Centerband-only Detection Of Exchange Research Articles

SARS-CoV-2 Envelope Protein Forms Clustered Pentamers in Lipid Bilayers.

The SARS-CoV-2 envelope (E) protein is a viroporin associated with the acute respiratory symptoms of COVID-19. E forms cation-selective ion channels that assemble in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment. The channel activity of E is linked to the inflammatory response of the host cell to the virus. Like many viroporins, E is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the E stoichiometry have led to inconclusive results and suggested mixtures of oligomers whose exact nature might vary with the detergent used. Here, we employ 19F solid-state nuclear magnetic resonance and the centerband-only detection of exchange (CODEX) technique to determine the oligomeric number of E’s transmembrane domain (ETM) in lipid bilayers. The CODEX equilibrium value, which corresponds to the inverse of the oligomeric number, indicates that ETM assembles into pentamers in lipid bilayers, without any detectable fraction of low-molecular-weight oligomers. Unexpectedly, at high peptide concentrations and in the presence of the lipid phosphatidylinositol, the CODEX data indicate that more than five 19F spins are within a detectable distance of about 2 nm, suggesting that the ETM pentamers cluster in the lipid bilayer. Monte Carlo simulations that take into account peptide–peptide and peptide–lipid interactions yielded pentamer clusters that reproduced the CODEX data. This supramolecular organization is likely important for E-mediated virus assembly and budding and for the channel function of the protein.

Probing Molecular Packing of Drug Substances in Nanometer Domains in Pharmaceutical Formulations Using 19F Magic Angle Spinning NMR

Structural details of molecular packing provide a fundamental understanding of the resulting material properties of pharmaceutical materials. For example, both the physical stability and dissolution rate of amorphous drugs are significantly enhanced by embedding into a polymeric network to form an amorphous solid dispersion. Despite the ubiquitous use of amorphous solid dispersions to formulate poorly water-soluble drugs, structural details elucidating the molecular mechanism of drug recrystallization remain elusive. In this study, solid-state NMR was used to quantitatively determine the size of the drug-rich domains in amorphous solid dispersions and determine the number of drug molecules within the polymer matrix by using 19F centerband-only detection of exchange (CODEX). Such results provide the highest resolution of oligomeric packing of drug molecules in the nanometer domain in an amorphous dispersion and offer a valuable structural basis for how drug–drug interaction induces recrystallization in a confined polymer matrix.

Centerband-Only Detection of Exchange NMR with Natural-Abundance Correction Reveals an Expanded Unit Cell in Phenylalanine Crystals.

The NMR pulse sequence CODEX (centerband‐only detection of exchange) is a widely used method to report on the number of magnetically inequivalent spins that exchange magnetization via spin diffusion. For crystals, this rules out certain symmetries, and the rate of equilibration is sensitive to distances. Here we show that for 13C CODEX, consideration of natural abundance spins is necessary for crystals of high complexity, demonstrated here with the amino acid phenylalanine. The NMR data rule out the C2 space group that was originally reported for phenylalanine, and are only consistent with a larger unit cell containing eight magnetically inequivalent molecules. Such an expanded cell was recently described based on single crystal data. The large unit cell dictates the use of long spin diffusion times of more than 200 seconds, in order to equilibrate over the entire unit cell volume of 1622 Å3.

UV Pretreatment Impairs the Enzymatic Degradation of Polyethylene Terephthalate.

