A set of biophysical methodologies for studying protein structure, kinetics, folding, protein-protein or protein-ligand interactions is available. The group has several anaerobic chambers equipped with stopped-flow apparatus and steady-state kinetics (diode-array), techniques that are also available outside the anaerobic chamber (along with a nanodrop). The labs are equipped with spectrometers and fluorimeters are also available, and there is the knowledge to use microcalorimetry and analytical ultracentrifugation under active collaborations.
Protein-protein interactions are also studied at computational level using an in house-program BiGGER, freely available to any user.
Bioelectrochemistry / Biofilms
Several of the proteins/enzymes under study in the lab are metalloproteins/metalloenzymes and thus there not only the redox potential of its metal centers but also the activity can be studied using bioelectrochemical techniques (such as cyclic voltammetry, differential pulse voltammetry).
Biofilm formation of Desulfovibrio species under different growth conditions to mimic environmental conditions are also available.
Recombinant Protein Production (Expression & Purification)
The aim is to design strategies to attain high level expression of soluble proteins and membrane proteins that can be used in the projects being developed, especially the ones involving Biomolecular NMR and X-ray crystallography. We have a vast collection of vectors (with different promoters and using the LIC methodology or restriction enzyme based cloning), from which the proteins can be produced with several tags (solubility, ease purification). Several strains, mainly derived from E.coli are also available. Specifically, we clone the gene of interest into expression vectors, rapidly screen for optimal combinations of vectors, strains and conditions and produce the protein in E. coli up to 10 L scale (from 37ºC to low temperature growth).
A multiple fermentation system is also available, as well as, fermentation of organisms like Desulfovibrio species and other anaerobic bacteria, Paracoccus pantotrophus and Marinobacter hydrocarbonolcasticus, and P. pastoris.
Homologous cloning of Desulfovibrio sp. proteins are also available in order to produce these proteins either homologously or heterologously in strictly anaerobic bacteria.
Proteins are then purified using standard chromatography techniques and quality control is assessed, as well as protein identity, purity, stability and folding, using MALDI TOF-TOF, 1H NMR, gel filtration chromatography and SDS-PAGE.
57Fe Mössbauer spectroscopy is the technique of choice to study iron-containing proteins/enzymes as well as relevant inorganic compounds. A Low-field (600 G) Mössbauer spectrometer with variable temperature (4.5 - 300 K) is available. This system comprises a proven Janis cryostat SHI-850-5 cryostat system with a base temperature of less than 5 K. Such closed cycle system ensures even cooling and offers fast sample exchange at a reduced operation cost. The system is complemented by a SEE Co. VT104 velocity transducer, a W302 resonant gamma-ray spectrometer with integrated servo controller and dual multi-channel scalers, and a W202 Gamma-ray spectrometer with integrated high voltage power supply, charge sensitive pre-amp, shaping amp, and dual single-channel analyzers.
Electrochemical techniques applied to biological systems can be performed from classic stationary bulk voltammetric and amperometric techniques towards fast pulse techniques, hydrodynamic regimes through rotating disc and ring-disk apparatus (RDE and RRDE), spectroelectrochemistry, piezoelectric techniques using electrochemical quartz crystal microbalance (EQCM) and small scale electrochemistry using microeletrodes (also suitable for highly resistive media). Chemical and physical immobilization techniques of target biological molecules on different matrix are employed, such as using self-assembled monolayers (SAMs) and conducting polymers (CPs) or ionic liquids based hydrogel materials, to perform studies in diffusionless regimes and direct electron transfer conditions. Disposable screen-printed electrodes and correspondent interfaces are available for specific low-cost practical applications (e.g. biosensors). Also, electrochemical studies in strict anaerobic conditions are accessible using a MBraun LABmaster glovebox workstation. These electrochemical integrated techniques constitute an important setup that allows determination of mechanistic, kinetic and electrocatalytic properties of proteins and other biological systems.
Current equipment includes:
- Autolab PGSTAT128N Potenciostat/Galvanostat with BA (bipotenciostat), EQCM (electrochemical quartz crystal microbalance) and ECD (extreme low current) modules;
- CHI 440C Potentiostat/Galvanostat Time-Resolved Electrochemical Quartz Crystal Microbalance (EQCM);
- Pine Instruments RRDE system with exchangeable disks.
Rapid Kinetics and Spectroscopy Facility
Rapid Freeze-Quench (RFQ) techniques enabled the identification and characterization of several reaction intermediates. Correlation with UV/Visible and Fluorescence stopped-flow kinetic data is possible as well as parallel sample preparation (for several different spectroscopic uses such as Mössbauer, EPR, EXAFS and Resonance Raman spectroscopies). Both aerobic and anaerobic sample preparation is possible. For sample preparation a BioLogic Science Instruments SFM-300 setup capable of single and double mixing experiments, equipped with high-density Berger-Ball mixers and coupled to high-speed diode-array detectors (MOS-DA, 0,8 nm/ms) or single wavelength absorbance, fluorescence (excitation/emission) or 90° light scattering (MOS-250) spectrometers. Additionally, rapid-freeze quench can be perform using proven in-house developed mixing and cryogenic devices. For all cases, aerobic or anaerobic experiments can be performed using a MBraun LABmaster glovebox workstation.
The Glycan Microarray core provides the use of state-of-the-art microarray technology for high-throughput screening of glycan-recognition systems and for glycan-ligand discovery. The microarray Lab is set-up with conditions for microarray binding and analysis. There is collaboration with the Carbohydrate Microarray Facility at Imperial College London (http://www3.imperial.ac.uk/glycosciences) for microarray printing and glycan microarray development and for access to a unique glycan resource. Fluorescence microarray imaging is provided for the scanning, image acquisition and processing of microarray slides (for example of glycan, protein and DNA microarrays). User support and training can be provided on microarray binding experiments (glycan and protein microarrays).