Prosser, R. Scott

Research

We are interested in structural and dynamic features of membrane proteins, membrane model systems, 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 (O2). 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; more recently we have resorted to the use of paramagnetic nanoparticles at 50 μM concentrations, which tend to “label” water. 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 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. 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.

3) 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.

4) 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. 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.

5) Nanoparticles for Medical Imaging. 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 by now and we are pursuing focused applications in MRI, PET, CT, and cancer therapy.