Étude théorique de la structure et de la dynamique de l’hémoglobine tronquée N de Mycobacterium tuberculosis

Authors: Daigle, Richard
Advisor: Guertin, Michel; Lague, Patrick
Abstract: The truncated hemoglobin N from Mycobacterium tuberculosis (TrHbN) protects aerobic respiration of Mycobacterium bovis BCG cells from the inhibitory effect of •NO. In addition, TrHbN catalyses the very rapid dioxygenation of •NO into the innocuous NO3- ions (NOD reaction: TrHbN-Fe2+–O2 + •NO  TrHbN-Fe3+ + NO3-) with a bimolecular rate constant of 745 µM-1s-1 at 20°C. This high efficiency was largely associated to the presence of two hydrophobic tunnels visible in the 3D-structure of TrHbN. In this context, the main goal of this thesis is to study TrHbN structure and dynamics with bioinformatics tools, especially molecular dynamics simulations. Several molecular dynamics simulations of TrHbN under its deoxy, oxygenated and cyanomet forms were conducted. These simulations allowed to study dynamics of TrHbN backbone, that of the active site and especially, that of the tunnels. As a main result, our simulations revealed that tunnels are highly dynamics, more complex than anticipated from the 3D-structure and that they are hosted in a very rigid two-on-two fold. Other simulations, this time including free •NO molecules, highlighted the use of these tunnels to reach the buried active site. These simulations allowed to propose many hypotheses regarding the preferred routes and to propose diffusions mechanisms from the solvent to the active site. In order to validate our hypotheses and to push further our knowledge on TrHbN, other simulations coupled with some experimental approaches were performed. First, simulations on TrHbN under its cyanomet form coupled with a detailed NMR confirmed that the backbone of the protein is ridig. In addition, this work revealed i) the presence of µs-ms motions localized along B and G helices and ii) that the pre-A region is not structured in contrast to the alpha helice seen in the crystal structure. Finally, other simulations along with kinetics characterizations of obstructed tunnel mutants were conducted. As a main result, the latter work revealed that TrHbN core is quite plastic, allowing substrate diffusion despite the presence blocking mutations. Our comprehension on TrHbN is still incomplete, however the work presented in this thesis constitutes a considerable progress. Moreover, the work presented herein contributes to other fields of research, especially on globins, to tunnel-containing proteins and finally, to gaseous substrates diffusion inside proteins.
Document Type: Thèse de doctorat
Issue Date: 2012
Open Access Date: 18 April 2018
Permalink: http://hdl.handle.net/20.500.11794/23673
Grantor: Université Laval
Collection:Thèses et mémoires

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