Protein Structure and Dynamics -
Energy Landscapes and Folding Pathways

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Research Synopsis

We focus on problems relating to protein structure and dynamics using NMR. 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 fluorine and ¹³C NMR via tags and biosynthetic approaches to study protein structure and dynamics. We also utilize 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 gambit 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.

1) Nanoparticles for Medical Imaging. We are investigating the potential of a class of lanthanide trifluoride nanoparticle for medical imaging. 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 and we are pursuing focused applications in MRI, CT, and fluorescence imaging.

2) Conformation and Dynamics of ¹³C and ¹⁹F-tagged proteins. We are experimenting with methyl and CF₃ tags which are residue specific and which provide details on conformation and dynamics of proteins. We have several projects focused on the study of a GPCR and millisecond conformational dynamics related to the inactive and active conformations, in addition to a recent project in which protein folding is monitored in vivo.

3) Studies of protein topologies using dissolved oxygen (O₂). Dissolved O₂ causes distinct paramagnetic shifts in fluorine (¹⁹F) and carbon (¹³C) resonances. These shifts are proportional to the extent of solvent exposed surface area. 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.

4) Studies of membranes and membrane proteins using dissolved O₂. 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.

5) NMR studies of proteins, membranes, and disordered systems under pressure. The application of modest pressure (< 270 bar) is a useful means of studying packing, specific volumes, and compressibilities of membranes and even membrane proteins.

6) Studies of protein conformation and dynamics by ¹⁹F NMR. 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. 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.

Moriya, J., Sakakura, M., Tokunaga, Y., Prosser, R. S., and Shimada, I. 2009.
An NMR Method for the Determination of Protein Binding Interfaces using TEMPOL-Induced Chemical Shift Perturbations. Biochim. Biophys Acta (In Press)

Al-Abdul-Wahid, M. S., Neale, C., Pomès, R., and Prosser, R. S. 2009. A Solution NMR Approach to the Measurement of Amphiphile Immersion Depth and Orientation in Membrane Model Systems. J. Am. Chem. Soc. 131, 6452-6459.

Zhang, F., …Al-Abdul-Wahid, M. S., Prosser, R.S., Davidson, A., and Woolley, A. 2009. Structure-based approach to the photo-control of protein folding. J. Am. Chem. Soc. 131, 2283-2289.

Kitevski-LeBlanc, J.L., Al-Abdul-Wahid, M. S., and Prosser, R. S. 2009. A Mutagenesis-Free Approach to Assignment of 19F NMR Resonances in Biosynthetically Labeled Proteins. J. Am. Chem. Soc. 131, 2054-2055.

Kitevski-LeBlanc, J. L., Evanics, F., and Prosser, R. S. 2009. Approaches for the measurement of solvent exposure in proteins by 19F NMR. J. Biomol. NMR. 45, 255-264.

Bezsonova, I., Forman-Kay, J., and Prosser, R. S. 2008. Molecular Oxygen as a Paramagnetic NMR Probe of Protein Solvent Exposure and Topology. Concepts in Magnetic Resonance 32:239-253.

Prosser, R. S., Evanics, F., Kitevski, J. L., and Patel, S. 2007. The measurement of immersion depth and topology of membrane proteins by solution state NMR. Biochim. Biophys. Acta 1768: 3044-3051.

Bezsonova, I., Evanics, F., Marsh, J. A., Forman-Kay, J. D., and Prosser, R. S. 2007. Oxygen as a Paramagnetic Probe of Clustering and Solvent Exposure in Folded and Unfolded States of an SH3 Domain. J. Am. Chem. Soc. 129: 1826-1835.

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 19F and 1H NMR. Biochemistry 45: 14120-14128.

Evanics, F., Kitevski, J. L., Bezsonova, I., …and Prosser, R. S. 2007. 19F NMR Studies of Solvent Exposure and Peptide Binding to an SH3 Domain. Biochim. Biophys. Acta . 770: 221-230.

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.

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

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 contacts in solution NMR studies of PagP. J. Am. Chem. Soc. 128: 8256-8264.

Evanics, F., Diamente, P. R., van Veggel, F. C. J. M., Stanisz, G. J., and Prosser, R. S. 2006. Water-soluble GdF3 and GdF3/LaF3 nanoparticles - Physical characterization and NMR relaxation properties. Chem. Mat. 18: 2499-2505.

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

Bezsonova, I., …Prosser, R. S., Forman-Kay, J. D., Kay, L. E. 2006. Hydration and packing along the folding pathway of SH3 domains by pressure-dependent NMR. Biochemistry 45: 4711-4719.

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) pp. 471-479.