13C-13C and (15)N-(13)C correlation spectroscopy of membrane-associated and uniformly labeled human immunodeficiency virus and influenza fusion peptides: amino acid-type assignments and evidence for multiple conformations

Many viruses which cause disease including human immunodeficiency virus (HIV) and influenza are "enveloped" by a membrane and infection of a host cell begins with joining or "fusion" of the viral and target cell membranes. Fusion is catalyzed by viral proteins in the viral membrane. For HIV and for the influenza virus, these fusion proteins contain an approximately 20-residue apolar "fusion peptide" that binds to target cell membranes and plays a critical role in fusion. For this study, the HIV fusion peptide (HFP) and influenza virus fusion peptide (IFP) were chemically synthesized with uniform (13)C, (15)N labeling over large contiguous regions of amino acids. Two-dimensional (13)C-(13)C and (15)N-(13)C spectra were obtained for the membrane-bound fusion peptides and an amino acid-type (13)C assignment was obtained for the labeled residues in HFP and IFP. The membrane used for the HFP sample had a lipid headgroup and cholesterol composition comparable to that of host cells of the virus, and the (13)C chemical shifts were more consistent with beta strand conformation than with helical conformation. The membrane used for the IFP sample did not contain cholesterol, and the chemical shifts of the dominant peaks were more consistent with helical conformation than with beta strand conformation. There were additional peaks in the IFP spectrum whose shifts were not consistent with helical conformation. An unambiguous (13)C and (15)N assignment was obtained in an HFP sample with more selective labeling, and two shifts were identified for the Leu-9 CO, Gly-10 N, and Gly-10 Calpha nuclei. These sets of two shifts may indicate two beta strand registries such as parallel and antiparallel. Although most spectra were obtained on a 9.4 T instrument, one (13)C-(13)C correlation spectrum was obtained on a 16.4 T instrument and was better resolved than the comparable 9.4 T spectrum. More selective labeling and higher field may, therefore, be approaches to obtaining unambiguous assignments for membrane-associated fusion peptides.     

Solid-state NMR yields structural constraints on the V3 loop from HIV-1 Gp120 bound to the 447-52D antibody Fv fragment

Solid-state NMR measurements were performed on the complex of an 18-residue peptide derived from the V3 loop sequence of the gp120 envelope glycoprotein of the HIV-1 MN strain with Fv fragments of the human anti-gp120 monoclonal antibody 447-52D in a frozen glycerol/water solution. The peptide was uniformly (15)N- and (13)C-labeled in a 7-residue segment containing the conserved GPGR motif in the epitope. (15)N and (13)C NMR chemical shift assignments for the labeled segment were obtained from two-dimensional (13)C-(13)C and (15)N-(13)C magic-angle spinning NMR spectra. Reductions in (13)C NMR line widths and changes in chemical shifts upon complex formation indicate the adoption of a well-defined, antibody-dependent structure. Intramolecular (13)C-(13)C distances in the complex, which constrain the peptide backbone and side chain conformations in the GPGR motif, were determined from an analysis of rotational resonance (RR) data. Structural constraints from chemical shifts and RR measurements are in good agreement with recent solution NMR and crystallographic studies of this system, although differences regarding structural ordering of certain peptide side chains are noted. These experiments explore and help delineate the utility of solid state NMR techniques as structural probes of peptide/protein complexes in general, potentially including membrane-associated hormone/receptor complexes.     

