Project Work Package 3


Widening services for biophysical characterization of proteins and their interactions

Leader: Prof. Enrico Ravera

  • Improving the biophysical characterization to extend the structural biology services portfolio. (UNIFI-CERM)
  • Improving the understanding of biomolecular interactions and high-throughput screening. (UNIFI-CERM)
  • Accessing the coordination environment of metalloproteins. (UNIFI-CERM)
  • Expanding biophysical characterization capabilities @CNR. (CNR-IBPM-Roma)
  • Empowering molecular characterization capabilities. (CNR-ICB-Catania)
SEC-MALS is the gold standard for the absolute characterization of proteins and biopolymers, to complement the structural information at atomic detail obtained by NMR. This technique can improve sample preparation and overall sample quality for all structural techniques. The ITACA.SB SEC-MALS instrument has the following features:
1) The HPLC 1260 Bioinert module, that together with and asymmetric-flow field-flow fractionation accessory can separate and characterize nanoparticles, protein and polymers.
2) The DAWN unit, a compreised by 18 light scattering detectors, to determine the absolute molar mass of proteins, polymers and biopolymers (from hundreds of Da to hundreds millions of Da);
3) The WyattQELS unit, a dynamic light scattering (DLS) module, to measure the size of nano- and microparticles (from 0.5 nm to 1 μm);
4)The Eclypse unit, a refractometer module, to determine the concentration of proteins, oligonucleotides, polymers and other macromolecules.

The MicroCal PEAQ-iTC is a semi-automatic system capable of measuring the heat released or absorbed of a biomolecular binding event. This technique does not require molecular probes nor immobilization of the sample. This instrument can measure several thermodynamic parameters in a single measurement using only 5 μg of a macromolecule in 280 μL volume and can determine KD between millimolar to nanomolar range, under standard measurements, and between nanomolar and picomolar when using competitive assays. This instrument is also compatible with acid, bases and different solvents. This instrument has the following features:
1) A vacuum system for cleaning, to refill of the sample’s syringe and to remove samples’ bubbles;
2) A temperature module capable of operating from 2 °C to 80 °C, with a thermal stability of +/- 0,00012 °C at 25 °C.
3) A dispenser module to precisely injected volumes of 0.1 μL to 40 μL of titrant.

Varian Eclipse fluorimeter is equipped with a xenon flash lamp that allows fluorescence measurements between 200 to 900 nm even with the sample’s compartment open, without negatively impacting the experiments. This instrument can determine measure binding events, kinetic parameters and molecular dynamics by taking advantage of either intrinsic fluorescence or fluorophore probes. The instrument can be equipped with different accessories such as:
1) Single Cell Peltier, for reliable temperature control between 0° to 100°C with a resolution of 0.1°C;
2) A fiber optic probe, for direct fluorescence measures of surfaces, powders and solutions;
3) A solid sample probe, for the measurement of powder’s fluorescence at different angles;
4) A microplate reader for high sample throughput.

The SFM-4000 stopped-flow is a versatile instrument useful to a wide variety of rapid mixing techniques and applications, consisting of a mixing device coupled to a detection system. This mixing system can work with up to four solutions and can accurately ensure microliter precision at all flow rates, over a wide viscosity, temperature, and mixing ratio range. In SFM-4000 stopped-flow system has several modes that can be used to run experiments, from optical stopped-flow to chemical quench-flow, to freeze quench, and to cryo stopped-flow (with temperature range from -25 °C to 120 °C). These experiments can measure the kinetic parameters, such as velocities, association constants and stoichiometry, of biomolecular events.The acquisition modes of the SFM-4000 are:
1) Absorption mode, monitoring the changes at a single wavelength.
2) Fluorescence mode, exciting the sample at a specific wavelength and measuring the emitting wavelength;
3) Fluorescence anisotropy and 90° light scattering mode, to measure the scattered light or changes in fluorescence intensity.

To complement the NMR instrumentation, in ITACA.SB we acquired an ENDOR module for the EPR instrument. ENDOR is an EPR to reveal spatial proximity between the unpaired electron and neighboring nuclei through excitation of the nuclear spin transitions of those nuclei. Those nuclei are usually within the blind sphere of traditional paramagnetic NMR experiments: ENDOR detection is perfectly complementary to NMR to characterize the structure of the metal centers of metalloproteins. ENDOR is also complementary to DEER, because it enables distance measurements with site-selective tags containing 19F.

The scientific staff specifically hired for this purpose will test the equipment by using standard samples and other protein samples mimicking the expected samples from the users. These tests will result in a number of protocols for carrying the various experiments.