Department of Chemistry, Iowa State University  
Pulse Sequences
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Department of Chemistry
Iowa State University

Research Interests

The long-term objective of our research is to elucidate the structure and mechanism of action of membrane proteins and other macromolecules important in biology. Phospholipid membranes and the proteins embedded in them are universal components of cells and play key roles in many essential cellular functions such as ion transport, signal transduction, and cell-cell fusion. Despite the importance and abundance of membrane proteins, their high-resolution structures are challenging to determine by crystallography and solution NMR due to the inherent disorder and large molecular masses of protein-lipid complexes.

Solid-state NMR is an atomic-resolution spectroscopic technique uniquely suited to the study of non-crystalline materials and biological macromolecules. We develop and apply multidimensional magic-angle-spinning (MAS) solid-state NMR techniques to elucidate the structural basis for the function of various classes of membrane peptides, proteins, and other insoluble macromolecules. These studies are conducted in the near-native environments of the systems of interest, such as phospholipid bilayers for membrane proteins and intact cell walls for plant polysaccharides. Currently, we are interested in: 

Structure and dynamics of the influenza virus M2 protein

Structure and mechanism of cationic antimicrobial peptides and cell-penetrating peptides.

Structure-function relation of viral fusion proteins.  

Structure and intermolecular interactions of polysaccharides and glycoproteins in plant cell walls.

Solid-state NMR methods for determining molecular structure and dynamics.


1. The Influenza M2 proton channel

The M2 protein of the influenza virus forms a low-pH activated tetrameric proton channel that is important for the virus lifecycle 1. The influenza A M2 protein is the target of amantadine and rimantadine, which constitute one of only two classes of anti-influenza drugs. The emergence of widespread resistance to these M2-blocking drugs due to natural mutations in the transmembrane (TM) domain of the M2 protein makes it imperative to design new inhibitors that target the M2 variants. Elucidating the structural basis for drug inhibition and proton conduction is important not only for guiding the design of new anti-flu drugs but also for understanding fundamental aspects of proton transport in ion channels in general.


Fig. 1  Solid-state NMR studies of the influenza M2 protein elucidate the drug inhibition and proton conduction mechanisms. (a) High-resolution SSNMR structure of the amantadine-bound M2 transmembrane domain in DMPC bilayers. The drug binds with high affinity to the pore of the channel near S31. A second, low-affinity, binding site is present on the lipid-facing surface of the protein. (b) Key 13C-2H REDOR spectra that revealed drug binding at different concentrations. With one equivalent drug per tetramer, only the S31 signal shows intensity difference due to deuterated amantadine. With excess drug, D44 also shows difference intensity. (c) His37-water proton exchange constitutes the essential step in proton conduction across the M2 channel. (d) NMR dynamics data indicate reorientation of the His37 imidazolium ring at low pH. This motion shuttles protons into the virion.

We use solid-state NMR spectroscopy to determine the high-resolution structure of the M2 protein in lipid bilayers in multiple states: with and without the drug 2,3, at low and high pH (open versus closed channel) 4,5, and in model membranes versus virus-envelope mimetic mixed membranes 6,7.  We have elucidated  1) the drug binding site and inhibition mechanism 8, 2) conformational plasticity and its potential functional relevance 5, 3) backbone and sidechain motion of the protein in the lipid bilayer 6,9, 4) water-protein interactions 10, and 5) the structures of key functional residues for proton conduction and channel gating 4,11,12. A wide range of solid-state NMR techniques such as multidimensional correlation, distance measurements, orientation determination, dynamics and relaxation NMR, are employed to obtain a comprehensive understanding of this proton channel.


2. Structure and mechanism of cationic membrane peptides

Antimicrobial peptides (AMPs) are produced by many animals and plants to defend against microbial infections. They achieve this function by disrupting the cell membranes of the invading bacteria, fungi, or viruses. Understanding the mechanisms of action of AMPs is thus important for designing more potent and resistance-free antibiotics. We use solid-state NMR to investigate the structural topology, oligomeric assembly and lipid interactions of AMPs in phospholipid membranes, to understand how these molecules selectively destroy the microbial membrane 13,14. We have studied a number of beta-hairpin-rich AMPs, such as protegrin-1 (PG-1) 19,20, and alpha defensins 21,22. We found that the most active beta-hairpin AMPs insert into bacteria-mimetic anionic lipid membranes and induce toroidal pores, while the less active AMPs remain at the membrane-water interface. The lipid composition of the membrane has a strong influence on the AMP structural topology. Zwitterionic eukaryote-mimetic lipid membranes prevent or reduce AMP insertion (Fig. 2) 15.

Fig. 2 Oligomeric structure of PG-1 in different lipid membranes. (a) PG-1 forms transmembrane beta barrels in negatively charged lipid membranes, which mimic the bacterial cell membrane. Electron micrographs (left) indicate the dramatic cell morphology change upon the addition of PG-1. (b) PG-1 forms large beta sheets on the surface of zwitterionic lipid membranes containing cholesterol, which mimic the red-blood-cell membrane.

