Fluorescence spectroscopie (application to characterize the interaction between biological macromolecules and the helicase enzymatic activity studies)

Ce mémoire présente la mise en place des techniques de fluorescence en milieu biologique pour mieux comprendre le principe des interactions entre macromolécules biologiques ainsi que leurs mécanismes catalytiques. Dans ce contexte, nous avons appliqué trois méthodes d'analyse de la fluorescence. Une première technique d'analyse dynamique, la spectroscopie de cross-corrélation de fluorescence, basée sur des mesures en micro-volume et sur une faible concentration moléculaire, a essentiellement été appliquée à étudier l'activité hélicase en mesurant la corrélation croisée des fluctuations de fluorescence entre deux molécules d'ADN complémentaires. En particulier, l'activité d'hélicase de la protéine E.Coli RecQ et l'activité d'annealing pour la protéine RecQ5 humaine ont été étudiées. Les performances de la technique FCCS pour appréhender l'étude des activités enzymatiques de la famille des hélicases RecQ ont été validées par nos résultats. Une deuxième technique d'analyse dynamique qui nous permet de mesurer l'anisotropie de fluorescence statique a été appliquée pour comprendre l'effet de deux voies de résistances au Raltégravir (N155H et G140S/Q148H) sur la réplication virale du virus VIH-1 et sur les propriétés enzymatiques de l'intégrase du VIH (INs). Les applications de cette technique nous ont permis de démontrer la mutation Q148H joue un rôle prépondérant pour la résistance au Raltegravir, tandis que la mutation G140S augmente la fitness virale dans le contexte de la double mutation G140S/Q148H. Une troisième technique d'analyse dynamique, le déclin de la photo luminosité résolue en temps de la fluorescence, a été appliquée à caractériser les propriétés de fluorescence de nanocristaux de CdTe couronnés par MPA. Cette approche a confirmé les avantages des nanocristaux et leurs applications pour le marquage de fluorescence. En conclusion, ce mémoire inclus les principes et les applications variées des techniques de fluorescence qui sont engagées à intégrer la domaine différente (lumière, photoniques et biologie) dans la même domaine biophotonique.This thesis presents the applications of fluorescence detection approaches in understanding the fundamental principles of the light activation of biomolecules, bioassemblies, and their catalytic mechanisms. In this context, three frequently used fluorescent methods have been discussed. The first technique, the fluorescence cross-correlation spectroscopy, based on measurements in micro-volumes with weak molecular concentration, has been essentially applied to monitor the crosscorrelation of the fluorescence fluctuations of the two complementary DNA strands. In particular, the helicase activity of E.Coli RecQ enzyme and the strand annealing activity of human RecQ5 helicase have been monitored. Results proved that the FCCS approach is particularly well-suited for monitoring the RecQ helicase enzymatic activity. The second technique, the fluorescence steady-state anisotropy measurements, has been adopted to analyse impact of the two main Raltegravir resistance pathways (N155H and G140S/Q148H) on HIV viral replication and the catalytic properties of recombinant integrase (INs). Results demonstrated the Q148H mutation is responsible for predominant resistance to Raltegravir whereas the G140S mutation increases viral fitness in the context of double mutant G140S/Q148H. The third technique, the time-resolved photoluminescence decay measurement, has been conducted to characterise the fluorescent properties of MPA capped CdTe quantum dots (QDs). Results confirmed the advantages of QDs and their promising applications in fluorescent labelling. In conclusion, this thesis encompasses the fundamentals and various applications involving the integration of light, photonics and biology into biophotonics.CACHAN-ENS (940162301) / SudocSudocFranceF

Disulfide-Linked Integrase Oligomers Involving C280 Residues Are Formed In Vitro and In Vivo but Are Not Essential for Human Immunodeficiency Virus Replication

The human immunodeficiency virus type 1 integrase (IN) forms an oligomer that integrates both ends of the viral DNA. The nature of the active oligomer is unclear. Recombinant IN obtained under reducing conditions is always in the form of noncovalent oligomers. However, disulfide-linked oligomers of IN were recently observed within viral particles. We show that IN produced from a baculovirus expression system can form disulfide-linked oligomers. We investigated which residues are responsible for the disulfide bridges and the relationship between the ability to form covalent dimers and IN activity. Only the mutation of residue C280 was sufficient to prevent the formation of intermolecular disulfide bridges in oligomers of recombinant IN. IN activity was studied under and versus nonreducing conditions: the formation of disulfide bridges was not required for the in vitro activities of the enzyme. Moreover, the covalent dimer does not dissociate into individual protomers on disulfide bridge reduction. Instead, IN undergoes a spontaneous multimerization process that yields a homogenous noncovalent tetramer. The C280S mutation also completely abolished the formation of disulfide bonds in the context of the viral particle. Finally, the replication of the mutant virus was investigated in replicating and arrested cells. The infectivity of the virus was not affected by the C280S IN mutation in either dividing or nondividing cells. The disulfide-linked form of the IN oligomers observed in the viral particles is thus not required for viral replication

HIV-1 Integrase Catalytic Core: Molecular Dynamics and Simulated Fluorescence Decays

ABSTRACT Two molecular dynamics simulations have been carried out on the HIV-1 integrase catalytic core starting from fully determined crystal structures. During the first one, performed in the absence of divalent cation (6-ns long), the catalytic core took on two main conformations. The conformational transition occurs at approximately 3.4 ns. In contrast, during the second one, in the presence of Mg 2 � (4-ns long), there were no such changes. The molecular dynamics simulations were used to compute the fluorescence intensity decays emitted by the four tryptophan residues considered as the only chromophores. The decay was computed by following, frame by frame, the amount of chromophores that remained excited at a certain time after light absorption. The simulation took into account the quenching through electron transfer to the peptide bond and the fluorescence resonance energy transfer between the chromophores. The fit to the experimental intensity decays obtained at 5°C and at 30°C is very good. The fluorescence anisotropy decays were also simulated. Interestingly, the fit to the experimental anisotropy decay was excellent at 5°C and rather poor at 30°C. Various hypotheses such as dimerization and abnormal increase of uncorrelated internal motions are discussed