The biocatalytic degradation of polyethylene terephthalate (PET) emerged recently as a promising alternative plastic recycling method. However, limited activity of previously known enzymes against post-consumer PET materials still prevents the application on an industrial scale. In this study, the influence of ultraviolet (UV) irradiation as a potential pretreatment method for the enzymatic degradation of PET was investigated. Attenuated total reflection Fourier transform infrared (ATR-FTIR) and 1H solution nuclear magnetic resonance (NMR) analysis indicated a shortening of the polymer chains of UV-treated PET due to intra-chain scissions. The degradation of UV-treated PET films by a polyester hydrolase resulted in significantly lower weight losses compared to the untreated sample. We also examined site-specific and segmental chain dynamics over a time scale of sub-microseconds to seconds using centerband-only detection of exchange, rotating-frame spin-lattice relaxation (T1ρ), and dipolar chemical shift correlation experiments which revealed an overall increase in the chain rigidity of the UV-treated sample. The observed dynamic changes are most likely associated with the increased crystallinity of the surface, where a decreased accessibility for the enzyme-catalyzed hydrolysis was found. Moreover, our NMR study provided further knowledge on how polymer chain conformation and dynamics of PET can mechanistically influence the enzymatic degradation.

Novel approaches to probe the binding of recoverin to membranes.

Recoverin is a protein involved in the phototransduction cascade by regulating the activity of rhodopsin kinase through a calcium-dependent binding process at the surface of rod outer segment disk membranes. We have investigated the interaction of recoverin with zwitterionic phosphatidylcholine bilayers, the major lipid component of the rod outer segment disk membranes, using both 31P and 19F solid-state nuclear magnetic resonance (NMR) and infrared spectroscopy. In particular, several novel approaches have been used, such as the centerband-only detection of exchange (CODEX) technique to investigate lipid lateral diffusion and 19F NMR to probe the environment of the recoverin myristoyl group. The results reveal that the lipid bilayer organization is not disturbed by recoverin. Non-myristoylated recoverin induces a small increase in lipid hydration that appears to be correlated with an increased lipid lateral diffusion. The thermal stability of recoverin remains similar in the absence or presence of lipids and Ca2+. Fluorine atoms have been strategically introduced at positions 4 or 12 on the myristoyl moiety of recoverin to, respectively, probe its behavior in the interfacial and more hydrophobic regions of the membrane. 19F NMR results allow the observation of the calcium-myristoyl switch, the myristoyl group experiencing two different environments in the absence of Ca2+ and the immobilization of the recoverin myristoyl moiety in phosphatidylcholine membranes in the presence of Ca2+.

Characterization of the Slow Molecular Dynamics of Poly(l‐Lactic Acid) in α and α′ Phases, in a Glassy State, and in a Complex with Poly(d‐Lactic Acid) by Solid‐State NMR

AbstractRecently, slow molecular dynamics of poly(l‐lactic acid) (PLLA) by using 1D and 2D exchange NMR are investigated. In this work, slow molecular dynamics of PLLA chains in the α′, a stereocomplex (SC) with poly(d‐lactic acid), and glassy states are investigated in terms of centerband‐only detection of exchange (CODEX) NMR. The mixing‐time dependence of the CODEX data demonstrates that the molecular dynamics of stems become slower in the order of α′, α, and SC. The temperature dependence of the correlation time 〈τc〉 of the helical jump motions in the α and SC phases simply exhibits Arrhenius behaviors, with activation energy, Ea, values of 91 ± 1 and 97 ± 1 kJ mol−1, respectively. In contrast, the temperature dependence of 〈τc〉 in the α′ sample exhibits two Arrhenius lines with substantially different Ea values of 273 ± 12 and 16 ± 14 kJ mol−1 at temperatures below and above 84 °C. The obtained kinetics of molecular dynamics not only establish the relationship between packing structure and dynamics in PLLA polymorphs and in the SC, but also allow for an understanding of the coupled dynamics between the crystalline and amorphous regions at approximately Tg.

