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Content archived on 2024-06-16

Intraprotein signalling in heme-based sensors: the oxygen sensor FixL

Objective

This project aims at elucidating the mechanisms of intra-protein signalling and gaseous ligand discrimination in a heme-based gas sensor. As a model system, I propose to study the bacterial oxygen sensor FixL.

This protein contains a sensory PAS domain, which regulates the activity of a histidine kinase domain via the binding/release of oxygen to the heme. Structures of the O2-bound and unliganded isolated heme domain are available and provide models for the start- and endpoints of the intra-domain signalling process.

However, information on the intermediate pathway is lacking. In addition, the transmission to, and the influence of, the enzymatic domain, have not been taken into account. To address these issues, this proposal describes an approach integrating molecular biology, biochemical, ultrafast spectroscopic, and computational techniques.

Based on structural information and simulations, substitutions of residues potentially involved in the signalling pathway are designed. The initial set of mutations concerns residues in the heme pocket and in the 'FG loop', which is located at the presumed interface between the constituents of the functional homodimer.

The corresponding proteins will be over-expressed, as isolated sensor domain, as well as full-length proteins. Strategies are devised to address the challenging task of isolating stable and sufficient full-length proteins for spectroscopic analysis.

To generate intermediates in the signalling pathway, the unique possibility to photocleave the heme-ligand bond will be exploited. Using ultrafast absorption and vibrational (Raman and infrared) spectroscopy, this allows the characterization of the electronic and structural properties of transient states and their dynamics, with femtosecond time resolution.

Along with molecular dynamics modelling and with the perspective of ultrafast time resolved crystallography, pathways will be mapped and general features for intraprotein signal propagation proposed.

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Keywords

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Topic(s)

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Call for proposal

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FP6-2005-MOBILITY-7
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Funding Scheme

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IIF - Marie Curie actions-Incoming International Fellowships

Coordinator

ECOLE POLYTECHNIQUE
EU contribution
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Total cost

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