Dye decolorizing peroxidases
We are interested in development of improved peroxidase based biosensors for detection of hydrogen peroxide. The optimization of the devices is achieved by sensitive and simultaneous monitoring of the structure of the immobilized enzymes and electrocatalysis under working conditions.
Among peroxidases, the novel dye decolorizing peroxidases (DyPs) represent particularly interesting candidates for the job, due to their stability, versatility, relatively easy genetic manipulation and capacity to couple reduction of hydrogen peroxide with efficient oxidation of structurally different molecules, including numerous industrially relevant and environmentally hazardous dyes. We have recently demonstrated that immobilized DyPs can be used for development of 3rd generation biosensor of hydrogen peroxide, which outperforms currently existing commercially available devices based on HRP. In parallel we explore DyP-based devices for biotransformation of environmentally harmful dyes, such as azo and antharquinone dyes.
We explore DyPs from different organisms, which have distinct substrate specificities, catalytic efficiencies, pH optima and redox potentials (which define the range of substrates that they can oxidize), and therefore offer a variety of options for improvement and fine tuning of the DyP based biotechnological devices. The selectivity, sensitivity, stability and substrate inhibition properties can be further optimized by employing variants engineered towards specific objectives. Collaboration with Ligia Martins, ITQB and and Marco Fraaije, RUG, the Netherlands.
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Virus detection by SE(R)RS
We are interested in Raman based detection of biomolecules, which takes advantage of the molecular specificity of the technique and the enhancement of the signal intensity of the bioanalyte by plasmonic substrate. We are developing improved procedures for adeno associated virus (AAV) production in insect cells, based on SER detection along different stages of AAV development. In parallel, we are designing SER substrates: bare and functionalized nanoparticles (NPs), which allow for indirect and direct detection of AAV, respectively. The former is based on the characteristic spectral fingerprinting of AAV capsid proteins obtained upon adsorption onto plasmonic NPs, and it´s appropriate for later stages of AAV development. The indirect detection relies on the signal of a specific Raman label attached to NPs, which simultaneously carry antibodies for specific binding of the AAV at various stages of AAV production. SERS detection of AAV, which is used for gene therapy, will ensure process optimization and improved yields. Collaboration with Inês Isidro, IBET and Chris Maycock, ITQB.
Structure
RR fingerprint of novel Fe-S cluster in HdrB, heterodisufide reductase
We employ RR to learn about structural intricacies of exotic non-cubane 4Fe–4S cluster present in HdrB subunit of HdrABC heterodisufide reductase. The complex reduces CoM-S-S-CoB, generated together with CH4 in the terminal step of methanogenesis back to thiolated coenzymes; its physiological substrate in not known in non-methanogens. RR spectra, obtained upon excitation into S → Fe CT electronic transition band, which are sensitive to Fe–S cluster type, geometry and nature of the ligands are used to reveal the properties of this non-cubane 4Fe–4S cluster which is unique in nature. We aim to assign the bridging (Fe–S)b and terminal (Fe–S)t vibrational modes that involve inorganic and cysteinyl sulfur ligands of the cluster and use them as internal probes for monitoring interactions that involve the cluster, such as the hypothetical substrate biding to HdrB. Collaboration with Inês Pereira, ITQB.
Function
We employ advanced vibrational spectro-electrochemistry to probe biophysical properties of DNA repair enzyme Endonuclease III (EndoIII). This enzyme is a DNA glycosylase and it is crucial for removal of oxidation damaged bases in DNA. It possesses [4Fe–4S] cluster of still unknown role; both, structural / regulatory and redox state linked roles in substrate recognitions have been suggested, both of which were based either on spectroscopic or on electrochemical evidence only. We use SERR and SEIRA spectro-electrochemistry to probe the cluster in EndoIII, which is electronically coupled to metal electrodes modified with different bifunctional alkanethiol-based SAMs, including normal/damaged DNA terminated SAMs that mimic the substrate.
We have observed that the cluster of the immobilized EndoIII is prone to reduction, which is not necessarily DNA-mediated. The same strategy is used to study the role and the properties of the cluster in the mammalian analogue of EndoIII, hNTH1, which is the central enzyme for repair of ROS damage in humans. Collaboration with Elin Moe, ITQB.
SERR fingerprint of catalytic intermediates
We use SERR to identify and characterize catalytic intermediates of immobilized enzymes based on the respective vibrational-spectroscopic fingerprint. We actually provided the first report of a catalytic intermediate formed in the reaction between an immobilized peroxidase (DyP) and hydrogen peroxide, obtained by SERR spectroscopy. This demonstrates that SERR can be regarded as an important alternative for RR spectroscopic studies of the catalytic cycle of peroxidases when fluorescence impedes solution measurements. SERR moreover requires only small (sub-micromolar) amounts of protein, due to high sensitivity which results from both the plasmonic and resonance enhancements of the signal, it is applicable to the EPR-silent Compound II and does not require cryogenic temperatures. As a drawback, SERR spectroscopic identification of intermediates is restricted to species with relatively long lifetimes.
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Raman label free chemical imaging of cells
We take advantage of Raman label-free imaging, which provides simultaneous spectral and spatial information about all Raman active chemical species in the sample (i.e. it does not require external labeling of molecules) to visualize yeast cells and mutants with impaired cell wall. The images are directly derived from Raman fingerprint spectra of the molecular constituents of the sample, e.g. lipids, proteins, nucleic acids, chromophores, etc. We have employed 3- color excitation to image simultaneously the spatial distribution of polysaccharides characteristic for cell wall, lipids and proteins and specifically heme proteins (using RR enhancement). The sample is screened through point-by-point acquisition of Raman spectra and the image is reconstructed from the spatial distribution of selected Raman band(s). In this manner identification of the specific chemical species can be achieved and their distribution/localization visualized inform of 2D maps. Collaboration with Catarina Pimentel, ITQB and Daniel Murgida UBA, Argentina.
