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Prof. Mei Hong NMR Spectroscopy, Biophysical Chemistry, Structural Biology
Research interests The broad aim of our research is to elucidate the structure and dynamics of membrane proteins, fibrous protein aggregates, and other insoluble macromolecules important in biology. Biological membranes and the proteins embedded in them are universal components of cells and play key roles in a large number of processes such as photosynthesis, ion transport, and cell signaling. Many fibrous proteins (e.g. elastin, collagen) are the essential structural components of tissues. Despite the importance and abundance of these naturally insoluble proteins, little is known about their three-dimensional structure and dynamics due to the difficulties of crystallizing and solubilizing these molecules. We develop and apply multi-dimensional, high-resolution solid-state NMR spectroscopy to determine the structure of these insoluble proteins.
Current projects of interest are: 1. Topological and oligomeric structures of b-sheet antimicrobial peptides and their relation to membrane-disruptive mechanisms. 2. Three-dimensional structure of a human defensin in the lipid membrane. 3. Structure of cell-penetrating peptides and the energetics of cationic protein transport across lipid membranes 4. Oligomeric structure of the influenza viral M2 proton channel. 5. Conformation and dynamics of collagen. 6. Solid-state NMR techniques for investigating the oligomeric structure and functional motions of membrane proteins.
I. Structure and dynamics of antimicrobial peptides Antimicrobial peptides (AMPs) are produced by many animals, including humans, as an innate immune response against bacteria, fungi, and viruses. They achieve this by disrupting the cell membranes of pathogens. To understand the mechanism of action of these peptides for eventual rational designs of potent antibiotics, we investigate the topological structure, depth of insertion and orientation, oligomeric assembly and dynamics of these proteins in the lipid membrane. We determine protein orientation using a variety of NMR probes and both uniaxially aligned membranes and unoriented liposome samples. Orientation-dependent dipolar couplings or chemical shift anisotropies are measured and then converted to the protein orientation in the membrane. Examples of orientation-determined AMPs include PG-1 (1), retrocyclin-2 (2), and TP-I (3). Antimicrobial peptides often do not act alone, but form oligomeric assemblies in the membrane to bring on membrane disruption. The size and structure of these oligomers in the membrane are difficult to determine by most other spectroscopic techniques due to the nanometer length scale of interest and the membrane-bound state of these molecules. We have developed a 19F spin diffusion technique to determine the oligomeric number and intermolecular distances (up to ~15 Å) of membrane proteins (4) and have applied this to the b-hairpin antimicrobial peptide PG-1 (5). We found that PG-1 forms transmembrane beta-barrels in anionic lipid membranes that mimic the bacterial cell membrane composition, but surface-bound beta-sheets in neutral cholesterol-containing membranes that mimic the red blood cell membrane (Figure 1). This study provided the first view of the structure of the protein in the so-called toroidal pore model. It also provided a structural basis for the selectivity of this class of AMPs against microbial cells but not host eukaryotic cell.
Figure 1. Oligomeric structure of PG-1 in anionic and neutral cholesterol-containing membranes. (a) Top and side views of the PG-1 b-barrel in POPE/POPG membranes. For clarity only two dimers are shown in the side view. (b) PG-1 b-sheets on the POPC/cholesterol membrane surface.
A question of fundamental and general interest is how highly charged proteins such as these cationic AMPs transport the charged residues across the hydrophobic part of the lipid membrane. For example, the free energy penalty of transferring an Arg residue from water to the center of the membrane is ~2.6 kcal/mol. We are using protein-lipid 13C-31P distance experiments to address this intriguing thermodynamic question (6). In PG-1, we found that the Arg residues both in the middle of the beta-strand and at the beta-turn are 4-6 Å from the 31P atom of the lipid headgroup. Since the peptide is transmembrane based on independent experiments, this indicates that some of the lipid headgroups must be embedded in the hydrophobic region of the membrane (Figure 2). This directly supports the toroidal pore model, where the membrane bends onto itself and the two leaflets merge (7). It appears that guanidinium-phosphate ion pairs are formed to reduce the free energy of insertion of Arg residues, in so doing also causing the toroidal pore defects.
Figure 2. (a) 13C-31P distances from R4 and R11 in PG-1 to the 31P of POPE/POPG membrane. (b) Toroidal pore model of the lipid membrane to explain the 13C-31P distances. The peptide forms ordered beta-barrels, surrounded by disordered lipids. Some lipid headgroups become embedded in the center of the membrane to offset the free energy of guanidinium insertion. (c) Bidentate complex between the lipid phosphate groups and the Arg guanidinium ion.
