Computational biology and protein engineering for design and development of novel photosynthesis-based biomediators in biosensor technology

 

TITLE

Computational biology and protein engineering for design and development of novel photosynthesis-based biomediators in biosensor technology

STAFF

Maria Teresa Giardi
Giuseppina Rea
Francesca Vergari
Amina Antonacci*
Sandro Pastorelli*
Maya Lambreva*
Davide Raffi*
Arianna Tibuzzi*
Viviana Scognamiglio*
Gianni Pezzotti*
Ittalo Pezzotti*
Daniela Giannini*
Juan Bernardo Cano*
[*] Personale non strutturato

KEYWORDS

random and site directed mutagenesis, chlamydomonas, D1 protein, plastoquinones, herbicide affinity and resistance

COLLABORATIONS

 

DESCRIPTION

Photosynthetic systems are supramolecular arrangements of proteins and pigments that are able to harvest light energy and funnel it towards building up biomass and release oxygen from water. The hierarchical organization of these pigment-proteins complex is at the basis of their unique efficiency. Fundamental and applied research have made it possible to integrate biological photosystems or their functional sub-structures into artificial assemblies in order to get them to carry out their tasks in a controlled environment for specific applications. Some herbicides, such as atrazine, a potent and persistent environmental contaminant causing severe hormonal damages (endocrine disruptor chemical) can bind to photosystems and modify their fluorescence properties, inhibiting the photo-induced electron flow in a specific way. These effects can be transduced in an output signal in biosensors devices.
Currently available structural and functional information on photosynthetic proteins allows the design and development of engineered photosynthetic microorganisms with improved sensitivity and stability features to be used as bio-recognition elements for the detection of environmental contaminants.
The green alga Chlamydomonas reinhardtii and the purple bacterium Rhodobacter sphaeroides were selected for our purposes. The aim is the modification of the photosynthetic reaction centre (RC) proteins as they have a crucial role in electron tunnelling-mediated charge separation and transmembrane electric field generation, principally through (plasto)quinones reduction, release and migrations.
In silico studies including molecular modeling and dynamic simulations are used to predict mutations which can improve specificity, sensitivity and binding affinity for herbicides using atrazine as model compound. Promising chlamydomonas mutants were produced by chloroplast transformation through particle gun bombardments. In addition, as bacterial RC are more stable compared to eukaryotic orthologs, the feasibility of constructing hybrid or chimeric RC is being studied through comparative protein analysis, molecular modeling and analysis of the structural compatibility of the predicted forms. Finally, as opposite to rational protein design, a directed evolution strategy was undertaken to obtain a pool of random mutated D1 strains to be selected for the desired requirement of sensitivity or resistance to herbicides.

1) Barber J. (2006) Photosystem II: an enzyme of global significance. Biochemical Society transactions; 34(5):619-31.
2) Rea G, Polticelli F, Antonacci A, Scognamiglio V, Katiyar P, Kulkarni SA, Johanningmeier U, Giardi MT, Structure-based design of novel Chlamydomonas reinhardtii D1-D2 photosynthetic proteins for herbicide monitoring. Protein Sci. 2009 Oct;18(10):2139-51.
3) Giardi MT, Scognamiglio V, Rea G, Rodio G, Antonacci A, Lambreva M, Pezzotti G, Johanningmeier U., Optical biosensors for environmental monitoring based on computational and biotechnological tools for engineering the photosynthetic D1 protein of Chlamydomonas reinhardtii. Biosens Bioelectron. 2009 Oct 15;25(2):294-300. Epub 2009 Jul 17.
4) V. Scognamiglio, D. Raffi, A. Antonacci, M. Lambreva, G. Rea, A. Tibuzzi, G. Pezzotti, U. Johanningmeier, M.T. Giardi, "Chlamydomonas reinhardtii genetic variants as probes for fluorescence multi-biomediator sensing system in pollutants detection", Analytical and Bioanalytical Chemistry, 2009 394(4), 1081-1087.
5) A. Mallardi, M. Giustini, F. Lopez, M. Dezi, G. Venturoli, G. Palazzo, Functionality of photosynthetic reaction centers in polyelectrolyte multilayers: toward an herbicide biosensor J Phys Chem B 111:3304-14.
6) Johanningmeier U, Heiss S. Plant Mol Biol. 1993 Apr;22(1):91-9. Construction of a Chlamydomonas reinhardtii mutant with an intronless psbA gene.

Computational biology and protein engineering for design and development of novel photosynthesis-based biomediators in biosensor technology  - Img

 

CONTACTS


Giuseppina Rea
Email: giuseppina.reaATic.cnr.it
Tel.: +390690672631

 

Last Updated (Tuesday, 21 December 2010 16:04)