Neurons are among the most highly polarized cells in the human body. This polarization allows the neuron to participate in the transfer of chemical and electrical signals which are crucial to the survival of the organism. As part of polarization, each neuron develops a dendritic arbor and an axon. To ensure the survival of the cell, materials synthesized in the cell body must be trafficked through the axon for delivery throughout ultimately ending at the synaptic termini. The bulk of this cargo transport is microtubule-based fast axonal transport which is molecular motor mediated and tightly regulated though many pathways. Motor based transport is established early in development and maintained for the life of the cell. The kinesin motor protein family plays an integral role in fast axonal transport and the regulation of these motors is essential to proper cargo delivery. Regulation occurs through auto-inhibition, motor interactions with microtubule associated proteins (MAPs) and complex signaling pathways which control the post-translational modification of MAPs, the microtubule track and the motors. The disruption of cargo transport is linked to neurodegeneration and disease state development. Of particular interest in this process is the MAP Tau which has been implicated in a number of neurodegenerative diseases including Alzheimer’s Disease. Tau is expressed at all stages of neural development and has been shown to participate in signaling cascades, modulate microtubule dynamics and preferentially inhibit kinesin-1 motility. Though Tau is involved in these processes, the non-disease state regulation of this MAP and it’s inhibition of kinesin-1 is not well understood. Tau has been shown to bind the microtubule surface in a static-diffusive state equilibrium which differs with isoform and lattice. Previous work demonstrates that the static state is more inhibitory to kinesin-1 than the diffusive state. These different binding behaviors with their different effects on kinesin-1 motility, suggest that cellular regulation of Tau’s static-diffusive binding equilibrium may control inhibition of kinesin-1 and that structural changes may underlie Tau binding to the microtubule surface. Cellular regulation of Tau’s structure and therefore its behavior on the microtubule surface points to a means by which Tau is regulated in the non-disease state. Additionally, this would highlight how early changes lead to disease state development. Using a combination of molecular biology, biochemical techniques and imaging strategies including Total Internal Reflection Fluorescence, single molecule Fluorescence Resonance Energy Transfer (smFRET) and Alternating Laser Excitation, we show that Tau’s static-diffusive state equilibrium is regulated by non-disease state phosphorylation at tyrosine 18. Phospho-mimetics are shifted towards diffusive binding and have decreased affinity for the microtubule surface which in turn reduces inhibition of kinesin-1 motility. These results further demonstrate that Tau undergoes long range structural change while bound to the microtubule surface. We performed smFRET assays and found that Tau binds the microtubule surface in distinct conformations which underlie static and diffusive binding. This work ties the regulation of Tau’s structure and binding behavior to its function and paves the way for our understanding of how cellular regulation acts on multiple levels to fine tune axonal transport.

Les récepteurs métabotropes au glutamate (mGluR) sont des RCPG de classe C. Ils sont exprimés dans le système nerveux central où, suite à l'activation par le glutamate, ils participent à la modulation de la transmission nerveuse. En raison de leur rôle essentiel dans la régulation de l'activité synaptique, ils représentent des cibles potentielles pour le développement de médicaments contre les troubles neurologiques et psychiatriques telles que la schizophrénie, l'épilepsie, l'anxiété et la douleur. Mon projet de recherche de doctorat a porté sur l'étude du mécanisme d'activation du domaine extracellulaire de liaison au ligand du mGluR (ECD), avec un accent particulier sur ce qui différencie au niveau moléculaire un agoniste partiel d'un agoniste total. A cette fin, j'ai utilisé une méthode innovante à l'échelle de la molécule unique appelée Transfert d'Energie par Résonance de Forster, développé pour l'étude de la dynamique conformationnelle des molécules individuelles à l'échelle de la nanoseconde. J'ai réussi à montrer que le dimère d'ECD oscille entre une conformation active et une conformation de repos sur une échelle de temps de ~100μsec et que les ligands influencent les vitesses de transition entre ces états avec des vitesses intermédiaires pour les agonistes partiels. Ces résultats sont validés par l'utilisation de mutants spécifiques et indiquent clairement que le rôle des ligands n'est pas de stabiliser une conformation donnée mais de modifier le comportement dynamique du récepteur. L'ensemble de ces résultats contribuent à une meilleure description du mécanisme d'activation des mGluRs, et ouvrent potentiellement la voie à la compréhension des RCPG en général. / Metabotropic Glutamate Receptors (mGluRs) are class C GPCRs, expressed throughout the central nervous system. They participate in the long term modulation of neural transmission following activation by the excitatory neurotransmitter glutamate. This critical role in the regulation of synaptic activity makes them promising targets in the development of drugs for the treatment of various neurologic and psychiatric disorders such as schizophrenia, epilepsy, anxiety and pain relief. My Ph.D. research project has focused on the study of the activation mechanism of the mGluR extracellular ligand binding Venus-Flytrap domain (VFT), with particular emphasis on the differences between partial and full agonists on a molecular level. To this aim, I have used a state-of-the-art single molecule Förster Resonance Energy Transfer (smFRET) approach, developed for the study of conformational dynamics of single molecules on the nanosecond to millisecond timescale. I have managed to show that the VFT-dimer constantly oscillates between an active and a resting conformation on a ~100µsec timescale. I also discovered that the role of ligands is to influence the transition rate between these boundary states, and that partial agonists display intermediate transition rates. My results, supported by the use of specific mutants, clearly indicate that the role of ligands is not to stabilize a given conformation but to modify the overall dynamic of the receptor, which favors a conformational selection mechanism. Altogether, these results represent a most-valuable contribution to the better understanding of the activation mechanism of mGluRs, and potentially GPCRs in general.