Alamethicin Supramolecular Organization in Lipid Membranes from 19F Solid-State NMR

Alamethicins (ALMs) are antimicrobial peptides of fungal origin. Their sequences are rich in hydrophobic amino acids and strongly interact with lipid membranes, where they cause a well-defined increase in conductivity. Therefore, the peptides are thought to form transmembrane helical bundles in which the more hydrophilic residues line a water-filled pore. Whereas the peptide has been well characterized in terms of secondary structure, membrane topology, and interactions, much fewer data are available regarding the quaternary arrangement of the helices within lipid bilayers. A new, to our knowledge, fluorine-labeled ALM derivative was prepared and characterized when reconstituted into phospholipid bilayers. As a part of these studies, C19F3-labeled compounds were characterized and calibrated for the first time, to our knowledge, for 19F solid-state NMR distance and oligomerization measurements by centerband-only detection of exchange (CODEX) experiments, which opens up a large range of potential labeling schemes. The 19F-19F CODEX solid-state NMR experiments performed with ALM in POPC lipid bilayers and at peptide/lipid ratios of 1:13 are in excellent agreement with molecular-dynamics calculations of dynamic pentameric assemblies. When the peptide/lipid ratio was lowered to 1:30, ALM was found in the dimeric form, indicating that the supramolecular organization is tuned by equilibria that can be shifted by changes in environmental conditions.

Probing the two-stage transition upon crossing the glass transition of polystyrene by solid-state NMR

A two-stage transition upon crossing the glass transition of polystyrene with increasing temperature was precisely determined and interpreted by using solid-state nuclear magnetic resonance (SSNMR), 1H-1H dipolar couplings based double quantum-filtered (DQF) and dipolar filter (DF) experiments and 13C chemical shift anisotropy (CSA) based centerband-only detection of exchange (CODEX) experiment are used to fully characterize the time scale of molecular motions during the glass transition. While differential scanning calorimetry (DSC) and CODEX experiment predicted the first stage of glass transiton, DQF and DF experiments provided the evidence for the second stage transition during which the time scale of molecular motions changed from very slow (t > ms) to very fast (t < µs). The first stage of glass transition begins with the occurrence of remarkable slow re-orientation motions of the polymer backbone segments and ends when the degree of slow motion reaches maximum. The onset and endpoint of the conventional calorimetric glass transition of polystyrene can be quantitatively determined at the molecular level by SSNMR. In the second stage, a subsequent dramatic transition associated with the melting of the glassy components was observed. In this stage liquid-like NMR signals appeared and rapidly increased in intensity after a characteristic temperature T f (~1.1T g). The signals associated with the glassy components completely disappeared at another characteristic temperature T c (~1.2T g).

Insights into the Molecular Dynamics in Polysulfone Polymers from 13C Solid-State NMR Experiments

The molecular and segmental motions in three different grades of ductile polysulfone polymers; poly(ether sulfone) (PESU), polysulfone (PSU), and poly(phenyl sulfone) (PPSU) are probed using (13)C solid-state NMR experiments. Polarization inversion spin exchange at magic angle (PISEMA) experiments indicates that the phenyl rings in the polymers are undergoing π-flip motions on the order of 100 kHz. The temperature dependent PISEMA experiments show that the fraction of mobile regions that undergoes aromatic π-flips is higher in PPSU than in the other two polymers. The center band only detection of exchange (CODEX) experiments was carried out and was unable to detect any slow segmental motions in the chains. A combination of (13)C spin-lattice relaxation time (T1) and T1-filtered PISEMA experiments show that the mobile regions in all the polymers are dynamically heterogeneous.

Determination of Local Packing Structure of Mesomorphic Form of Isotactic Polypropylene by Solid-State NMR.

Understanding the local packing structures of a disordered mesomorphic phase is a challenging issue in polymer characterization. In this work, 13C-13C through-space interactions, as well as a molecular dynamics analysis based on the reorientation of chemical shift anisotropy (CSA), were proposed for the evaluation of the local packing structure of the mesomorphic form of isotactic polypropylene (iPP). 13C-13C double quantum (DQ) buildup curves of 13C 15% CH3 selectively labeled iPP and spin-dynamics simulations demonstrated that the local packing structures in the mesomorphic form were very similar to the packing in the β phase. Moreover, centerband only detection of exchange (CODEX) NMR proved that the correlation time ⟨τc⟩ of the overall stem dynamics in the mesomorphic form followed the same Arrhenius line observed for the β phase, but it deviated from that for the α phase. Based on both structural and dynamic results, it was concluded that the local packing structure in the mesomorphic form was exceedingly close or the same as that of the β phase.

