The Mittermaier lab uses a variety of biochemical and biophysical methodologies to prepare and characterize proteins and nucleic acids. Our main techniques are nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry (ITC), and differential scanning calorimetry (DSC), which provide different yet highly complementary views of biomolecular dynamics and energetics. NMR quantifies internal motions over a wide range of time scales with atomic resolution. Calorimetry is extremely sensitive to the energetics of conformational transitions and macromolecular interactions. Together, they can yield detailed, quantitative descriptions of biomolecular function that are inaccessible to any of the techniques alone.
Nuclear Magnetic Resonance Spectroscopy
NMR spectroscopy measures the energy of interaction between the magnetic dipoles of atomic nuclei and a strong applied magnetic field. It simultaneously detects separate signals for up to hundreds of individual atoms within a macromolecule, which can be visualized as peaks in one, two, or more dimensions, as shown above. The shapes of the peaks and correlations between peaks give detailed information on the local environments of individual atoms, as well as revealing how the environment fluctuates on timescales from seconds to picoseconds. Thus NMR rapidly gives atomic-resolution maps of biomacromolecular structure and dynamics. The Mittermaier lab uses 800 MHz and 500 MHz spectrometers located in the Quebec/Eastern Canada High Field NMR Facility, as well as 500 MHz NMR spectrometers located in the McGill Chemistry Department.
Isothermal Titration Calorimetry
ITC measures the heat that is released or absorbed when a ligand is titrated into a sample of its partner as a function of time, thereby yielding the full thermodynamic signature of binding in a single experiment. In the ITC isotherm on the right, each peak corresponds to an injection of ligand, the sizes of the peaks give the binding enthalpy and the shape of the saturation curve gives the affinity. ITC is very sensitive to events that are coupled to binding such as folding/unfolding or protonation/deprotonation equilibria. Furthermore, the shapes of the peaks yield kinetic information on binding and enzyme catalysis. Thus ITC provides extremely detailed information on biomacromolecular interactions that can be incorporated into quantitative models of biological function.
Differential Scanning Calorimetry
DSC measures the amount of heat required to raise the temperature of biomacromolecule as it is thermally denatured. In the thermogram to the right, the low-termparature and hight-temperature baselines give the heat capacity of the folded and unfolded states, respectively, while the excess heat capacity peak corresponds to the heat absorbed during the denaturation process. DSC is extremely sensitive to the presence of folding intermediates, and can therefore be combined with spectroscopic and/or other calorimetric data to explain the role of exchange among different conformational states in biological function.
Sample Preparation and Characterization
The Mittermaier lab is equipped to express proteins in bacteria (autoclave, incubators, -80C freezer, etc.), to purify protein and nucleic acid samples (sonifier, liquid chromatography, HPLC, FPLC, etc.) and to perform biochemical characterization (acrylamide and agarose electrophoresis, UV-Vis spectrophotometry, etc.) In addition, we have a MicroCal VP-ITC, a TA Instruments nanoDSC, and will soon be acquiring a MicroCal ITC-200.
