GREEN PHOTOCATALYTIC PRODUCTION OF FUEL-LIKE HYDROCARBONS USING FATTY ACID PHOTODECARBOXYLASE

A PhD position in bio-photocatalysis at the I2BC/Paris-Saclay University & BIAM/Aix Marseille University in France is available for a dynamic and enthusiastic candidate. Funded by the French National Research Agency (ANR), the interdisciplinary project focuses on developing innovative biocatalysts for photo-production of hydrocarbon fuels of non-fossil origin.
The PhD project, entitled 'Green photocatalytic production of fuel-like hydrocarbons using Fatty Acid Photodecarboxylase,' aims to optimize the natural Fatty Acid Photodecarboxylase (FAP) enzyme for higher photostability and more efficient binding and photodecarboxylation of short fatty acid substrates, yielding liquid hydrocarbons.
Key responsibilities include preparing wild-type and mutant FAP proteins, conducting screening and tests of photoenzymatic activity, participating in enzyme evolution, characterizing new mutants using spectroscopic techniques, contributing to data analysis, writing scientific publications, and presenting results at conferences.
Requirements for applicants include a Master’s or engineering degree in relevant fields, practical laboratory skills, ability to work independently, proficiency in English and/or French, and readiness to relocate from Provence to Ile-de-France during the PhD.
The position offers the opportunity to contribute to sustainable energy research while gaining expertise in molecular biology, biochemistry, and optical spectroscopy. The PhD will be conducted within a collaborative ANR project involving four teams with diverse expertise and skills. The ideal candidate should integrate seamlessly into a multidisciplinary environment.
Supervisor will be Pavel Müller (pavel.muller@i2bc.paris-saclay.fr) and co-supervisor will be Damien Sorigué (damien.sorigue@cea.fr).Workplaces include the Institute of Biosciences and Biotechnologies of Aix-Marseille and CEA Saclay/Institut de Biologie Intégrative dela Cellule.
The contract begins between October 2024 and February 2025 and lasts for three years, with a monthly salary of 2400 € (brut). Applicants passionate about addressing energy challenges are encouraged to apply!

LIGHT-NMR: A POWERFUL TOOL FOR UNDERSTANDING AND IMPROVING THE PROPERTIES OF PHOTOSWITCHABLE FLUORESCENT PROTEINS

The recruited student will investigate the photophysical mechanisms of reversibly photoswitchable fluorescent proteins (RSFPs) employing solution NMR spectroscopy coupled with in-situ illumination and variable oxygen pressure. RSFPs are capable to switch between a fluorescent on-state and a nonfluorescent off-state under specific light illumination, and have fostered many types of imaging applications including super-resolution methods. Multidimensional NMR spectroscopy is a particularly powerful atomic resolution technique providing detailed information on conformational protein dynamics, as well as the local chemistry (protonation states, H-bonding interactions, …) involved in the photophysics of the chomophore within the protein scaffold. In the proposed PhD project, we intend to further improve our NMR in-situ illumination device by adding capabilities such as additional wavelengths of emitting light sources, fluorescence detection, and oxygen pressure control. This will allow to directly correlate the conformational dynamics of various states with their photophysical properties, as well as the effect of oxygen on triplet state formation and photobleaching. We will apply this NMR methodology to several green and red model RSFPs, as well as FAST systems. The goal will be to contribute fundamental knowledge of these fluorescent markers and to design improved variants.

Understanding reversibly switchable red fluorescent proteins

Fluorescence imaging is essential to unlocking the secrets of life and has benefited greatly from the discovery of fluorescent proteins (FPs). Reversibly switchable fluorescent proteins (RSFPs, https://doi.org/10.1002/iub.1023) are capable of switching from a fluorescent "on-state" to a non-fluorescent "off-state" upon specific illumination, and have fostered many imaging applications, including some super-resolution methods. However, RSFPs are still imperfect: for example, their brightness is limited, their switching kinetics is dependent on environmental conditions, their resistance to irreversible photobleaching is insufficient. In particular, whereas green RSFPs are performing relatively well, red RSFPs have been lagging behind. The switching performances of green and red RSFPs are linked with their intrinsic or light-activated protein-dynamics properties and can be studied by combining structural biology approaches, such as kinetic X-ray crystallography, with optical spectroscopy and fluorescence imaging (doi: 10.1038/s41592-019-0462-3). In the proposed PhD project, those techniques will be used to better understand red RSFPs and facilitate their rational engineering towards brighter and more photo-resistant variants. The recruited student will work in close collaboration with another PhD student to be hired, who will approach the same questions by employing NMR.

Candidates should have a strong interest to work at the interface between physics, chemistry and biology. Knowledge of advanced fluorescence microscopy and/or X-ray crystallography is required. Preliminary experience in image analysis, biochemistry, cell biology and/or molecular biology will be appreciated.