Centerband-only-detection-of-exchange (31)P nuclear magnetic resonance and phospholipid lateral diffusion: theory, simulation and experiment.

Centerband-only-detection-of-exchange (CODEX) (31)P NMR lateral diffusion measurements were performed on dimyristoylphosphatidylcholine (DMPC) assembled into large unilamellar spherical vesicles. Optimization of sample and NMR acquisition conditions provided significant sensitivity enhancements relative to an earlier first report (Q. Saleem, A. Lai, H. Morales, and P. M. Macdonald, Chem. Phys. Lipids, 2012, 165, 721). An analytical description was developed that permitted the extraction of lateral diffusion coefficients from CODEX data, based on a Gaussian-diffusion-on-a-sphere model (A. Ghosh, J. Samuel, and S. Sinha, Europhys. Lett., 2012, 98, 30003-p1) as relevant to CODEX (31)P NMR measurements on a population of spherical unilamellar phospholipid bilayer vesicles displaying a distribution of vesicle radii.

Chain Mobility, Thermal, and Mechanical Properties of Poly(ethylene furanoate) Compared to Poly(ethylene terephthalate)

Poly(ethylene furanoate) (PEF), the furan-derived analogue to poly(ethylene terephthalate) (PET), can provide a fully biosourced alternative to PET with greatly improved barrier properties and attractive thermal and mechanical properties. The improved barrier for PEF compared to PET is unexpected due to the higher free volume of PEF vs PET. Segmental motions related to penetrant diffusion in both polyesters were studied using dynamic mechanical analysis, 13C–CP/MAS solid-state NMR variable contact-time experiments, and centerband-only detection of exchange (CODEX) measurements. Unlike the active phenyl ring-flipping mechanism in PET, furan ring-flipping is greatly suppressed, thereby reducing β relaxation motions and diffusion in PEF due to the energy penalty associated with the nonlinear axis of ring rotation and ring polarity. Preliminary work also shows similar oxygen solubilities for PEF and PET, thereby proving that the drastic permeability reduction results from a decrease in diffusion coefficient c...

Detection of slow dynamics by solid‐state NMR: Application to L‐phenylalanine hydrochloride

AbstractNew methods for probing dynamical properties of biological macromolecules on the millisecond timescale will enable a better understanding of the structure–function relationship. In this article, three solid‐state NMR detection methods, line‐shape analysis, two‐dimensional exchange experiments, and a variant of the center‐band only detection of exchange (CODEX) experiment called R‐CODEX, are used to characterize a crystalline amino acid that serves as a model for motions in solid proteins, L‐phenylalanine hydrochloride. The millisecond ring flip motion of the aromatic ring in L‐phenylalanine hydrochloride is characterized in detail for the first time. Limitations of these experiments for processes involving submillisecond timescales are also discussed. © 2013 Wiley Periodicals, Inc. Concepts Magn Reson Part A 42A: 14–22, 2013.

Molecular Dynamics of Poly(Ethylene Glycol) Intercalated in Clay, Studied Using 13C Solid-State NMR.

In this study, Cross-Polarization Magic-angle Spinning CP/MAS, 2D Exchange, Centerband-Only Detection of Exchange (CODEX), and Separated-Local-Field (SLF) NMR experiments were used to study the molecular dynamics of poly(ethylene glycol) (PEG) inside Hectorite/PEG intercalation compounds in both single- and double-layer configurations. The results revealed that the overall amplitude of the motions of the PEG chain in the single-layer configuration is considerably smaller than that observed for the double-layer intercalation compound. This result indicates that the effect of having the polymer chain interacting with both clay platelets is to produce a substantial decrease in the motional amplitudes of those chains. The presence of these dynamically restricted segments might be explained by the presence of anchoring points between the clay platelets and the PEG oxygen atoms, which was induced by the Na+ cations. By comparing the PEG motional amplitudes of the double-layered nanocomposites composed of polymers with different molecular weights, a decrease in the motional amplitude for the smaller PEG chain was observed, which might also be understood using the presence of anchoring points.

