Macromolecular analysis
Equipment
Spectrofluorometer: Carry Eclipse, is a photon counting, computer controlled spectrometer equipped with a double grating excitation and a single grating emission monochromator. The instrument can be used for intrinisic tryptophan fluorescence analysis of proteins (e.g. to follow a protein conformation), FRET studies involving green fluorescence proteins (e.g. to follow interactions and proximity between two interacting partners), binding studies of fluorescently-labeled ligands etc. The instrument is equipped with very accurate thermostating due to a Peltier temperature-control element and a four-position cuvette so that multipe samples can be run simultaneously under identical conditions. Temperature can be ramped up at specific rates thus allowing for thermal denaturation experiments and the determination of apparent melting temperatures. Expect to use proteins up to low micromolar range concentration in the cuvettes. For fluors with intense emission these amounts can be reduced several fold.
Size Exclusion Chromatography coupled with multi-angle laser light scattering (SEC/MALLS), refractive index (RI), and UV detection provides a universal approach for determination of the native molar mass, oligomeric state and protein-protein complex formation in solution of native as well as glycosylated proteins or membrane proteins solubilized in non-ionic detergents. Since glycosylated proteins, protein-detergent complexes and non-globular elongated proteins show anomalous migration behavior on SEC, they present a challenge in terms of determining their molar mass and oligomeric state in solution. In the SEC/MALLS/RI/UV approach, SEC serves solely as a fractionation step while the responses from the three detectors are utilized to calculate the molar mass for the polypeptide portion of the native or modified protein in each "slice" of the SEC chromatogram. The amount of sugar, lipid, or detergent bound to the polypeptide chain can also be estimated from the SEC-UV/MALLS/RI analysis. Expect to load small (10-30 kDa) proteins in the low micromolar range concentration for accurate measurements. Large proteins due to profuse scattering can be measured in nanomolar amounts.
Isothermal Calorimetry (ITC) provides a universal tool for monitoring bimolecular binding interactions because it measures the heat/enthalpy change associated with practically all binding interactions. During an ITC experiment a "ligand" in a syringe is titrated into a measurement cell containing a solution of the "macromolecule". As the two molecules interact, heat is released or absorbed. When the macromolecule becomes saturated with ligand, the heat signal diminishes to only the background heat of dilution. The analysis is performed at equilibrium, in solution phase, and without any labeling or need for a fluorescent or other probe. ITC provides direct information about the thermodynamics of binding. The enthalpy of binding is measured directly and when combined with the equilibrium binding constant (also measured directly) yields the entropy change. ITC can also determine the mass ratio between interacting species; however, the absolute stoichiometry of the final complex needs to be verified by other methods. Expect to use proteins in the tens of micromolar range concentration in the measuring cell and ligand in the millimolar range in the injection syringe.
Surface Plasmon Resonance (SPR) Sensor: The IBISII Biosensor is a universal tool for studying macromolecular interactions in a label-free state. SPR detects binding in real time by monitoring changes in mass concentration at the chip surface; the association and dissociation rate constants are determined directly from the reaction traces. Samples ranging from small molecules to crude extracts, lipid vesicles, viruses (and under some conditions even whole bacteria and eukaryotic cells) can be studied in real-time, without labels and with little sample preparation. Ideally, the partner with the smaller mass (e.g. a peptide, an oligonucleotide) is immobilized on the detection surface of the chip so that substantial mass changes can be expected when the large partner binds to the surface. Immobilization can involve covalent cross-linking, metal affinity binding, biotin-avidin chemistries etc. Expect to immobilize small amounts (ng range) of material. Contact Person
Links Sideris, D.P., Petrakis, N., Katrakili, N., Mikropoulou, D., Gallo, A., Ciofi-Baffoni, S., Banci, L., Bertini, I., and Tokatlidis, K.* (2009) A novel targeting signal primes precursors for correct cysteine docking onto Mia40 in the mitochondrial intermembrane space J. Cell Biol. 187(7), 1007-1022 Karamanou, S., Bariami, V., Papanikou, E., Kalodimos, C. and Economou, A. (2008) Vergnolle, M.A.S., Alcock, F.H., Petrakis, N. and Tokatlidis K*. (2007) Mutation of conserved charged residues in mitochondrial TIM10 subunits precludes TIM10 complex assembly, but does not abolish growth of yeast cells, J. Mol. Biol. 371(5), 1315-1324 Baud, C., de Marcos-Lousa, C. and Tokatlidis, K*. (2007) Molecular interactions of the mitochondrial Tim12 translocase subunit Prot. Pept. Lett. 14(6), 597-600 Vergnolle, M.A.S, Baud C., Golovanov, A.P., Alcock, F., Luciano, P., Lian. L.Y., and Tokatlidis, K*. (2005) Distinct domains of small Tims involved in subunit interaction and substrate recognition J. Mol. Biol. 351, 839-849. Papanikou, E., Baud, C., Karamanou, S., Frank, M., Sianidis, G., Keramissanou, D., Kalodimos, C.G., Kuhn, A. and Economou, A. (2005) Identification of the preprotein binding domain of SecA. Journal of Biological Chemistry 280 , 43209-43217 |
