Welcome! Our group encompasses undergraduate and graduate students, and senior researchers affiliated with the departments of Chemistry and Biochemistry at the University of Toronto. Our wet lab and NMR lab is located on the University's Mississauga campus, with some of our collaborative research performed on the main campus and at Sunnybrook Hospital.
We focus on problems relating to protein structure and dynamics using NMR and much of our recent efforts have centered on new methods to study membrane protein topology and dynamics and protein folding. In addition to the normal regimen of solution NMR approaches we make full use of 19F and 13C NMR via tags and biosynthetic approaches to study protein structure and dynamics. We also commonly make use of pressure effects and paramagnetic effects from dissolved oxygen as a “molecular contrast agent” in studies of membrane protein and disordered protein topologies. It’s fair to say that we cover the gamut of molecular biology, biochemistry, NMR, physical chemistry, organic chemistry, with a pinch of biophysics. Much of ongoing work in the lab is also focused on a new project involving the use of nanoparticle contrast agents for medical imaging. The work naturally ties in with MRI, Computed Tomography (CT), epi-fluorescence and confocal microscopy, and related cell- and small animal imaging. Click one of the areas below for more details:
We are investigating the potential of a class of lanthanide trifluoride nanoparticle for medical imaging, which boast the largest mass relaxivities for MRI ever reported. Our goal is to co-develop a common platform such that the nanoparticles may be used for a variety of medical imaging and therapeutic applications. We have succeeded in producing dramatically uniform nanoparticles we are pursuing focused applications in MRI, CT, and fluorescence imaging.
In situations where biosynthetic labeling is prohibitive, we are experimenting with methyl and CF3 tags which are residue specific and which provide details on conformation and dynamics of proteins. This is a very active research niche for us at the moment. We have several projects focused on the study of a GPCR and millisecond conoformational dynamics related to the inactive and active conformations, in addition to a recent project in which protein folding is monitored in vivo.
At partial pressures of 20-60 bar, dissolved O2 causes distinct paramagnetic shifts in fluorine (19F) and carbon (13C) resonances. Moreover, these shifts are generally in proportion to the extent of solvent exposed surface area. Similarly, significant paramagnetic effects from dissolved O2 may be observed in protons (1H) via relaxation rates, allowing the entire protein to be studied in great detail. Proteins can also be interrogated with hydrophilic paramagnetic additives; in some cases, isotope shifts from water/D2O exchange give us a perspective of solvent exposure. Together, a detailed mapping of paramagnetic shifts or rates from dissolved O2 and separate measures of solvent isotope effects provides information at atomic resolution of the surface topology and surface potentials of proteins.
In membranes (lipid bilayers and micelles) O2 adopts a pronounced concentration gradient from the water interface to the hydrophobic center. The resulting paramagnetic gradient can be used to measure immersion depth with unprecedented detail, particularly when a second complementary paramagnetic additive is used. The experiments may be used to refine membrane protein structures and understand their topologies. The phenomenon of passive oxygen transport and the distribution of O2 at atomic resolution across lipid bilayers are also of great interest to cell physiologists. Until now, it was only possible to study oxygen distributions in membranes using bulky fluorescent or ESR probe molecules. Our current studies require no probes and provide atomic resolution (we can 'see' every carbon atom in a lipid) of the transmembrane oxygen distribution.
The application of modest pressure (< 270 bar) is a useful means of studying packing, specific volumes, and compressibilities of membranes and even membrane proteins
Over the past few years we have invested a significant effort in developing ways of biosynthetically tagging proteins with 19F labels. The most interesting aspects of protein biochemistry invariably involve “change” and 19F NMR is one of the most sensitive means of studying kinetics, binding, enzymatic processes, or intra/intermolecular dynamics. Our innovation in this field involves the use of 15N,13C-enriched fluorinated amino acids. By doubly tagging the fluorinated amino acids, each signal in the biosynthetically labeled protein becomes a unique reporter. Our goal is to 'marry' 19F NMR to the modern regimen of protein solution NMR techniques. We hope to apply the 19F NMR techniques under development in our lab to studies of membrane proteins and intrinsically disordered proteins, which represent two of the most interesting and challenging niches in structural biology.
Research Facilities: Our NMR suite consists of a 4-channel 600 MHz spectrometer plus a cryogenic probe designed for either 19F NMR or {1H,13C,15N} NMR with the option for deuterium decoupling. The HFCN quad probe and HX probes are also often used in studies of bicelles, or labeled proteins, where exotic 19F,13C,1H experiments, 31P, or 2H NMR experiments are employed to assign peaks, study phase equilibria, or study orientational order and dynamics. We also have a fully equipped chemistry lab for nanoparticle synthesis, protein expression (> 40 L) and purification, and wet chemistry. The Mississauga campus has three research PIs with a focus in NMR, which makes for stimulating interactions and weekly group meetings.
