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Biotechnology

Rational protein engineering

The research in the Rational Protein Engineering (RPE) group is focused in the design of enzymes with increased activities, new specificities and improved selectivities. Computational biochemistry methods, which include Molecular Docking, Molecular Dynamics and Quantum Mechanics/Molecular Mechanics (QM/MM) simulations are employed using our developed protocols in the study of enzyme binding, catalysis and dynamics and in the design of new variant enzymes with the desired properties.

Keywords

Enzymes

Rational design

Quantum Mechanics/Molecular Mechanics (QM/MM)

Molecular Dynamics (MD)

Research Lines

1 - Development of genetic tools.

We are developing cutting-edge genetic tools for gene editing and control of gene expression. Our models include different endonucleases, ranging from simple Restriction Enzymes (REs) that recognize 8 bps sequences, to the more complex CRISP-Cas systems that are revolutionizing genetic editing. We are also studying bacterial allosteric transcription factors (TFs) in collaboration with the Prather lab (Massachusetts Institute of Technology, MIT). In this case, we are interested in regulating gene expression by changing the effector domain specificity. We are using computational and synthetic biology approaches. Finally, we are studying drugs that can modulate TFs binding, using as a model system, the DNA binding domain of the p53 protein in collaboration with the Molecular Pharmacology and Cancer Therapeutics group (I3S) and molecules that can potentially bind to RNA Polymerase II and prevent the toxic effect of alpha-amanitin in collaboration with the Toxicology group of the Faculty of Pharmacy (Porto University).

2 - Development of enzymes for the biosynthesis of biodegradable polyesters.

We are exploring the free energy surfaces for enzymatic polyester synthesis and hydrolysis. Detailed atomic and electronic characterisation of these profiles allows us to change key residues in the enzyme to increase activity and improve enzyme stability. We applied state-of-the-art Quantum Mechanics/Molecular mechanics (QM/MM) Molecular Dynamics (MD) simulations in the determination of these catalytic mechanisms. New variants are rationally designed using our own protocols. The projects include not only the in silico rational design of enzymes, but also in vitro validation by managing the generation the enzyme variants, their expression and the kinetics analysis.

GROUP LEADER AND PRINCIPAL INVESTIGATOR

Alexandra Carvalho, PhD

ORCID

Ciência ID

Alexandra Carvalho is a researcher at CNC and the leader of the Rational Protein Engineering group. She has a licentiate degree in Biochemistry and a Ph.D. degree in Chemistry with Maria Ramos (Porto University) in collaboration with Matthias Bickelhaupt (Vrije University). She did post-doctoral work at Porto University, Girona University with Marcel Swart and Uppsala University with Lynn Kamerlin. From 2015-2016 she worked for Almac Sciences in the development of new biocatalysts. Her group is focused on enzyme design.

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Funded by ERDF - European Regional Development Fund through COMPETE 2020 and National Funds via FCT - Fundação para a Ciência e Tecnologia, under projects POCI-01-0145-FEDER-007440 e UIDB/04539/2020.