Conformation and Lipid Interaction of the Fusion Peptide of the Paramyxovirus PIV5 in Anionic and Negative-Curvature Membranes from Solid-State NMR

Viral fusion proteins catalyze the merger of the virus envelope and the target cell membrane through multiple steps of protein conformational changes. The fusion peptide domain of these proteins is important for membrane fusion, but how it causes membrane curvature and dehydration is still poorly understood. We now use solid-state NMR spectroscopy to investigate the conformation, topology, and lipid and water interactions of the fusion peptide of the PIV5 virus F protein in three lipid membranes, POPC/POPG, DOPC/DOPG, and DOPE. These membranes allow us to investigate the effects of lipid chain disorder, membrane surface charge, and intrinsic negative curvature on the fusion peptide structure. Chemical shifts and spin diffusion data indicate that the PIV5 fusion peptide is inserted into all three membranes but adopts distinct conformations: it is fully α-helical in the POPC/POPG membrane, adopts a mixed strand/helix conformation in the DOPC/DOPG membrane, and is primarily a β-strand in the DOPE membrane. ³¹P NMR spectra show that the peptide retains the lamellar structure and hydration of the two anionic membranes. However, it dehydrates the DOPE membrane, destabilizes its inverted hexagonal phase, and creates an isotropic phase that is most likely a cubic phase. The ability of the β-strand conformation of the fusion peptide to generate negative Gaussian curvature and to dehydrate the membrane may be important for the formation of hemifusion intermediates in the membrane fusion pathway

Immobilized molybdenum acetylacetonate complex on expanded starch for chemoselective oxidation of sulfides to sulfoxides with <i>t</i>-BuOOH at room temperature

<p></p> <p>We explored a highly efficient protocol for the oxidation of alkyl aryl sulfides to sulfoxides with high selectivities catalyzed by a molybdenum acetylacetonate complex immobilized on expanded corn starch (ECS-MoO₂(acac)₂) in the presence of 70% <i>t</i>-BuOOH solution (water). We obtained predominantly the monooxygenated product. The resulting products were obtained in good to excellent yields within a reasonable time. The catalyst could readily be separated from the reaction mixture and reused for several runs without significant loss in catalytic efficiency.</p

¹⁵N and ¹H Solid-State NMR Investigation of a Canonical Low-Barrier Hydrogen-Bond Compound: 1,8-Bis(dimethylamino)naphthalene

Strong or low-barrier hydrogen bonds have often been proposed in proteins to explain enzyme catalysis and proton-transfer reactions. So far ¹H chemical shifts and scalar couplings have been used as the main NMR spectroscopic signatures for strong H-bonds. In this work, we report simultaneous measurements of ¹⁵N and ¹H chemical shifts and N–H bond lengths by solid-state NMR in ¹⁵N-labeled 1,8-bis­(dimethylamino)­naphthalene (DMAN), which contains a well-known strong NHN H-bond. We complexed DMAN with three different counteranions to examine the effects of the chemical environment on the H-bond lengths and chemical shifts. All three DMAN compounds exhibit significantly elongated N–H distances compared to the covalent bond length, and the ¹H^N chemical shifts are larger than ∼17 ppm, consistent with strong NHN H-bonds in the DMAN cation. However, the ¹⁵N and ¹H chemical shifts and the precise N–H distances differ among the three compounds, and the ¹⁵N chemical shifts show opposite dependences on the proton localization from the general trend in organic compounds, indicating the significant effects of the counteranions on the electronic structure of the H-bond. These data provide useful NMR benchmarks for strong H-bonds and caution against the sole reliance on chemical shifts for identifying strong H-bonds in proteins since neighboring side chains can exert influences on chemical shifts similar to those of the bulky organic anions in DMAN. Instead, N–H bond lengths should be measured, in conjunction with chemical shifts, as a more fundamental parameter of H-bond strength

Pectin–Cellulose Interactions in the <i>Arabidopsis</i> Primary Cell Wall from Two-Dimensional Magic-Angle-Spinning Solid-State Nuclear Magnetic Resonance

The primary cell wall of higher plants consists of a mixture of polysaccharides whose spatial proximities and interactions with each other are not well understood. We recently obtained the first two-dimensional (2D) and three-dimensional high-resolution magic-angle-spinning ¹³C solid-state nuclear magnetic resonance spectra of the uniformly ¹³C-labeled primary cell wall of <i>Arabidopsis thaliana</i>, which allowed us to assign the majority of ¹³C resonances of the three major classes of polysaccharides: cellulose, hemicellulose, and pectins. In this work, we measured the intensity buildup of ¹³C–¹³C cross-peaks in a series of 2D ¹³C correlation spectra to obtain semiquantitative information about the spatial proximities between different polysaccharides. Comparison of 2D spectra measured at different spin diffusion mixing times identified intermolecular pectin–cellulose cross-peaks as well as interior cellulose–surface cellulose cross-peaks. The intensity buildup time constants are only modestly longer for cellulose–pectin cross-peaks than for interior cellulose–surface cellulose cross-peaks, indicating that pectins come into direct contact with the cellulose microfibrils. Approximately 25–50% of the cellulose chains exhibit close contact with pectins. The ¹³C magnetization of the wall polysaccharides is not fully equilibrated by 1.5 s, indicating that pectins and cellulose are not homogeneously mixed on the molecular level. We also assigned the ¹³C signals of cell wall proteins, identifying common residues such as Pro, Hyp, Tyr, and Ala. The chemical shifts indicate significant coil and sheet conformations in these structural proteins. Interestingly, few cross- peaks were observed between the proteins and the polysaccharides. Taken together, these data indicate that the three major types of polysaccharides in the primary wall of <i>Arabidopsis</i> form a single cohesive network, while structural proteins form a relatively separate domain