Solid-state NMR spectroscopy of human immunodeficiency virus fusion peptides associated with host-cell-like membranes: 2D correlation spectra and distance measurements support a fully extended conformation and models for specific antiparallel strand registries

The human immunodeficiency virus (HIV) is "enveloped" by a membrane, and infection of a host cell begins with fusion between viral and target cell membranes. Fusion is catalyzed by the HIV gp41 protein which contains a functionally critical approximately 20-residue apolar "fusion peptide" (HFP) that associates with target cell membranes. In this study, chemically synthesized HFPs were associated with host-cell-like membranes and had "scatter-uniform" labeling (SUL), that is, only one residue of each amino acid type was U-(13)C, (15)N labeled. For the first sixteen HFP residues, an unambiguous (13)C chemical shift assignment was derived from 2D (13)C/(13)C correlation spectra with short mixing times, and the shifts were consistent with continuous beta-strand conformation. (13)C-(13)C contacts between residues on adjacent strands were derived from correlation spectra with long mixing times and suggested close proximity of the following residues: Ala-6/Gly-10, Ala-6/Phe-11, and Ile-4/Gly-13. Specific antiparallel beta-strand registries were further tested using a set of HFPs that were (13)CO-labeled at Ala-14 and (15)N-labeled at either Val-2, Gly-3, Ile-4, or Gly-5. The solid-state NMR data were fit with 50-60% population of antiparallel HFP with either Ala-14/Gly-3 or Ala-14/Ile-4 registries and 40-50% population of structures not specified by the NMR experiments. The first two registries correlated with intermolecular hydrogen bonding of 15-16 apolar N-terminal residues and this hydrogen-bonding pattern would be consistent with a predominant location of these residues in the hydrophobic membrane interior. To our knowledge, these results provide the first residue-specific structural models for membrane-associated HFP in its beta-strand conformation.     

Isotropic chemical shifts in magic-angle spinning NMR spectra of proteins

Here we examine the effect of magic-angle spinning (MAS) rate upon lineshape and observed peak position for backbone carbonyl (C') peaks in NMR spectra of uniformly-(13)C,15N-labeled (U-(13)C,15N) solid proteins. 2D N-C' spectra of U-(13)C,15N microcrystalline protein GB1 were acquired at six MAS rates, and the site-resolved C' lineshapes were analyzed by numerical simulations and comparison to spectra from a sparsely labeled sample (derived from 1,3-(13)C-glycerol). Spectra of the U-(13)C,15N sample demonstrate large variations in the signal-to-noise ratio and peak positions, which are absent in spectra of the sparsely labeled sample, in which most 13C' sites do not possess a directly bonded 13CA. These effects therefore are a consequence of rotational resonance, which is a well-known phenomenon. Yet the magnitude of this effect pertaining to chemical shift assignment has not previously been examined. To quantify these effects in high-resolution protein spectra, we performed exact numerical two- and four-spin simulations of the C' lineshapes, which reproduced the experimentally observed features. Observed peak positions differ from the isotropic shift by up to 1.0 ppm, even for MAS rates relatively far (a few ppm) from rotational resonance. Although under these circumstances the correct isotropic chemical shift values may be determined through simulation, systematic errors are minimized when the MAS rate is equivalent to approximately 85 ppm for 13C. This moderate MAS condition simplifies spectral assignment and enables data sets from different labeling patterns and spinning rates to be used most efficiently for structure determination.     

Simple organofluorine compounds giving field-dependent 13C and 19F NMR spectra with complex patterns: higher order effects and cross-correlated relaxation

The CF(3) signals in the (13)C{(1)H} spectrum of 1,1,1,3,3,3-hexafluoroisopropyl alcohol and the (CF(3))(2) CH signals in the corresponding triflate exhibit much greater complexity than might first be expected. The same holds for the (13)C satellites in the (19)F spectra. Complex patterns appear because of higher order effects resulting from the combination of a relatively large four-bond (19)F-(19)F J coupling in the ((13)CF(3))(12)CH((12)CF(3))-containing isotopomer and a typical large one-bond (13)C/(12)C isotope effect on the (19)F chemical shift. This complexity cannot be eliminated at very high magnetic field strengths. The triflate (CF(3))(2)CH-O-SO(2)CF(3) presents still additional complexity because of the presence of two different types of CF(3) groups exhibiting (6)J(FF) in any of the isotopomers and the chemical shift differences in hertz between the various (19)F signals in the two different (13)CF(3)-containing isotopomers. In addition, the presence of a small (5)J(CF) in the ((13)CF(3))((12)CF(3))(12)CH-O-SO(2) (12)CF(3) isotopomer is revealed only through simulations. The hexafluoroisopropyl CF(3) groups in the alcohol and triflate and the SO(2)CF(3) group in the triflate apparently provide the first examples of cross-correlated relaxation in (13)CF(3) groups. An analysis of the spectra in the context of previously reported work highlights the novel aspects of our findings. In particular, for each part of the complex hexafluoroisopropyl CF(3) quartet, peak height and linewidth variations resulting from cross-correlated relaxation are observed. These variations within a group of (13)C signals reflect different spin-lattice and spin-spin relaxation rates for the transitions within that group arising from higher order coupling effects.     

