In-situ monitoring of the regulation of photosynthesis in microalgae using in-cell real-time NMR.
Keyword: in-cell NMR, photosynthesis regulation, redox transition, Calvin Benson Basham cycle, structural NMR, diffusion ordered NMR spectroscopy.
The assimilation of CO2 by photosynthetic organisms thanks to the Calvin-Benson-Bassham (CBB) cycle is a key process of the carbon cycle in the biosphere and a prerequisite for the synthesis of biomolecules by these organisms.
New lessons emerge from this ancient process and recent genetic studies that are re-assessing the classic redox regulation. The applicant will monitor in-situ and in real-time the relative population of the reduced and oxidised states of known redox mediators including NADP/NADPH or GSSG/GSH on model micro-algae upon light-to-dark and dark-to-light transitions using NMR. Also, the redox transition of a well-known regulatory protein, CP12[2,3,4], will be investigated directly in the chloroplast. Different means will be employed to introduce NMR visible 15N-labelled CP12 in NMR invisible chloroplast. Besides, the effect of molecular crowding will be monitored using diffusion-ordered NMR spectroscopy. Despite the recent development and renewed interest for in-cell structural biology and real-time in-cell metabolomic, this approach has not yet been applied to photosynthetic cells using NMR.
The success of this approach will rely on the close collaboration between a group specialised in biochemistry: micro-algae culture, protein production and purification, enzymatic regulation[9,10] (BIP), and a group specialised in NMR methods development for the identification and characterisation of biomolecules in complex solution (BioSciences)[11,12]. The applicant will have an easy access to NMR spectrometers: 600MHz liquid, 600MHz with a cryo-probe, 400MHz HR-MAS, and to algal-culture and biochemistry laboratories.
1. Cejudo, F. J. et al. Chloroplast Redox Regulatory Mechanisms… Front. Plant Sci. 10, (2019) doi: 10.3389/fpls.2019.00380.
2. Gontero, B. & Maberly, S. C. CP12: jack of all trades… Biochem. Soc. Trans. 40, 995–999 (2012) doi: 10.1042/BST20120097.
3. Launay, H. et al. Intrinsically disordered regulatory protein CP12 in its reduced state. BBRC. 477, 20–26 (2016) doi: 10.1016/j.bbrc.2016.06.014.
4. Launay, H. et al. Encounter between conditionally disordered CP12 and GAPDH. J. Mol. Biol. 430, 1218–1234 (2018) doi: 10.1016/j.jmb.2018.02.020.
5. Luchinat, E. and Banci, L. In-cell NMR: a topical review. IUCrJ 4, 108-118 (2017) doi: 10.1107/S2052252516020625.
6. Weiss, M. Crowding, Diffusion, and Biochemical Reactions. in Int. Rev. of Cell and Mol. Biol. vol. 307 (2014). doi: 10.1016/B978-0-12-800046-5.00011-4.
7. Nishida, N. et al. In situ structural biology using in-cell NMR. Biochim. Biophys. Acta BBA 1864, 129364 (2020) doi: 10.1016/j.bbagen.2019.05.007.
8. Edison, A. S. et al. NMR: Unique Strengths That Enhance Modern Metabolomics. Anal. Chem. 93, 478–499 (2021) doi: 10.1021/acs.analchem.0c04414.
9. Launay, H. et al. Orchestration of algal metabolism by protein disorder. Arch. Biochem. Biophys. 672, 108070 (2019) doi: 10.1016/j.abb.2019.108070.
10. Launay, H. et al. Regulation of carbon metabolism: a perspective from diatoms. Front. Plant Sci. 11, 1033 (2020) doi: 10.3389/fpls.2020.01033.
11. Manjunatha Reddy, G. N. et al. Combined max.-quantum and DOSY 3D experiments. Magn. Reson. Chem. 55, 492–497 (2017) doi: 10.1002/mrc.4465.
12. Yemloul, M. et al. Characterization of illicit drug samples by NMR. Anal. Bioanal. Chem. 410, 5237–5244 (2018) doi: 10.1007/s00216-018-1175-7.
Benefit: The awarded doctoral student will be granted with a 3-year fellowship (salary of €1421 net / month) plus €4,000 to attend international conferences or to follow training related to their research or professional project. In addition, through the Plinius doctoral programme, they will benefit from personalised support to train in a wide range of cutting-edge technologies and prepare their professional project in an international and multidisciplinary environment
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