Beta-hairpin antimicrobial peptides of similar sequences abound; but they do not all act in the same fashion. Comparative studies of TP-I, a horseshoe crab AMP whose Arg distribution differs from that of PG-1, indicate an entirely different topological structure and dynamics. In contrast to PG-1, TP-I does not insert transmembrane into anionic lipid membranes but remain parallel to the membrane surface near the glycerol backbone region (8). It also undergoes extensive motion in the liquid-crystalline membrane, in contrast to the highly oligomerized and immobilized PG-1. These suggest a mobility-based antimicrobial mechanism (9). Sequence amphipathicity thus seems to have a significant effect on the mode of action of these proteins. We are extending the studies of synthetic beta-hairpin antimicrobial peptides to larger recombinant defensins with more complex secondary structures. The recent development in expressing defensins in E. coli makes it possible to determine the full three-dimensional structure of these mini-proteins in the lipid bilayer environment. We use 2D and 3D correlation experiments that aim to obtain full 13C and 15N resonance assignment and atomic-resolution conformational constraints of the protein.
II. Oligomeric structure of membrane proteins Structure determination of membrane proteins has so far mainly focused on intra-molecular structure. Yet the functional state of many membrane proteins involves oligomeric assemblies of multiple units, thus quaternary structure of membrane proteins is also important. We are investigating the dynamic structure of a tetrameric helical bundle, the M2 protein of the influenza A virus. The M2 protein forms a proton-conducting channel that acidifies the virus, which is an important step in the viral life cycle and replication. We use the 19F spin diffusion technique to determine the oligomeric number (10), measure interhelical distances between key sidechains, and probe the gating mechanism of this proton channel (Figure 3) (11). We are also interested in obtaining high-resolution backbone and sidechain structure of the protein in the absence and presence of a channel blocker, amantadine. 2D 13C and 15N NMR techniques are used to obtain conformational and dynamic constraints to elucidate the effect of amantadine on the M2 structure (12). The M2 helical bundle undergoes fast uniaxial rotational diffusion around the membrane normal, a property that we have characterized in detail. This rigid-body uniaxial rotation is extremely useful for deriving orientation information on the helices without the use of aligned samples (13).
Figure 3. (a) 19F CODEX data of Trp41 labeled M2, indicating a nearest neighbor F-F distance of 11.7 Å. (b) This distance depends mainly on the X1 and X2 torsion angles of Trp, which are constrained to (X1, X2) = (180˚, 90˚) by the CODEX data. The only other solution of (60˚, -100˚) causes steric conflict. (c) Top view of the M2 tetrameric helical bundle, showing the Trp sidechain conformation. This data suggests that Trp is not the gate of the channel.
III. Cell-penetrating peptides and cationic protein import into the membrane Cell-penetrating peptides are small cationic molecules that translocate across the lipid membrane without damaging them. Moreover they are able to do this while carrying large macromolecular cargoes, and thus are important drug-delivery agents. It is not understood how this membrane translocation occurs in light of the highly cationic nature of these sequences. We employ a number of solid-state NMR methods to probe the dynamic conformation and the depth of insertion of these peptides, to better understand the mechanism of action of these Trojan peptides.
IV. Conformation and dynamics of structural proteins Collagen and elastin constitute two of the most ubiquitous structural proteins in connective tissues. We are interested in functionally relevant conformational and dynamical features of these proteins. Our detailed studies of the torsion angles in an elastin-mimetic sequence (VPGVG)n revealed two major conformations, a beta-strand conformation (~2/3) and a beta-turn conformation (1/3), suggesting that the elasticity of the protein is built into the innate conformational propensity of the repeat sequence (14). The hydration dependence of the protein differs from typical globular proteins: above a threshold hydration level, the protein chains undergo near-isotropic large-amplitude motion (15). The conformational dynamics of the triple-helical collagen has been studied in various tissues using site-specific 13C and 2H labels and relaxation NMR. However, details of the motion of the Pro ring and the hydroproline (O) rings in the collagen sequence have not been probed. We are using site-resolved 13C NMR techniques to delineate in detail the ring motion in collagen and examine these as a function of various environmental parameters.
V. New solid-state NMR techniques for protein structure determination Nuclear spin interactions depend on the orientation of atoms in space and on inter-atomic distances. Thus, they act as atomic "spies" to the three-dimensional structures of solids and their time-dependent changes. Part of our research is to develop new solid-state NMR methods to facilitate protein structure determination. Of particular interest are long-range distance techniques (16, 17), methods for determining protein motion (18) and for exploiting motion to obtain structural information (3, 19), and sensitivity enhancement techniques in NMR (20, 21).
References
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