Applications & sample preparation BMA Geneva
1) Why are microcalorimetry & light scattering measurement useful to plant researchers?
The Bio-molecular analysis platform opens the door to technology, perhaps not previously considered within the plant science community. These methods are of special interest to scientists who are interested in characterizing protein complexes and protein in the context of an interaction partner. Micro-calorimetry is specifically important to scientists who are interested in ligand binding. Such molecules can be novel pesticides, plant hormones, and generally compounds that enhance plant growth under stress conditions (e.g. drought, salt, heat or cold, heavy metals). Interaction with other proteins and nucleic acid can be measured as well. Light scattering and micro-calorimetry - the methods offered by the BMA Geneva - are superior for analysing the size of a protein, for protein complex assessment, protein interactions and conformational changes, because they are non invasive and performed in solution under native conditions. These methods are powerful because data acquisition analysis is performed within hours.
2) Which biological information can be obtained by ITC micro-calorimetry and light scattering?
Micro-calorimetry is applied for the detailed quantification of the binding of a protein to other proteins, ligands/inhibitors or nucleic acids. Binding between small molecules or even different nucleic acid molecules is possible. The strength of interaction and the enthalpy of binding can be measured. Micro-calorimetry is also used to analyse enzyme kinetics. Light scattering is used to determine the molecular mass of a biological macromolecule in solution, the oligomeric state, the size of a macromolecular complex and possibly its stoichiometry. It is also a method to analyze the quality of a macromolecular sample. Both methods deliver different qualitative and quantitative information on the isolated proteins, in a non invasive, non destructive manner, and in their solution state.
When substances bind, heat is either generated or absorbed. ITC is a thermodynamic technique that directly measures the heat released or absorbed during a biomolecular binding event. Interaction of proteins with small molecule ligands, with other proteins or nucleic acids can be characterized. Further, measurements of interaction parameters between small molecules and between nucleic acid molecules are also possible. The ITC technique allows accurate determination of binding constants (KD), reaction stoichiometry (n), enthalpy (ΔH) and entropy (ΔS) of binding, thereby providing a complete thermodynamic profile of the molecular interaction in a single experiment. ITC also can be used to analyze enzyme kinetics without the need of colour-substrates and extensive assay development. Because it can elucidate the mechanism of the molecular interaction, it has become a method of choice for characterizing biomolecular interactions. The operating temperature is in the range of 2-80°C
Multi-Angle Light Scattering (MALS) coupled to size exclusion chromatography or other fractionation methods provide absolute means for measuring the molar mass, size, complex stoichiometry, conformation and distribution of biological macromolecules and polymers of all sorts. Mw - the weight averaged molecular mass - is directly measured in the MALS experiment. An estimation of the hydrodynamic radius and the second virial coefficient A2 of the gas equation can be calculated. The later is a measure for the self-attractive force of molecules and was shown to predict the ability of a protein to crystallize.
3) What are the sample requirements to perform light scattering and micro-calorimetry analysis?
First, researchers need to produce sufficient purified and homogeneous protein. The BMA Geneva offers consulting services in recombinant protein expression (host, vector, expression conditions) and in the purification of proteins and proteins in the context of interaction partners. For biophysical analysis of proteins, homogeneous samples need to be prepared at micro to millimolar concentrations. The concentration of the samples has to be determined very accurately. We recommend using the Extinction coefficient and absorbance measurement.
For ITC micro-calorimetry, the molecule of interest should be in the concentration range of 3x the binding constant. For high affinity binders, typically 0.005 mM to 0.015 mM is sufficient, for low affinity binders the concentration can be up to 10x to 100x higher. The concentration of the ligand molecule is 15-20 times the concentration of the target molecule. Both molecules need to be dissolved in exactly the same buffer. The minimal probe volume is 300 ul, the minimal volume of the ligand is 70 ul. For previously uncharacterized molecules, measurement at different concentrations might be required. An aliquot of exactly the same buffer as used to solubilise the probe and ligand should be provided to the platform for instrument equilibration and sample dilution. For tight binding events (nano-picomolar) competition experiments need to be performed with a lower affinity ligand. For light scattering, samples and buffers need to be filtered with 0.1 nm filtration membranes and be degassed, because any sort of particles in solution (air, microbes, and precipitates) affects proper data interpretation. For online measurements 120 ul of a filtered sample at a concentration of 1 mg/ml is needed. Sample buffer filtration is performed by the platform manager.
4) What information is required prior to ITS and light scattering?
For ITC-Mirco-calorimetry, a ligand has to be identified prior to the binding study, e.g. by thermal shift assay (increase in thermal stability by binding a ligand), gel shift, high throughput screening (inhibitory effect of a ligand on the activity), mass spectrometry, protein crystallography, bioinformatics, literature, experimental database search. The BMA can assist you in finding the appropriate method for ligand identification. For Light Scattering, no further information is required, except the Extinction coefficient and the refractive index increment if it is not a protein sample. It is a direct determination method of the mass & size of the molecule or complex of molecules in solution.