Examining Biomolecular Interactions using Microscale Thermophoresis

Renseignements sur le financement
Natural Sciences and Engineering Research Council of Canada
  • Type de subvention: Outils et instruments de recherche - catégorie 1 (<150 000$)
  • Année: 2013/14
  • Financement total: $150,000
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Sommaire du projet

As part of a team of 10 structural biologists at the University of Toronto (spanning several departments and faculties), we use biophysical tools such as Atomic Force Microscopy, NMR spectroscopy, X- ray crystallography and electron cryomicroscopy to analyze the biomolecular interactions between small molecules (including ions, toxins, ligands, etc), proteins, and nucleic acids. Herein we request NSERC RTI funds for the purchase of an innovative instrument, Monolith NT, that uses microscale thermophoresis to measure the affinities between a fluorescent (or intrinsic Trp Fluorescent) ligand and an analyte. Nanotemper technologies created the Monolith NT detector to monitor the change in migration rate of a fluorescent probe upon interaction with an additional molecule to inform on the affinity between two molecules. This method detects changes in the hydration shell, charge or size of molecules and thus allows measurement of a wide range of biomolecular interactions under close-to-native conditions: Label-Free and immobilization-Free, and in any buffer or complex bioliquid. The method also requires very low concentrations of protein (nM) and low sample consumption of less than 4 µl per sample and can measure a high dynamic range of dissociation constants between sub-nM to mM. Currently this instrument would be the first of its kind in Canada and one of only a handful in North America (as of Oct 15, 2013). Each of the 10 researchers has an NSERC Discovery Grant that primary deals with the interactions between proteins, small molecules, lipids, nucleic acids or ions. This instrument will not only benefit the training their ~74 HQP but will also increase their productivity. Furthermore the instrument fits well with this teams' goals towards an NSERC-CREATE grant on biomolecular interactions that is being developed for the upcoming competition.