey Knight. First principles and proceedings of action-FRET : probing the molecular conformation of gas phase proteins and macro-ions. Theoretical and/or physical chemistry. Université de Lyon, 2017. English. ⟨NNT : 2017LYSE1232⟩. ⟨tel-01767996⟩

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