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.
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:
dynamics of the influenza virus M2 protein.
mechanism of cationic antimicrobial peptides and
Structure-function relation of viral fusion proteins.
intermolecular interactions of polysaccharides and
glycoproteins in plant cell walls.
NMR methods for determining molecular structure and
The Influenza M2
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.
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
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
at low and high pH (open versus closed channel)
and in model membranes versus virus-envelope mimetic mixed
We have elucidated
1) the drug binding site and inhibition mechanism
conformational plasticity and its potential functional relevance
3) backbone and sidechain motion of the protein in the lipid
6,9, 4) water-protein interactions 10, and 5) the structures of key functional residues for proton conduction
and channel gating
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
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
and alpha defensins
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.
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.
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.
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
This lack of stable secondary structure may underlie the
transient interaction of these peptides with the lipid membrane.
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.
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
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
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
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.
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
to simplify resonance assignment
to determine membrane curvature
and to determine lipid-protein interactions
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