How do we study

How do we study

In order to study the function and structure of the different targeted metalloenzymes we use different organisms and several biochemical and spectroscopic techniques.

• Organisms

We study mainly prokaryotic organisms from the Bacteria and Archaea domains. Prokaryotes are very versatile and diverse organisms, thus increasing the diversity of sampling. This is a very important feature because if the organisms under study are from closely related phylogenetic groups, the conclusions of the analyses are strongly biased, i.e., an observation that could be of general relevance may in fact be the reflex of the phylogenetic relationship and be only important for a certain group of organisms. Thus, a good sampling has to ensure representability, including different living conditions and phylogenetic characteristics.

The organisms currently under study in the group are anaerobic and facultative bacterial pathogens, such as E. coli and Clostridiales, namely Clostridium difficile. But we also address other organisms, such as the cyanobacterium, Synechocystis sp . PCC6803 and sulfate-reducing bacteria from the Desulfovibrio genus, and several archaea, such as Ignicoccus hospitalis, Nanoarcheum equitans and Archaeoglobus fulgidus.

• Methodologies

In our Laboratory we cover the whole process of studying an electron transfer metalloenzyme, i.e., we start from growing the organisms, mainly using heterologous overexpression but also wild type strains, site directed mutants and chimeric constructs, isolate and purify the enzymes/proteins, through different chromatographic steps, and pursue with the biochemical, kinetic  and spectroscopic characterisation. For this, several in-Lab and ITQB facilities and a large number of equipment are available, such as glove boxes, UV-Visible and Electron Paramagnetic Resonance spectrometers, stopped-flow instrument, including a rapid-freeze quench apparatus, oxygen and nitric oxide specific electrodes. We resort to other techniques, such as Resonance Raman and NMR, through National or International collaborations.

Here are listed the common procedures performed in our lab:

Protein purification

. Cell growth and disruption
. Ultra-centrifugation (preparation of membrane and soluble extracts)
. Chromatographic steps
. Purity assessment

Characterization

. Cofactor analysis – extraction, identification and quantification
. Quaternary structure analysis
. Redox properties followed by potentiometry with visible and/or EPR spectroscopy
. Steady-state kinetics followed by UV-visible spectroscopy

. Fast kinetics by stopped flow

Spectroscopy

. UV-visible
. Electron Paramagnetic Resonance

Polarography & Amperometry – Clark-type Electrodes

. oxygen polarography
. nitric oxide amperometry