The influenza M2 protein not only forms a proton channel but also mediates membrane scission in a cholesterol-dependent manner to cause virus budding and release. The atomic interaction of cholesterol with M2, as with most eukaryotic membrane proteins, has long been elusive. We have now determined the cholesterol-binding site of the M2 protein in phospholipid bilayers using solid-state NMR spectroscopy. Chain-fluorinated cholesterol was used to measure cholesterol proximity to M2 while sterol-deuterated cholesterol was used to measure bound-cholesterol orientation in lipid bilayers. Carbon-fluorine distance measurements show that at a cholesterol concentration of 17 mol%, two cholesterol molecules bind each M2 tetramer. Cholesterol binds the C-terminal transmembrane (TM) residues, near an amphipathic helix, without requiring a cholesterol recognition sequence motif. Deuterium NMR spectra indicate that bound cholesterol is approximately parallel to the bilayer normal, with the rough face of the sterol rings apposed to methyl-rich TM residues. The distance- and orientation-restrained cholesterol-binding site structure shows that cholesterol is stabilized by hydrophobic interactions with the TM helix and polar and aromatic interactions with neighboring amphipathic helices. At the 1:2 binding stoichiometry, lipid P spectra show an isotropic peak indicative of high membrane curvature. This M2-cholesterol complex structure, together with previously observed M2 localization at phase boundaries, suggests that cholesterol mediates M2 clustering to the neck of the budding virus to cause the necessary curvature for membrane scission. The solid-state NMR approach developed here is generally applicable for elucidating the structural basis of cholesterol's effects on membrane protein function.

It is known that smooth, hydrophobic solid surfaces exhibit low ice adhesion values, which have been shown to approach a lower ice adhesion strength limit (∼150 kPa) defined by the water receding contact angle. To overcome this limit, we have designed self-lubricating icephobic coatings by blending polydimethylsiloxane (PDMS)-poly(ethylene glycol) (PEG) amphiphilic copolymers into a polymer matrix. Such coatings provide low ice adhesion strength values (∼50 kPa) that can substantially reduce the lower bound of the ice adhesion strength achieved previously on smooth, hydrophobic solid surfaces. Different molecular mechanisms are responsible for the low ice adhesion strength attained by these two approaches. For the smooth hydrophobic surfaces, an increased water depletion layer thickness at the interface weakens the van der Waals' interactions between the ice and the polymeric substrate. For the self-lubricating icephobic coatings, the PEG component of the amphiphilic copolymer is capable of strongly hydrogen bonding with water molecules. The surface hydrogen-bonded water molecules do not freeze, even at substantial levels of subcooling, and therefore serve as a self-lubricating interfacial liquid-like layer that helps to reduce the adhesion strength of ice to the surface. The existence of nonfrozen water molecules at the ice-solid interface is confirmed by solid-state nuclear magnetic resonance (NMR) spectroscopy.

Glucagon and insulin maintain blood glucose homeostasis and are used to treat hypoglycemia and hyperglycemia in diabetic patients, respectively. Whereas insulin is stable for weeks in its solution formulation, glucagon fibrillizes rapidly at the acidic pH required for solubility, and is therefore formulated as a lyophilized powder that is reconstituted in acidic solution immediately before use. Here we use solid-state NMR to determine the atomic-resolution structure of fibrils of synthetic human glucagon grown at pharmaceutically relevant low pH. Unexpectedly, two sets of chemical shifts are observed, indicating the coexistence of two β-strand conformations. Those two conformations have distinct water accessibilities and intermolecular contacts, indicating that they alternate and hydrogen-bond in an antiparallel fashion along the fibril axis. Two antiparallel β-sheets assemble with symmetric homodimer cross sections. This amyloid structure is stabilized by numerous aromatic, cation-π, polar and hydrophobic interactions, suggesting mutagenesis approaches to inhibit fibrillization to improve this important drug.

The influenza M2 protein forms a tetrameric proton channel that conducts protons from the acidic endosome into the virion by shuttling protons between water and a transmembrane histidine. Previous NMR studies have shown that this histidine protonates and deprotonates on the microsecond time scale. However, M2's proton conduction rate is 10-1000 s, more than 2 orders of magnitude slower than the histidine-water proton-exchange rate. M2 is also known to be conformationally plastic. To address the disparity between the functional time scale and the time scales of protein conformational dynamics and water dynamics, we have now investigated a W41F mutant of the M2 transmembrane domain using solid-state NMR. C chemical shifts of the membrane-bound peptide indicate the presence of two distinct tetramer conformations, whose concentrations depend exclusively on pH and hence the charge-state distribution of the tetramers. High-temperature 2D correlation spectra indicate that these two conformations interconvert at a rate of ∼400 s when the +2 and +3 charge states dominate, which gives the first experimental evidence of protein conformational motion on the transport time scale. Protein C-detected waterH T relaxation measurements show that channel water relaxes an order of magnitude faster than bulk water and membrane-associated water, indicating that channel water undergoes nanosecond motion in a pH-independent fashion. These results connect motions on three time scales to explain M2's proton-conduction mechanism: picosecond-to-nanosecond motions of water molecules facilitate proton Grotthuss hopping, microsecond motions of the histidine side chain allow water-histidine proton transfer, while millisecond motions of the entire four-helix bundle constitute the rate-limiting step, dictating the number of protons released into the virion.

The influenza B M2 protein forms a water-filled tetrameric channel to conduct protons across the lipid membrane. To understand how channel water mediates proton transport, we have investigated the water orientation and dynamics using solid-state NMR spectroscopy and molecular dynamics (MD) simulations. 13C-detected water 1H NMR relaxation times indicate that water has faster rotational motion in the low-pH open channel than in the high-pH closed channel. Despite this faster dynamics, the open-channel water shows higher orientational order, as manifested by larger motionally-averaged 1H chemical shift anisotropies. MD simulations indicate that this order is induced by the cationic proton-selective histidine at low pH. Furthermore, the water network has fewer hydrogen-bonding bottlenecks in the open state than in the closed state. Thus, faster dynamics and higher orientational order of water molecules in the open channel establish the water network structure that is necessary for proton hopping.