Investigation on the artificial exchange signals induced by the RIDER effect in CODEX experiments

The CODEX (center-band only detection of exchange) NMR experiment is widely used for the detection of slow motions in organic solids, especially polymers. However, the RIDER (relaxation-induced dipolar exchange with recoupling) effect may result in artificial exchange signals in the CODEX pure exchange spectrum, which greatly limits the application of CODEX method. Herein, we investigate the distance range that the RIDER effect can reach by performing CODEX experiments on two typical organic solids, hexadecyltrimethylammonium bromide (CTAB) and semi-crystalline polyamide-6 (PA6) where there are no slow molecular motions at room temperature. Our experimental results demonstrate that generally two-bond distance is far enough to ignore the RIDER effect resulted from the dipolar interactions between 13C and the fast relaxing heteronucleus 14N. From the built-up curve of RIDER signals as a function of recoupling time and mixing time, it is clearly revealed that the RIDER effect can greatly affect the signal from 13C directly bonded with 14N. However, this RIDER effect accounts less than 3% of the reference intensity for signals from 13C not directly bonded with 14N if typical recoupling (∼0.5ms) and mixing times (∼0.5s) are used for the investigation of slow motions. When longer recoupling and mixing time are used, there are small RIDER signals even for the 13C far away from the 14N. These signals, to a large degree, result from the spin diffusion effect and/or the special microscopic molecule arrangement. However, they are so small compared to the reference signal (∼5%) that they can be ignored. Finally, according to the simulation results, it is worth noting that the RIDER signal is still generally negligible compared to the signals due to slow motions if the chemical shift anisotropy reorientation during the mixing time is not too small(larger than 20°) under the condition of 4tr recoupling time at the magic-angle-spinning speed of 6.5kHz.

Correlations between Conjugation Length, Macromolecular Dynamics, and Photophysics of Phenylene-Vinylene/Aliphatic Multiblock Copolymers

This work reports a detailed spectroscopy study of a series of multiblock conjugated nonconjugated copolymers built by p-phenylene vinylene type units (PV) and octamethylene spacers, namely, poly(1,8-octanedioxy-2,6-dimethoxy-1,4-phenylene-1,2-ethenylene) (LaPPS18). The relative proportions of the PV and aliphatic segments were estimated on the basis of solid-state NMR and Raman spectroscopy. The overall structure was characterized by wide angle X-ray diffraction; (1)H wide-line dipolar chemical shift correlation (DIPSHIFT), and centerband-only detection of exchange (CODEX) NMR data, that together with glass transition temperatures allowed us to identify the groups involved in the molecular dynamics. These different structural properties were used to explain the photoluminescence properties in terms of peak position and spectral profile.

Chain Dynamics in Antiplasticized and Annealed Poly(ethylene terephthalate) Determined by Solid-State NMR and Correlated with Enhanced Barrier Properties

Using solid-state NMR, site-specific chain dynamics from the hertz to mid-kilohertz regime were examined in compression-molded films of poly(ethylene terephthalate) (PET), PET filled with 2 wt % acetanilide or phenacetin, and PET annealed below and above the glass transition temperature (Tg). More specifically, 13C relaxation time in the rotating frame, 1H–13C cross-polarization times, and 13C chemical-shift anisotropy exchange were measured at natural abundance. 13C CP/MAS spectra revealed that PET annealed above Tg was 28% crystalline, and all other PET samples were completely amorphous. Relaxation times in these polymers were analyzed using stretched exponentials which yielded characteristic time constants. The filled PET and PET annealed above Tg exhibited suppressed chain dynamics across the entire hertz to mid-kilohertz regime that were correlated with reduced CO2 and O2 permeabilities. Using the centerband-only detection of exchange (CODEX) sequence, the fraction of flipping phenylene rings was observed to decrease in these polymers, with the decrease in the semicrystalline sample consistent with the crystalline fraction.