A common feature of AMPs is their highly cationic and arginine-rich amino acid sequences. This sequence commonality raises the fundamental question of how cationic residues insert into the hydrophobic region of the lipid bilayer, as the measured transmembrane topology indicates. We have measured arginine-phosphate and arginine-water distances to show that the cationic guanidinium moiety of the arginines is stabilized by salt-bridge interaction with the lipid phosphate headgroups 17 and by a solvation shell 23. Salt bridge formation not only reduces the free energy of insertion but also provides a molecular mechanism for the formation of toroidal pores by AMPs. This guanidinium-phosphate and guanidinium-water interaction is now observed in many cationic membrane peptides 24-26.

Analogous to AMP in amino acid sequence but distinct in function are arginine-rich cell-penetrating peptides (CPP), which cross the membrane of eukaryotic cells alone as well as carrying macromolecular cargos. CPPs are useful as drug delivery agents (Fig. 3). Our studies of the HIV TAT peptide and penetratin of Drosophilia Antennapedia revealed short guanidinium-phosphate distances, similar to AMPs (Fig. 3). Interestingly, CPPs exhibit very different conformation and dynamics from AMPs: they do not form canonical hydrogen-bonded secondary structures (alpha helix or beta sheet) in the lipid membrane, but are either random coil or only partially ordered 24,27. This lack of stable secondary structure may underlie the transient interaction of these peptides with the lipid membrane.

Fig. 3 Arginine-rich cell-penetratin peptides carry macromolecular cargos into cells. Short 13C-31P distances have been measured between the guanidinium and the lipid phosphate, suggesting the importance of this ion-pair interaction for the membrane translocation of CPPs.


3. Plant cell walls

The cell walls of higher plants maintain the morphology, ionic balance, and allow the growth and development of plant cells. However, the molecular structure, dynamics and spatial proximity of polysaccharides and glycoproteins in the plant cell wall are still poorly understood because of the insoluble nature of the cell wall. Elucidating the molecular structure and dynamics of plant cell wall macromolecular structure is important both for fundamental advances in plant biochemistry and for harvesting the energy content of this biomaterial.

Traditional extraction-based chemical analysis, enzymatic hydrolysis, imaging and X-ray diffraction methods have revealed the chemical composition and some ultrastructural details of the plant cell wall. However, molecular-level structure information of the intact cell wall is almost non-existent so far. We are employing multidimensional MAS correlation NMR techniques to determine the intermolecular interactions among cell wall polysaccharides and structural proteins. By isotopically (13C and 15N) enriching whole plants, we have achieved sufficient sensitivity to obtain the first data in the literature on the spatial proximity between different wall polysaccharides 28. Our studies so far focus on the primary cell wall of Arabidopsis thaliana. Resonance assignment of the 13C signals of major polysaccharides has been accomplished 28. Cross peaks among these 13C signals are beginning to revise the existing paradigm about the interactions among cellulose, hemicellulose and pectins. Our data reveal that the surface of cellulose microfibrils are not extensively coated by hemicellulose, while pectic polysaccharides have direct non-covalent contact with cellulose, rather than forming a separate network. These findings revise the prevailing model of the three-dimensional architecture of the primary cell wall. We are using this approach to investigate the structure and intermolecular interactions of other cell wall components in a variety of plants.


4. Viral fusion peptides

The generation of membrane curvature underlies many biological processes such as virus entry into cells, virus budding, pore formation by antimicrobial peptides, and membrane vesicularization. One of the most widely studied membrane deformation events is the entry of enveloped viruses into cells, in which fusogenic proteins of the viruses catalyze the merger of the viral membrane and the cell membrane by undergoing a series of large conformational changes. Although a basic conceptual framework of viral membrane fusion has been well known based on extensive crystallographic and electron microscopy data of the water-soluble part of various fusion proteins, the structure of a membrane-bound fusion domain and its evolution during the fusion process remain largely a mystery. We are investigating the structure and dynamics of the fusion peptide of a class-I fusion protein, that of the paramyxovirus parainfluenza virus 5, to understand how this and other related fusion peptides generate membrane curvature to cause virus-cell fusion.


5. Solid-state NMR methods for biomolecular structure determination

To address the above biological questions, we have to constantly innovate and enhance existing solid-state NMR techniques. We have been particularly interested in methods to increase the distance reach of solid-state NMR (to 10-15 Å) 29,30, to probe molecular motion 31, to measure protein orientation in lipid membranes with or without alignment 6,32, to simplify resonance assignment 33, to determine membrane curvature 34,35, and to determine lipid-protein interactions 17,36.



(1)       Cady, S. D.; Luo, W. B.; Hu, F.; Hong, M. Biochemistry 2009, 48, 7356-7364.

(2)       Cady, S. D.; Hong, M. Proc. Natl. Acad. Sci. U.S.A 2008, 105, 1483-1488.

(3)       Cady, S. D.; Mishanina, T. V.; Hong, M. J. Mol. Biol. 2009, 385, 1127-1141.

