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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 and other insoluble macromolecular complexes 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 cellular processes such as ion transport and signal transduction. Despite the importance and abundance of membrane proteins, their high-resolution three-dimensional structures are extremely difficult to determine by conventional methods of X-ray crystallography and solution NMR, due to the inherent disorder of the lipid membranes, which obstructs crystallization, and the large sizes of the protein-membrane complexes, which prevent their solubilization. Solid-state NMR spectroscopy is a powerful and versatile technique to determine the atomic-resolution structures of membrane proteins in their native environment of lipid bilayers. In addition to membrane proteins, we have been interested in the conformation and dynamics of structural proteins (e.g. elastin, collagen), and are currently interested in the structure of polysaccharide-rich plant cell walls. We employ a wide range of solid-state NMR methods, many of which developed by our laboratory, to answer these biological questions. Specific current research topics are:
1. Influenza virus M2 proton channel The influenza A virus M2 protein is a pH-gated tetrameric proton channel that is important for the life cycle of the virus. Inhibition of the M2 proton channel by amantadine and rimantadine constitutes one of only two anti-influenza drug treatments available. Various natural mutations of the M2 transmembrane domain cause drug resistance. Thus, elucidating the molecular basis for M2 proton channel function and inhibition is of high public health significance. We use solid-state NMR to determine the high-resolution structure of the M2 protein in lipid bilayers in multiple states, including in the absence and presence of drugs, at different pH corresponding to the closed and open states, and in simple model lipid membranes versus complex virus-envelope mimetic membranes. We are particularly interested in 1) the amantadine-bound protein structure, 2) conformational heterogeneity of M2, 3) segmental and global motion of the protein in the lipid bilayer, 4) interaction of M2 with water, which underlies the proton channel activity, and 5) the structures of key functional residues involved in gating and channel activation. This ensemble of structural information is difficult to obtain in native lipid bilayers by most other biophysical techniques, and structural differences in detergent-solubilized proteins have been reported in the literature. We use a wide variety of NMR techniques, such as multidimensional correlation experiments, spin diffusion, orientation measurements, lineshape and relaxation experiments, and distance measurements, to reach a comprehensive picture of the structure and dynamics of the M2 protein. 2. Structure, topology and mechanism of action of antimicrobial peptides Antimicrobial peptides (AMPs) are immune-system peptides of many animals and plants against microbial infections. They achieve their function by disrupting the cell membranes of the invading bacteria, fungi, or viruses. Thus, understanding the mechanisms of action of AMPs is important for rational designs of potent new antibiotics. We use solid-state NMR to investigate the topological structure of AMPs in the lipid membrane. These topological structural features include the depth of insertion, orientation, and oligomeric assembly of AMPs. Our studies of a number of beta-hairpin AMPs, such as protegrins, tachyplesins, rhesus theta-defensins, and retrocyclin, led to the findings that 1) sequence amphipathicity determines the peptide¡¯s degree of insertion into the membrane; 2) the most amphipathic AMPs are able to insert across the membrane and induce toroidal pore defects, which cause the most significant membrane disruption; 3) less amphipathic beta-hairpin AMPs adopt a surface-bound or interfacial immersed structure, and can use rapid in-plane rotational diffusion as a means to disrupt the lipid membrane; 4) the lipid composition of the membrane has a determining effect on AMP structures, and eukaryote-mimetic lipid membranes strongly prevent AMP insertion. We are pursuing structure determination of human defensins, which are relatively large and complex AMPs with multiple b-strands and disulfide bonds. High-resolution structures of several human defensins have been solved by X-ray crystallography, however in water rather than lipid-mimetic detergents. Our solid-state NMR studies will provide a wealth of information about the membrane-bound structure and lipid interaction of these human defensins. 3. Cell penetrating peptides and cationic domains of membrane proteins A common feature of antimicrobial peptides is their highly cationic and arginine-rich amino acid sequences, which raises the fundamental question of how proteins carrying multiple positively charged residues are able to insert into the hydrophobic region of lipid bilayers. While biological measurements of membrane protein translocation across the lipid membrane have yielded fascinating information about the extent and free energy of translocation of positively charged residues, little direct structural information is available on the membrane-bound state of these proteins. We are investigating the membrane-bound structures of a number of Arg-rich peptides such as cell-penetrating peptides (CPP), and Arg-rich domains of voltage-gated potassium channels. Cell-penetrating peptides transport macromolecular cargos such as DNAs and proteins across the cell membrane into cells, and thus are efficient drug delivery vehicles. Potassium channels play a central role in many cellular processes such as the production of electrical impulses in the nervous system and the control of heart rate. The role of the large number of Arg residues in these proteins is not understood. A general approach of our study is to determine peptide - lipid headgroup distances, to determine if the Arg guanidinium groups use lipid counterions to reduce the free energy cost of membrane insertion. We have found short guanidinium Cz - lipid 31P distances (~4.0 Å) in several cases, such as the antimicrobial peptide PG-1 and the cell-penetrating peptide penetratin. For PG-1, these distances provided the first direct structural evidence for toroidal pore formation, which is directly related to the mechanism of membrane disruption by this antimicrobial peptide. Comparative studies of the structure and dynamics AMPs and CPPs, which have similar amino acid sequences, also provide insight into the structural differences that lead to membrane disruption by the former but the retention of membrane integrity by the latter. 4. Plant cell wall structure The cell walls of both land and oceanic plants are polysaccharide-rich complex materials with high energy contents. The cell wall structures of plants are of interest on a fundamental level, because so little is known about how polysaccharides, proteins and other biopolymers organize themselves to provide the material properties ¨C both strength and flexibility for growth - for plant cells. They are also important on a practical level, because harvesting the energy-rich cellulose from lignocellulosic materials can help to alleviate global energy shortages. To understand how cellulose, hemicellulose, pectin, lignin, and proteins in plant cell walls interact in three dimension, as well as how some of these molecules are chemical linked, we resort to solid-state NMR, which is the only high-resolution technique to study the cell wall structure without chemical treatment that severs the intermolecular linkages of interest. Central to our SSNMR investigation is the use of multidimensional correlation experiments on isotope-labeled plant cell walls. 5. Solid-state NMR methods for biomolecular structure determination In our wish to answer various biological questions, we often find it necessary to develop new as well as improve upon existing SSNMR methods. Recent examples of these developments for protein structure determination include:
¡¡ (updated 09/2009) |
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Please send comments or suggestions about the website to: tuowang@iastate.edu
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