Nucleases are integral to all DNA processing pathways. The exact nature of substrate recognition and enzymatic specificity in structure-specific nucleases that are involved in DNA replication, repair and recombination has been under intensive debate. The nucleases that rely on the contours of their substrates, such as 5’ nucleases, hold a distinctive place in this debate. How this seemingly blind recognition takes place with immense discrimination is a thought-provoking question. Pertinent to this question is the observation that even minor variations in the substrate provoke extreme catalytic variance. Increasing structural evidence from 5’ nucleases and other structure-specific nuclease families suggest a common theme of substrate recognition involving distortion of the substrate to orient it for catalysis and protein ordering to assemble active sites. Using three single-molecule (sm)FRET approaches of temporal resolution from milliseconds to sub-milliseconds, along with various supporting techniques, I decoded a highly sophisticated mechanism that show how DNA bending and protein ordering control the catalytic selectivity in the prototypic system human Flap Endonuclease 1 (FEN1). Our results are consistent with a mutual induced-fit mechanism, with the protein bending the DNA and the DNA inducing a protein-conformational change, as opposed to functional or conformational selection mechanism. Furthermore, we show that FEN1 incision on the cognate substrate occurs with high efficiency and without missed opportunity. However, when FEN1 encounters substrates that vary in their physical attributes to the cognate substrate, cleavage happens after multiple trials During the course of my work on FEN1, I found a novel photophysical phenomena of protein-induced fluorescence quenching (PIFQ) of cyanine dyes, which is the opposite phenomenon of the well-known protein-induced fluorescence enhancement (PIFE). Our observation and characterization of PIFQ led us to further investigate the general mechanism of fluorescence modulation and how the initial fluorescence state of the DNA-dye complex plays a fundamental role in setting up the stage for the subsequent modulation by protein binding. Within this paradigm, we propose that enhancement and quenching of fluorescence upon protein binding are simply two different faces of the same process. Our observations and correlations eliminate the current inconvenient arbitrary nature of fluorescence modulation experimental design.

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Beta-barrel outer membrane proteins (OMPs) present on the outer membrane of Gram-negative bacteria are vital to cell survival. Their biogenesis is a challenging process which is tightly regulated by protein-chaperone interactions at various stages. Upon secretion from the inner membrane, OMPs are solubilized by periplasmic chaperones seventeen kilodalton protein (Skp) and survival factor A (SurA) and maintained in a folding competent state until they reach the outer membrane. As periplasm has an energy deficient environment, thermodynamics plays an important role in fine tuning these chaperone-OMP interactions. Thus, a complete understanding of such associations necessitates an investigation into both structural and thermodynamic aspects of the underlying intercommunication. Yet, they have been difficult to discern because of the conformational heterogeneity of the bound substrates, fast chain dynamics and the aggregation prone nature of OMPs. This demands for use of single molecule spectroscopy techniques, specifically, single molecule Förster resonance energy transfer (smFRET). In this thesis, upon leveraging the conformational and temporal resolution offered by smFRET, an exciting insight is obtained into the mechanistic and functional features of unfolded and Skp/SurA - bound states of two differently sized OMPs: OmpX (8 β-strands) and outer membrane phospholipase A (OmpLA – 12 β-strands). First, it was elucidated that the unfolded states of both the proteins exhibit slow interconversion within their sub-populations. Remarkably, upon complexing with chaperones, irrespective of the chosen OMP, the bound substrates expanded with localised chain reconfiguration on a sub-millisecond timescale. Yet, due to the different interaction mechanisms employed by Skp (encapsulation) and SurA (multivalent binding), their clients were found to be characterised by distinct conformational ensembles. Importantly, the extracted thermodynamic parameters of change in enthalpy and entropy exemplified the mechanistically dissimilar functionalities of the two chaperones. Furthermore, both Skp and SurA were found to be capable of disintegrating aggregated OMPs rather cooperatively, highlighting their multifaceted chaperone activity. This work is of significant fundamental value towards understanding the ubiquitous chaperone-protein interactions and opens up the possibility to design drugs targeting the chaperone-OMP complex itself, one step ahead of the OMP assembly on the outer membrane.

DNA nanotechnology uses the Watson-Crick base-pairing of DNA to self-assemble structures at the nanoscale. DNA nanomachines are active structures that take energy from the system to drive a programmed motion. In this thesis, a new design for a reversible DNA motor and an automatically regenerating track is presented. Ensemble fluorescence measurements observe motors walking along the same 42nm track three times. A second new motor was designed to allow motors on intersecting tracks to block each other, which can be used to perform logical computation. Multiple design approaches are discussed. The chosen approach showed limited success during ensemble fluorescence measurements. The 'burnt bridges' motor originally introduced by Bath et al. 2005 was also sent down tracks placed along the inside of stacked origami tubes that are able to polymerise to micrometre lengths. Preliminary optical microscopy experiments show promise in using such a system for observing micrometre motor movement. Scaffold-based DNA origami is the technique of folding a long single-stranded DNA strand into a specific shape by adding small staple strands that hold it in place. Extended staple strands can be modified to functionalise the origami surface. In this thesis, the threading of staple extensions through a freely-floating origami tile was observed using single-molecule Förster resonance energy transfer (smFRET). Threading was reduced by bracing the bottom of the extension or by using a multilayered origami. smFRET was also used to investigate the process of staple repair, whereby a missing staple is added to a pre-formed origami missing the staple. This was found to be successful when the staple is single-stranded, and imperfect when partially double-stranded. Finally the idea for a new "DNA cage", a dynamic octahedron called the "Holliday Octahedron", is presented. The octahedron is made of eight strands, one running around each face. Mobile Holliday junctions at each face allow the stands to rotate causing a conformational change.

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