Distinguishing Bicontinuous Lipid Cubic Phases from Isotropic Membrane Morphologies Using ³¹P Solid-State NMR Spectroscopy

Nonlamellar lipid membranes are frequently induced by proteins that fuse, bend, and cut membranes. Understanding the mechanism of action of these proteins requires the elucidation of the membrane morphologies that they induce. While hexagonal phases and lamellar phases are readily identified by their characteristic solid-state NMR line shapes, bicontinuous lipid cubic phases are more difficult to discern, since the static NMR spectra of cubic-phase lipids consist of an isotropic ³¹P or ²H peak, indistinguishable from the spectra of isotropic membrane morphologies such as micelles and small vesicles. To date, small-angle X-ray scattering is the only method to identify bicontinuous lipid cubic phases. To explore unique NMR signatures of lipid cubic phases, we first describe the orientation distribution of lipid molecules in cubic phases and simulate the static ³¹P chemical shift line shapes of oriented cubic-phase membranes in the limit of slow lateral diffusion. We then show that ³¹P <i>T</i>₂ relaxation times differ significantly between isotropic micelles and cubic-phase membranes: the latter exhibit 2 orders of magnitude shorter <i>T</i>₂ relaxation times. These differences are explained by the different time scales of lipid lateral diffusion on the cubic-phase surface versus the time scales of micelle tumbling. Using this relaxation NMR approach, we investigated a DOPE membrane containing the transmembrane domain (TMD) of a viral fusion protein. The static ³¹P spectrum of DOPE shows an isotropic peak, whose <i>T</i>₂ relaxation times correspond to that of a cubic phase. Thus, the viral fusion protein TMD induces negative Gaussian curvature, which is an intrinsic characteristic of cubic phases, to the DOPE membrane. This curvature induction has important implications to the mechanism of virus–cell fusion. This study establishes a simple NMR diagnostic probe of lipid cubic phases, which is expected to be useful for studying many protein-induced membrane remodeling phenomena in biology

Paramagnetic Cu(II) for Probing Membrane Protein Structure and Function: Inhibition Mechanism of the Influenza M2 Proton Channel

Paramagnetic Cu­(II) ions enhance nuclear spin relaxation in a distance-dependent fashion and can be used as a structural probe of proteins. Cu­(II) can also serve as a functionally important ligand in proteins. Here we investigate the structural basis of Cu­(II) inhibition of the influenza M2 proton channel through Cu­(II)-induced paramagnetic relaxation enhancement (PRE). ¹³C <i>T</i>₁ relaxation rates of the central residues of the transmembrane (TM) domain of M2 are significantly enhanced by Cu­(II), and pronounced spectral broadening is observed for the proton-selective residue, His37. These data yielded quantitative distances of ¹³C spins to the Cu­(II) center and identified the Cu­(II) binding site to be Nε2 of His37. This binding site is surrounded by four imidazole rings from the top and four indole rings of Trp41 from the bottom, thus explaining the high affinity of Cu­(II) binding. Bound at this location, Cu­(II) can inhibit proton currents by perturbing histidine–water proton exchange, preventing histidine conformational dynamics, and interfering with His-Trp cation−π interaction. The Cu­(II) binding site is distinct from the binding site of the hydrophobic drug amantadine, which is about 10 Å N-terminal to His37. Consistently, Cu­(II) and amantadine induce distinct conformational changes at several key residues, suggesting the possibility of designing new drugs that target the His37 site to inhibit amantadine-resistant mutant M2 proteins. In addition to the high-affinity His37 binding site, we also examined the weaker and nonspecific binding of Cu­(II) to membrane–surface lipid phosphates and the extent of the resulting PRE to surface–proximal protein residues. This study demonstrates the feasibility of NMR studies of paramagnetic-ion-complexed membrane proteins, where the ion serves as both a functional ligand and a distance probe

Transport-Relevant Protein Conformational Dynamics and Water Dynamics on Multiple Time Scales in an Archetypal Proton Channel: Insights from Solid-State NMR

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^–1, 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. ¹³C 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^–1 when the +2 and +3 charge states dominate, which gives the first experimental evidence of protein conformational motion on the transport time scale. Protein ¹³C-detected water ¹H T₂ 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

