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Understanding signaling in photoreceptors involved in the regulation of bacterial photosynthesis genes using serial crystallography and time-resolved spectroscopy.

Project description

Shedding light on photoactivation events in energy-flexible bacteria

Despite all we know about photosynthesis, there are many details of the complex signal transduction pathways that remain to be illuminated. This becomes even more complex in 'facultative' photosynthetic organisms in which photosynthesis is optional – in other words, they can switch energy sources for survival. The expression of photosynthetic genes in the facultatively photosynthetic bacterium Rhodobacter sphaeroides is controlled by both oxygen and light levels. For more than two decades, scientists have been studying two photoreceptor proteins, AppA and CryB, involved in this regulation. However, it is still unclear how changes in these proteins in response to light affect the downstream signalling cascade. The EU-funded DynAppACry project plans to shed light on these light-induced events with the application of high-tech biochemical, computational, spectroscopic and structural methods. The resulting time-resolved molecular 'movie' could help us figure out how to use light to non-invasively control biological processes.

Objective

Photosynthetic organisms need to sense environmental light effectively in order to regulate cellular processes. AppA (Activation of Photopigment and PUC A protein) and CryB (cryptochrome B) are two flavin (FAD) containing photoreceptors found in the facultatively photosynthetic bacterium Rhodobacter sphaeroides and depending on the oxygen levels can regulate the expression of photosynthetic genes by light. Photoexcitation of AppA and CryB is accompanied by changes on their conformation and their affinity to partner binding proteins to initiate signaling transduction processes. The mechanisms by which AppA and CryB regulate downstream signaling events is not known. Thus there is substantial interest in capturing the conformational landscape that lead to the signaling state and in understanding how absorption of light by the flavin is coupled to these conformational changes. The aim of the project is to provide atomic level characterization of the photoactivation mechanism of AppA and structural information on the photocycle of CryB. To achieve our objectives, we will use a combination of biochemical methods, ultrafast transient absorption spectroscopy, QM/MM (hybrid quantum mechanics/molecular mechanisms) calculations and time-resolved structural methods. The latter include TR-serial crystallography (SX) at synchrotrons (SSX) and X-ray free electron lasers (SFX) and TR-solution scattering (small-angle, SAXS and wide-angle X-ray scattering, WAXS). Ultimately, this project will provide a molecular movie of the AppA that features the structural changes occurring upon blue-light illumination and significant information on the photocycle of CryB. The proposed research lies on the implementation of new instrumentation and novel approaches to advance our knowledge on how photoreceptors function in order to engineer novel systems that use light as a tool to achieve noninvasive control of biological processes with high spatiotemporal resolution.

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Coordinator

COMMISSARIAT A L ENERGIE ATOMIQUE ET AUX ENERGIES ALTERNATIVES
Net EU contribution
€ 184 707,84
Address
Rue leblanc 25
75015 Paris 15
France

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Region
Ile-de-France Ile-de-France Paris
Activity type
Research Organisations
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Other funding
€ 0,00