(4)       Hu, F.; Schmidt-Rohr, K.; Hong, M. J. Am. Chem. Soc, 2012, 134, 3703-3713.

(5)       Hu, F.; Luo, W.; Cady, S. D.; Hong, M. Biochim. Biophys. Acta 2011, 1808, 415-423.

(6)       Cady, S. D.; Goodman, C.; C.Tatko; DeGrado, W. F.; Hong, M. J. Am. Chem. Soc. 2007, 129, 5719-5729.

(7)       Luo, W.; Cady, S. D.; Hong, M. Biochemistry 2009, 48, 6361-6368.

(8)       Cady, S. D.; Schmidt-Rohr, K.; Wang, J.; Soto, C. S.; DeGrado, W. F.; Hong, M. Nature 2010, 463, 689-692.

(9)       Cady, S. D.; Hong, M. J. Biomol. NMR 2009, 45, 185-196.

(10)     Luo, W.; Hong, M. J. Am. Chem. Soc. 2010, 132, 2378-2384.

(11)     Hu, F.; Luo, W.; Hong, M. Science 2010, 330, 505-508.

(12)     Luo, W.; Mani, R.; Hong, M. J. Phys. Chem. 2007, 111, 10825-10832.

(13)     Hong, M.; Su, Y. Protein Sci. 2011, 20, 641-655.

(14)     Hong, M. Acc. Chem. Res. 2006, 39, 176-183.

(15)     Mani, R.; Cady, S. D.; Tang, M.; Waring, A. J.; Lehrer, R. I.; Hong, M. Proc. Natl. Acad. Sci. USA 2006, 103, 16242-16247.

(16)     Mani, R.; Tang, M.; Wu, X.; Buffy, J. J.; Waring, A. J.; Sherman, M. A.; Hong, M. Biochemistry 2006, 45, 8341-8349.

(17)     Tang, M.; Waring, A. J.; Hong, M. J. Am. Chem. Soc. 2007, 129, 11438-11446.

(18)     Tang, M.; Waring, A. J.; Lehrer, R. I.; Hong, M. Angew. Chem. Int. Ed. Engl. 2008, 47, 3202-3205.

(19)     Doherty, T.; Waring, A. J.; Hong, M. Biochemistry 2006, 45, 13323-13330.

(20)     Doherty, T.; Waring, A. J.; Hong, M. Biochemistry 2008, 47, 1105-1116.

(21)     Zhang, Y.; Lu, W.; Hong, M. Biochemistry 2010, 49, 9770-9782.

(22)     Zhang, Y.; Doherty, T.; Li, J.; Lu, W.; Barinka, C.; Lubkowski, J.; Hong, M. J. Mol. Biol. 2010, 397 408-422.

(23)     Li, S.; Su, Y.; Luo, W.; Hong, M. J. Phys. Chem. B 2010, 114, 4063-4069.

(24)     Su, Y.; Waring, A. J.; Ruchala, P.; Hong, M. Biochemistry 2010, 49, 6009-6020.

(25)     Su, Y.; Doherty, T.; Waring, A. J.; Ruchala, P.; Hong, M. Biochemistry 2009, 48, 4587-4595.

(26)     Doherty, T.; Su, Y.; Hong, M. J. Mol. Biol. 2010, 401, 642-652.

(27)     Su, Y.; Mani, R.; Doherty, T.; Waring, A. J.; Hong, M. J. Mol. Biol. 2008, 381, 1133-1144.

(28)     Dick-P¨¦rez, M.; Zhang, Y.; Hayes, J.; Salazar, A.; Zabotina, O. A.; Hong, M. Biochemistry 2011, 50, 989-1000.

(29)     Luo, W.; Hong, M. J. Am. Chem. Soc. 2006, 128, 7242-7251.

(30)     Schmidt-Rohr, K.; Hong, M. J. Am. Chem. Soc. 2003, 125, 5648-5649.

(31)     Hong, M.; Yao, X. L.; Jakes, K.; Huster, D. J. Phys. Chem. B 2002, 106, 7355-7364.

(32)     Hong, M.; Doherty, T. Chem. Phys. Lett. 2006, 432, 296-300.

(33)     Yao, X. L.; Schmidt-Rohr, K.; Hong, M. J. Magn. Reson. 2001, 149, 139-143.

(34)     Marasinghe, P. A. B.; Buffy, J. J.; Schmidt-Rohr, K.; Hong, M. J. Phys. Chem. B 2005, 109, 22036-44.

(35)     Wang, T.; Cady, S. D.; Hong, M. Biophys. J. 2012, 102, 787-794.

(36)     Huster, D.; Yao, X. L.; Hong, M. J. Am. Chem. Soc. 2002, 124, 874-883.


(updated 09/2012)

Professor Mei Hong
Department of Chemistry
0219 Hach Hall, Iowa State University
Ames, Iowa 50011-3111
Tel: 515-294-3521
Fax: 515-294-0105

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