NMR Detection of pH-Dependent Histidine–Water Proton Exchange Reveals the Conduction Mechanism of a Transmembrane Proton Channel

The acid-activated proton channel formed by the influenza M2 protein is important for the life cycle of the virus. A single histidine, His37, in the M2 transmembrane domain (M2TM) is responsible for pH activation and proton selectivity of the channel. Recent studies suggested three models for how His37 mediates proton transport: a shuttle mechanism involving His37 protonation and deprotonation, a H-bonded imidazole–imidazolium dimer model, and a transporter model involving large protein conformational changes in synchrony with proton conduction. Using magic-angle-spinning (MAS) solid-state NMR spectroscopy, we examined the proton exchange and backbone conformational dynamics of M2TM in a virus-envelope-mimetic membrane. At physiological temperature and pH, ¹⁵N NMR spectra show fast exchange of the imidazole ¹⁵N between protonated and unprotonated states. To quantify the proton exchange rates, we measured the ¹⁵N <i>T</i>₂ relaxation times and simulated them for chemical-shift exchange and fluctuating N–H dipolar fields under ¹H decoupling and MAS. The exchange rate is 4.5 × 10⁵ s^–1 for Nδ1 and 1.0 × 10⁵ s^–1 for Nε2, which are approximately synchronized with the recently reported imidazole reorientation. Binding of the antiviral drug amantadine suppressed both proton exchange and ring motion, thus interfering with the proton transfer mechanism. By measuring the relative concentrations of neutral and cationic His as a function of pH, we determined the four p<i>K</i>_a values of the His37 tetrad in the viral membrane. Fitting the proton current curve using the charge-state populations from these p<i>K</i>_a’s, we obtained the relative conductance of the five charge states, which showed that the +3 channel has the highest time-averaged unitary conductance. At physiologically relevant pH, 2D correlation spectra indicated that the neutral and cationic histidines do not have close contacts, ruling out the H-bonded dimer model. Moreover, a narrowly distributed nonideal helical structure coexists with a broadly distributed ideal helical conformation without interchange on the sub-10 ms time scale, thus excluding the transporter model in the viral membrane. These data support the shuttle mechanism of proton conduction, whose essential steps involve His–water proton exchange facilitated by imidazole ring reorientations

Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain

The fusion peptide (FP) and transmembrane domain (TMD) of viral fusion proteins play important roles during virus–cell membrane fusion, by inducing membrane curvature and transient dehydration. The structure of the water-soluble ectodomain of viral fusion proteins has been extensively studied crystallographically, but the structures of the FP and TMD bound to phospholipid membranes are not well understood. We recently investigated the conformations and lipid interactions of the separate FP and TMD peptides of parainfluenza virus 5 (PIV5) fusion protein F using solid-state nuclear magnetic resonance. These studies provide structural information about the two domains when they are spatially well separated in the fusion process. To investigate how these two domains are structured relative to each other in the postfusion state, when the ectodomain forms a six-helix bundle that is thought to force the FP and TMD together in the membrane, we have now expressed and purified a chimera of the FP and TMD, connected by a Gly-Lys linker, and measured the chemical shifts and interdomain contacts of the protein in several lipid membranes. The FP–TMD chimera exhibits α-helical chemical shifts in all the membranes examined and does not cause strong curvature of lamellar membranes or membranes with negative spontaneous curvature. These properties differ qualitatively from those of the separate peptides, indicating that the FP and TMD interact with each other in the lipid membrane. However, no ¹³C–¹³C cross peaks are observed in two-dimensional correlation spectra, suggesting that the two helices are not tightly associated. These results suggest that the ectodomain six-helix bundle does not propagate into the membrane to the two hydrophobic termini. However, the loosely associated FP and TMD helices are found to generate significant negative Gaussian curvature to membranes that possess spontaneous positive curvature, consistent with the notion that the FP–TMD assembly may facilitate the transition of the membrane from hemifusion intermediates to the fusion pore

Hydrogen-Bonding Partner of the Proton-Conducting Histidine in the Influenza M2 Proton Channel Revealed From ¹H Chemical Shifts

The influenza M2 protein conducts protons through a critical histidine (His) residue, His37. Whether His37 only interacts with water to relay protons into the virion or whether a low-barrier hydrogen bond (LBHB) also exists between the histidines to stabilize charges before proton conduction is actively debated. To address this question, we have measured the imidazole ¹H^N chemical shifts of His37 at different temperatures and pH using 2D ¹⁵N–¹H correlation solid-state NMR. At low temperature, the H^N chemical shifts are 8–15 ppm at all pH values, indicating that the His37 side chain forms conventional hydrogen bonds (H-bonds) instead of LBHBs. At ambient temperature, the dynamically averaged H^N chemical shifts are 4.8 ppm, indicating that the H-bonding partner of the imidazole is water instead of another histidine in the tetrameric channel. These data show that His37 forms H-bonds only to water, with regular strength, thus supporting the His–water proton exchange model and ruling out the low-barrier H-bonded dimer model