The Hong group develops and applies high-resolution solid-state NMR spectroscopy to elucidate the structure and dynamics of biological macromolecules, with an emphasis on membrane proteins. We design magic-angle-spinning NMR techniques that measure inter-atomic distances and molecular motions, and apply these techniques to problems in biology, pharmacology, and biomaterials. We have a long-standing interest in ion channels and curvature-inducing membrane proteins. We are also investigating the polysaccharide structures in energy-rich plant cell walls and amyloid proteins involved in neurodegenerative diseases. We study these complex noncrystalline proteins and carbohydrates in their native environments, at atomic resolution, with both structural and dynamical details, from which we obtain mechanistic insights that are rarely available from other structural techniques.
Influenza virus M2 proteins
Ion channels and transporters provide conduits for the passage of ions and polar compounds across the hydrophobic barrier of lipid membranes. The M2 protein of influenza viruses acts as a proton channel that enables virus uncoating; it also mediates membrane scission in the last step of virus budding. As such influenza M2 is an excellent model for larger ion channels, synthetic proton conductors, as well as curvature-inducing membrane proteins.
The M2 protein of influenza A viruses, which are responsible for all flu pandemics in history, is the target of the amantadine class of antiviral drugs, but currently circulating seasonal flu viruses have evolved amantadine-resistant mutations. Influenza B virus M2 is so far not druggable, thus structure elucidation of AM2 and BM2 is important for developing new antiviral drugs against flu. We investigate the structure and dynamics of the M2 protein to understand how it conducts protons, how it binds drugs, and how it induces membrane curvature. Solid-state NMR spectroscopy is used to determine the oligomeric assembly and three-dimensional structure of the protein; elucidate the equilibria and kinetics of proton transfer between water and protein sidechains; characterize the motion of the proton-selective residue and the gating residue of the channel; reveal protein-drug and protein-cholesterol binding sites; and investigate water dynamics in the channel. The structural and dynamical information obtained from these studies help to guide rational design of new antiviral drugs to inhibit M2 and prevent future flu pandemics.
Viral fusion proteins
Membrane curvature is essential to many biological processes such as endocytosis, vesicle trafficking, and cell division. Proteins can sense, stabilize, and induce membrane curvature. For example, cationic antimicrobial peptides cause membrane curvature to disrupt the integrity of lamellar phospholipid bilayers. An important class of membrane proteins that induce membrane curvature is viral fusion proteins, which merge the virus envelope and the target membrane to enable virus entry into cells. They accomplish this task by undergoing complex conformational rearrangements, as seen in X-ray crystal structures of water-soluble ectodomains of these proteins. These protein conformational changes presumably lower the free energy barriers for membrane dehydration and membrane structural changes from the lamellar state to hemifused intermediates to the final fused state. However, the current framework of virus-cell fusion largely excludes two key hydrophobic domains: the N-terminal fusion peptide domain and the C-terminal transmembrane domain, which are suspected to play important roles in destabilizing the lamellar structures of the two lipid membranes. We seek to elucidate the conformations and oligomeric structures of the hydrophobic fusion peptide and transmembrane domains in lipid membranes using solid-state NMR and complementary techniques. By coupling protein structure measurements with observation of membrane morphology by 31P NMR and detection of water-lipid and water-protein interactions, we couple protein structure information with membrane morphology and membrane hydration information, thus obtaining a comprehensive view of the protein and membrane structural transformations along the fusion pathway. The fusion proteins of parainfluenza virus and HIV are currently studied.
Plant cell walls
Plant cell walls provide mechanical strength to cells while also allowing the cells to expand during plant growth. This energy-rich material consists of a mixture of polysaccharides and proteins, whose composition varies between primary and secondary cell walls and between different plant organisms. Although the composition of plant cell walls is relatively well known, the three-dimensional structures and interactions of wall polymers have long been elusive due to the lack of high-resolution structural techniques for characterizing the insoluble and noncrystalline cell wall. We have pioneered the use of multidimensional 13C solid-state NMR to elucidate the structures and dynamics of the polysaccharides of intact plant cell walls. By enriching whole plants during growth with 13C, we are able to employ 2D and 3D correlation SSNMR techniques to detect and resolve the signals of cellulose, hemicellulose and pectins, determine their intermolecular interactions and their mobilities. Moreover, by sensitivity-enhancing dynamic nuclear polarization and paramagnetic relaxation enhancement approaches, we can elucidate how proteins bind polysaccharides to loosen the cell wall. Using model plants of both dicot (e.g. Arabidopsis thaliana) and grass (e.g. Brachypodium distachyon and Zea mays) families, we have shown that cellulose, hemicellulose and pectins form a single three-dimensional network instead of two separate networks, thus changing the long-held view of the cell wall architecture. With our collaborators, we investigate the structural polymorphism of cellulose microfibrils, hydration of wall polysaccharides, interactions of cellulose with matrix polysaccharides, and the effects of genetic mutations on the cell-wall network structure.
Amyloid fibrils are highly aggregated β-sheet structures formed by many peptides and proteins. Amyloid fibrils are involved in many neurodegenerative disorders as well as in biological functions. We are investigating the structures and assemblies of tau and Aβ peptides of the Alzheimer’s disease and amyloid fibrils formed by the peptide hormone glucagon. These structural studies aim to understand how amino acid sequences dictate the three-dimensional fold and oligomeric assembly of the fibrils, the origin of molecular structural polymorphism, how water impacts fibril formation, and how metal ions stabilize the fibril structures.
Solid-state NMR techniques for biomolecular structure determination
To answer important biological questions, we constantly expand the capability of solid-state NMR for characterizing molecular structure and dynamics. We have a long-standing interest in increasing the distance reach of NMR from angstroms to nanometers, by exploiting nuclear spins with high gyromagnetic ratios and multi-spin effects. This longer distance reach allows us to determine the oligomeric structure and large conformational changes of proteins, and to elucidate protein-ligand and protein-carbohydrate binding. To determine motional amplitudes and rates, we develop anisotropic-isotropic correlation NMR experiments involving dipolar as well as quadrupolar interactions under fast MAS and in high magnetic fields. Finally, we innovate new isotopic labeling approaches and computational methods to simplify and speed up resonance assignment of protein NMR spectra.