Chain Packing in Glassy Polymers by Natural-Abundance 13C−13C Spin Diffusion Using 2D Centerband-Only Detection of Exchange

The proximities of specific subgroups of nearest-neighbor chains in glassy polymers are revealed by distance-dependent (13)C-(13)C dipolar couplings and spin diffusion. The measurement of such proximities is practical even with natural-abundance levels of (13)C using a 2D version of centerband-only detection of exchange (CODEX). Two-dimensional CODEX is a relaxation-compensated experiment that avoids the problems associated with variations in T(1)(C)'s due to dynamic site heterogeneity in the glass. Isotropic chemical shifts are encoded in the t(1) preparation times before and after mixing, and variations in T(2)'s are compensated by an S(0) reference (no mixing). Data acquisition involves acquisition of an S(0) reference signal on alternate scans, and the active control of power amplifiers, to achieve stability and accuracy over long accumulation times. The model system to calibrate spin diffusion is the polymer itself. For a mixing time of 200 ms, only (13)C-(13)C pairs separated by one or two bonds (2.5 Å) show cross peaks, which therefore identify reference intrachain proximities. For a mixing time of 1200 ms, 5 Å interchain proximities appear. The resulting cross peaks are used in a simple and direct way to compare nonrandom chain packing for two commercial polycarbonates with decidedly different mechanical properties.

Direct Observation of Millisecond to Second Motions in Proteins by Dipolar CODEX NMR Spectroscopy

We present a site-resolved study of slow (ms to s) motions in a protein in the solid (microcrystalline) state performed with the use of a modified version of the centerband-only detection of exchange (CODEX) NMR experiment. CODEX was originally based on measuring changes in molecular orientation by means of the chemical shift anisotropy (CSA) tensor, and in our modification, angular reorientations of internuclear vectors are observed. The experiment was applied to the study of slow (15)N-(1)H motions of the SH3 domain of chicken alpha-spectrin. The protein was perdeuterated with partial back-exchange of protons at labile sites. This allowed indirect (proton) detection of (15)N nuclei and thus a significant enhancement of sensitivity. The diluted proton system also made negligible proton-driven spin diffusion between (15)N nuclei, which interferes with the molecular exchange (motion) and hampers the acquisition of dynamic parameters. The experiment has shown that approximately half of the peaks in the 2D (15)N-(1)H correlation spectrum exhibit exchange in a different extent. The correlation time of the slow motion for most peaks is 1 to 3 s. This is the first NMR study of the internal dynamics of proteins in the solid state on the millisecond to second time scale with site-specific spectral resolution that provides both time-scale and geometry information about molecular motions.

Staphylococcus aureus Peptidoglycan Tertiary Structure from Carbon-13 Spin Diffusion

The cell-wall peptidoglycan of Staphylococcus aureus is a heterogeneous, highly cross-linked polymer of unknown tertiary structure. We have partially characterized this structure by measuring spin diffusion from (13)C labels in pentaglycyl cross-linking segments to natural-abundance (13)C in the surrounding intact cell walls. The measurements were performed using a version of centerband-only detection of exchange (CODEX). The cell walls were isolated from S. aureus grown in media containing [1-(13)C]glycine. The CODEX spin diffusion rates established that the pentaglycyl bridge of one peptidoglycan repeat unit of S. aureus is within 5 A of the glycan chain of another repeat unit. This surprising proximity is interpreted in terms of a model for the peptidoglycan lattice in which all peptide stems in a plane perpendicular to the glycan mainchain are parallel to one another.