Internal dynamics of heme-based sensor proteins studied using advanced time-resolved optical spectroscopy

Abstract : Heme-proteins are involved in a large range of biological functions, including respiration, oxygen transport and xenobiotic detoxification. Importantly, bacterial heme-based sensor proteins exploit the ligation properties of heme to sense environmental gases. This thesis focuses on internal dynamics studies of the 6-coordinate heme-based gas sensor proteins CooA, from Rhodospirillum rubrum and DNR from Pseudomonas aeruginosa that are involved in adaptation of the metabolism of the organism to their environment. CooA and DNR, belonging to the important family of catabolite gene activator proteins, are transcription factors that bind DNA upon gas activation, thus enabling transcription of specific genes. Both sensor proteins are thought to undergo a large and delocalized conformational change upon binding of the physiological ligand to the heme (CO for CooA and NO for DNR). Here advanced optical spectroscopy techniques are used to investigate the mechanism and molecular pathway of activation/desactivation in this class of proteins. DNA-protein interactions were studied with steady-state and femtosecond ultrafast time resolved fluorescence techniques, using labeled DNA substrates. Physiological ligand-sensitive DNA binding in the nanomolar affinity range was deduced from anisotropy experiments. Quenching of the fluorescence label by energy transfer to the native heme in the protein moiety of the complex was observed, and the rate of this process, reflecting the heme-substrate distance, was determined directly from the measured fluorescence decays. This observation opens the perspective of mapping out the global protein conformational changes using time-resolved FRET techniques. The primary processes in heme-based sensor switching mechanisms concern ligand binding and ligand dissociation from the heme. Femtosecond transient absorption experiments were performed in order to study the ligand dynamics in CooA and DNR in the vicinity of the heme. In DNR, upon photodissociation of NO, particularly fast and efficient geminate recombination was observed. This strongly strengthens the hypothesis that NO-sensors act as ligand traps. Also, the energetic barriers involved in CO migration have been determined in both sensor proteins by temperature dependence studies. All 6-coordinate heme-based sensor proteins that function via the exchange of an internal residue and the gas molecule as a heme ligand, display barrierless recombination and a thermally activated CO-escape out of the heme pocket. By contrast, the barrier for the CO-escape appears smaller or absent for 5-coordinate systems, as has been found for the mycobacterial heme-sensor DosT. These findings point to a general mechanism, where similar protein motions are required for both, ligand exchange and ligand escape. For reasons of comparison, the energetic barriers have also been studied in ligand binding variants of the electron transfer protein cytochrome c. Here, a more complex mechanism of multiple barriers in the ligand escape pathway was deduced. This feature is proposed to reflect the rather rigid nature of this non-sensor protein, which contains a 6-coordinate heme and is devoid of ligand entry pathways in the native state. Finally, the primary processes occurring in the wild type and mutant heme domains of the recently discovered oxygen sensor YddV from Escherichia coli were investigated. In particular, an important role in the ligand dynamics was elucidated for the distal tyrosine residue. This residue hydrogen bonds to heme-bound O2 and NO molecules and was found to have a remarkably discriminating effect: after respective dissociation from the heme, it strongly promotes O2 rebinding, but favors NO escape from the heme pocket.
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Laura Lobato. Internal dynamics of heme-based sensor proteins studied using advanced time-resolved optical spectroscopy. Biological Physics [physics.bio-ph]. Ecole Polytechnique X, 2013. English. ⟨pastel-00866894⟩

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