This part of the CMS uses various biophysical methods to characterize the interactions of DNA, small molecules as well as new biological entities with protein targets. The most important techniques available in CMS are the following: circular dichroism, surface plasmon resonance, microscale thermophoresis (including label-free thermophoresis), differential scanning calorimetry, isothermal calorimetry, tyrosine and tryptophan fluorescence measurements, dynamic light scattering, precision UV/visible spectrophotometry. We also use these versatile techniques to investigate protein and DNA structure and stability in different conditions.
We design and optimize techniques to perform measurements, and analyse and interpret the results for the user. The results of our measurements are important for determination of suitable crystallisation conditions of a given protein or nucleic acid and for their optimization, for determination of molecular interactions between biomolecules, both macromolecules and small molecule ligands, and for studies of structure-function relationships including determination of enzyme kinetics.
Crystallization of proteins and nucleic acids, Diffraction techniques
This part of the Centre of Molecular Structure in BIOCEV provides dedicated, state-of-the-art equipment for crystallisation (including evaluation of the crystallization experiments) and testing of X-ray diffraction potential, X-ray diffraction data collection and analysis for 3D structure determination. This provides a unique platform both for experienced macromolecular crystallographers, and for biochemists and molecular biologists to embark on structural biology research. The facility possesses the equipment and know-how to guide investigators through all the steps in crystal structure determination, from initial crystallisation of a target to the 3D structure.
We collaborate with teams at the “Extreme light infrastructure” (ELI Beamlines) European large scale facility in Dolní Břežany. This collaboration will be focused on the use of the unique ELI X-ray sources driven by ultrashort laser pulses hitting plasmas for the investigation of biomolecular crystals, using e.g. serial femtosecond crystallography. This work should allow data collection from nanometer- to micrometer-sized objects (including crystals) in a ‘diffraction-before-destruction’ approach. This is viewed as essential for the structural characterization of biomolecules that are difficult to crystallize.
Structural mass spectrometry
The Structural mass spectrometry core facility within CMS is equipped with high-end instrumentation including the 15T-solariX XR FT-ICR mass spectrometer. The core facility provides different types of analyses such as protein identification from gels or from solutions, determination of precise molecular weight of proteins and their sequencing by fragmentation techniques (collision induced dissociation, electron transfer/capture dissociation). Further services provided by CMS including characterization of posttranslation modifications (glycosylation, phosphorylation, disulphide bonds…), structural mass spectrometry (hydrogen-deuterium exchange, chemical cross-linking, covalent labelling,..) for monitoring of protein-protein interaction, antibody-antigen interaction and protein folding.
The core facility for structural mass spectrometry cooperates with several academic laboratories, universities and commercial sphere to design and develop new strategies for analyte detection. We are improving hardware, workflows and processing features for hydrogen-deuterium exchange experiments, chemical cross-linking and epitope mapping.