Deciphering the photoactivation mechanism of the orange carotenoid protein by time-resolved serial femtosecond crystallography

The Orange Carotenoid Protein (OCP) is a photoactive protein involved in photoprotection of Cyanobacteria. Recently, a photoactivation mechanism was proposed for OCP, in which the initially excited S2 state yields multiple ps-lived excited states (ICT, S1, S*), but structural evidence is missing. Importantly, only one of these leads to the biologically active OCP-red state on the second timescale. We propose to conduct an ultra-fast time-resolved crystallography experiment at an X-ray free electron laser (XFEL) on OCP to accurately characterize the structures of the short-lived photo-intermediates on the femtosecond to millisecond timescale. In parallel, we will use the new time-resolved crystallography beamline of the ESRF to determine the structures of the later intermediates forming on the millisecond to second timescale. By allowing to visualize protein conformational changes upon photoactivation from the photochemical (hundreds of femtoseconds) to the photobiological timescales (seconds), our integrated structural biology project will allow to (i) test mechanistic hypotheses, (ii) pave the way to a detailed understanding the photophysical properties of OCP, and (iii) open avenues towards its exploitation as an optogenetic component or a regulator of light-uptake in biomimetic photosynthetic systems.

Exploring chemotaxis in magnetotactic bacteria

Magnetotactic bacteria (MTB) are a diverse group of bacteria characterized by their capacity to biomineralize magnetite nanoparticles called magnetosomes. The latter allow MTB to passively align along magnetic field lines. This feature makes MTB of great interest to develop magnetic-guided microrobots used for medical applications such as targeted drug delivery. To make the latter efficient, it is not only essential to understand MTB magnetic behavior but also how MTB react to diverse chemical stimuli.
The aim of this internship is to broaden our understanding of chemotaxis in MTB. Several MTB species can be grown in the lab and will be investigated during the thesis. Typically, tethered cells and motility assays involving the use of microfluidics, microscopy and image analysis approaches will be developed to investigate, on a single-cell and population level, the chemotaxis responses of the strains to different chemical stimuli. These responses will be studied with bacteria grown in different growth conditions and in the absence or presence of a magnetic field using a custom-made magnetic microscope. Altogether, the data generated will give first insight into how MTB give an integrated response to chemical and magnetic stimuli and will therefore open new routes for the further development of targeted drug delivery.

Role of excited state vibrational modes of chlorophylls in photosynthesis

Photosynthesis empowers the entire biosphere and is arguably the most important biological process on earth. The quantum efficiency of excitation energy transfer (EET) in photosynthetic light-harvesting complexes can reach almost unity. This high efficiency is even more puzzling if we take into account that the high excitation energy transfer through hundreds of pigments in a disordered energetic landscape cannot be explained with the current models. Currently, there are two main hypotheses to explain the ultrafast energy transfer: “quantumness” and “vibrational assistance” (see context section). To validate these hypotheses, it is necessary to characterize the electronic and vibrational properties of the excited states of the cofactors involved in the ETT process. We have designed an interdisciplinary project, in which the student will be trained in biochemical techniques for protein purification and ultrafast photophysics techniques to analisys of the excitation energy transfer. The use of different detergents for purifying light-harvesting complexes leads to disturbed systems with differences in the excitation energy pathways. The differences between light-harvesting complexes from each purification will be characterized by resonance Raman and time-resolved fluorescence. Then, we will use ultrafast techniques such as fs-transient absorption, 2-D electronic spectroscopy (2DES) and femtosecond stimulated Raman Spectroscopy (FSRS). This data will be employed to develop new models for photosynthetic energy transfer. The student will be involved in the analysis and discussions for the theoretical modeling of this process but learning modeling techniques is out of the scope of the thesis.

Molecular dynamics and disorder in the viral replication machinery of SARS CoV 2

The nucleoprotein (N) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for genome replication, encapsidating the viral genome and regulating gene transcription. The central disordered domain is essential to the function of this highly dynamic protein, containing a number of important mutations that are responsible for enhanced viral fitness, and comprising a region that is hyperphosphorylated during the viral cycle. NMR spectroscopy is the tool of choice for studying the conformational behaviour of intrinsically disordered proteins, an abundant class of proteins that are functional in their disordered form. They represent 40% of the proteome and are too dynamic to be studied by crystallography or electron microscopy. The host lab has developed a large number of unique NMR-based tools to help understand the function of this class of proteins at atomic resolution. We will use NMR, paramagnetic NMR, small angle scattering, single molecule FRET and electron microscopy, in combination with molecular dynamics simulation, to describe the interactions of N with viral partner proteins and viral RNA. Post-translational modification, in particular phosphorylation, is thought to play an important functional role, that remains poorly understood, we will investigate the impact of phosphorylation on conformational dynamics and relate this to modifications in function. The results will be correlated with light end electron microscopy, carried out in collaboration.

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