Scott Prosser

Academic Title: UTM Associate Chair Professor

Phone: 905-828-3802

Office: SB 4052

Email:

Research Homepage: http://www.utm.utoronto.ca/mbiotech/page/prosser/

We study structural and dynamic features of membrane model systems, membrane proteins and intrinsically disordered proteins from the perspective of modern solution-state NMR. Structural and dynamical details of membrane proteins and in particular, G protein-coupled receptors (GPCRs) are essential to our understanding of the principals of membrane protein folding, misfolding, signal transduction, and drug membrane protein interactions. While nearly one third of all proteins are membrane associated, intrinsically disordered proteins (IDPs) or proteins bearing a significant unstructured domain ( i.e. > 50 residues) constitute 30-50% of all eukaryotic proteins and directly relate to protein regulation and properties of protein-protein and protein-ligand interactions. Both classes of protein have thus far proven somewhat intractable in terms of detailed structure studies by X-ray crystallography. Membrane proteins and IDPs exhibit significant conformational exchange and a narrow range of chemical shifts which also hampers their study by traditional NMR methods. We have therefore developed several experimental approaches aimed at better understanding structure and function of membrane proteins and IDPs.

1) Studies of protein topologies using dissolved oxygen (O 2 ). At partial pressures of 20-60 bar, dissolved O 2 causes distinct paramagnetic shifts in fluorine ( 19 F) and carbon ( 13 C) resonances. Moreover, these shifts are generally in proportion to the extent of solvent exposed surface area. Similarly, significant paramagnetic effects from dissolved O 2 may be observed in protons ( 1 H) via relaxation rates, allowing the entire protein to be studied in great detail. Proteins can also be interrogated with hydrophilic paramagnetic additives. Together, a detailed mapping of such paramagnetic shifts or rates provides information at atomic resolution of the surface topology and surface potentials of proteins.

2) Studies of membranes and membrane proteins using dissolved O 2 . In membranes (lipid bilayers and micelles) O 2 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 O 2 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.

3) NMR studies of proteins, membranes, and disordered systems under pressure. The above experiments which use dissolved O 2 are made possible through pressure apparatus and sapphire NMR tubes designed to tolerate pressures as high as 270 bar. The application of modest pressure (< 270 bar) is a useful means of studying packing, specific volumes, and compressibilities of membranes and even proteins.

4) Studies of protein conformation and dynamics by 19 F NMR. Over the past few years we have invested a significant effort in developing ways of biosynthetically tagging proteins with 19 F labels. The most interesting aspects of protein biochemistry invariably involve “change” and 19 F 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 15 N, 13 C-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” 19 F NMR to the modern regimen of protein solution NMR techniques. We hope to apply the 19 F 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.

5) Miscellaneous. In collaboration with Prof Frank van Veggel ( University of Victoria ) we are investigating the potential of a class of lanthanide trifluoride nanoparticle for medical imaging. We became interested in these nanoparticle systems because of our prior focus on paramagnetic additives. 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 by now and we are pursuing focused applications in MRI, PET, CT, and cancer therapy.

Bezsonova, I., Evanics, F., Marsh, J. A.,  Forman-Kay, J. D. and Prosser, R. S. Oxygen as a Paramagnetic Probe of Clustering and Solvent Exposure in Folded and Unfolded States of an SH3 Domain J. Am. Chem. Soc. (accepted, Dec 1 2006)

Prosser, R. S. and Evanics, F. 2006. Paramagnetic Effects of Dioxygen in Solution NMR - Studies of Membrane Immersion Depth, Protein Topology, and Protein Interactions. Handbook of Magnetic Resonance (chemistry volume). ( In Press , Oct, 2006).

Evanics, F., Kitevski, J. L., Bezsonova, I. , Forman-Kay, J., and Prosser, R. S. 2006. 19 F NMR Studies of Solvent Exposure and Peptide Binding to an SH3 Domain. Biochimica Biophysica Acta ( In Press ).

Evanics, F., Bezsonova, I. , Marsh, J., Kitevski, J. L., Forman-Kay, J. D. and Prosser, R. S. 2006. Tryptophan Solvent Exposure in Folded and Unfolded States of an SH3 Domain by 19 F and 1 H NMR. Biochemistry ( In Press ).

Prosser R.S., Evanics, F., Kitevski, J.L., and Al-Abdul-Wahid, M.S. 2006. Current applications of bicelles in NMR studies of membrane-associated amphiphiles and proteins. Biochemistry 45: 8453-8465.

Al-Abdul-Wahid, M. S., Yu, C. H., Batruch, I. , Evanics, F., Pomes, R., and Prosser, R.S. 2006. A combined NMR and molecular dynamics study of the transmembrane solubility and diffusion rate profile of dioxygen in lipid bilayers. Biochemistry 45: 10719-10728.

Evanics, F., Diamente, P. R., van Veggel, F. C. J. M., Stanisz, G. J., and Prosser, R. S. 2006. Water-Soluble GdF 3 and GdF 3 /LaF 3 Nanoparticles – Physical Characterization and NMR Relaxation Properties. Chem. Mater. 18: 2499-2505.

Bezsonova, I. , Korzhnev, D. M., Prosser, R. S., Forman-Kay, J. D., and Kay, L. E. 2006. Hydration and Packing Along the Folding Pathway of SH3 Domains by Pressure-Dependent NMR. Biochemistry 45: 4711-4719.

Korzhnev, D. M., Bezsonova, I., Evanics, F., Taulier, N., Zhou, Z., Bai, Y., Chalikian, T. V., Prosser, R. S., and Kay, L. E. 2006. Probing the transition state ensemble of a protein folding reaction by pressure. J. Am. Chem. Soc. 128: 5262-5269.

Evanics, F., Hwang, P. M., Cheng, Y., Kay, L. E. and Prosser, R. S. 2006. Topology of an Outer Membrane Enzyme – Measuring Oxygen and Water Contact in Solution NMR Studies of PagP. J. Am. Chem. Soc. 128: 8256-8264.

Sakakura, M., Noba, S., Luchette, P. A., Shimada, I. and Prosser, R. S. 2005. An NMR Method for the Determination of Protein Binding Interfaces Using Dioxygen-Induced Spin-Lattice Relaxation Enhancement. J. Am. Chem. Soc. 127: 5826-5832.

Evanics, F. and Prosser, R.S. 2005. Discriminating Binding and Positioning of Amphiphiles to Lipid Bilayers by 1 H NMR. Analytica Chimica Acta 534: 21-29.