Spin diffusion driven by R-symmetry sequences: applications to homonuclear correlation spectroscopy in MAS NMR of biological and organic solids

We present a family of homonuclear (13)C-(13)C magic angle spinning spin diffusion experiments, based on R2(n)(v) (n = 1 and 2, v = 1 and 2) symmetry sequences. These experiments are well suited for (13)C-(13)C correlation spectroscopy in biological and organic systems and are especially advantageous at very fast MAS conditions, where conventional PDSD and DARR experiments fail. At very fast MAS frequencies the R2(1)(1), R2(2)(1), and R2(2)(2) sequences result in excellent quality correlation spectra both in model compounds and in proteins. Under these conditions, individual R2(n)(v) display different polarization transfer efficiency dependencies on isotropic chemical shift differences: R2(2)(1) recouples efficiently both small and large chemical shift differences (in proteins these correspond to aliphatic-to-aliphatic and carbonyl-to-aliphatic correlations, respectively), while R2(1)(1) and R2(2)(2) exhibit the maximum recoupling efficiency for the aliphatic-to-aliphatic or carbonyl-to-aliphatic correlations, respectively. At moderate MAS frequencies (10-20 kHz), all R2(n)(v) sequences introduced in this work display similar transfer efficiencies, and their performance is very similar to that of PDSD and DARR. Polarization transfer dynamics and chemical shift dependencies of these R2-driven spin diffusion (RDSD) schemes are experimentally evaluated and investigated by numerical simulations for [U-(13)C,(15)N]-alanine and the [U-(13)C,(15)N] N-formyl-Met-Leu-Phe (MLF) tripeptide. Further applications of this approach are illustrated for several proteins: spherical assemblies of HIV-1 U-(13)C,(15)N CA protein, U-(13)C,(15)N-enriched dynein light chain DLC8, and sparsely (13)C/uniformly (15)N enriched CAP-Gly domain of dynactin. Due to the excellent performance and ease of implementation, the presented R2(n)(v) symmetry sequences are expected to be of wide applicability in studies of proteins and protein assemblies as well as other organic solids by MAS NMR spectroscopy.     

Temperature effect on the 13C NMR chemical shift of pressurized CO2 containing selected modifiers

Temperature dependence of 13C NMR chemical shift of pressurized CO2 containing modifiers has been studied. Benzene, C6F6, C6F5OH, (CF3)2CHOH and [(CH3)2N]3PO were used as the modifiers. The 13C chemical shift of CO2 was found to show a different temperature dependence in the presence of selected modifiers.     

Shiftless nuclear magnetic resonance spectroscopy

The acquisition and analysis of high resolution one- and two-dimensional solid-state nuclear magnetic resonance (NMR) spectra without chemical shift frequencies are described. Many variations of shiftless NMR spectroscopy are feasible. A two-dimensional experiment that correlates the dipole-dipole and dipole-dipole couplings in the model peptide , (15)N labeled N-acetyl-leucine is demonstrated. In addition to the resolution of resonances from individual sites in a single crystal sample, the bond lengths and angles are characterized by the two-dimensional powder pattern obtained from a polycrystalline sample.     

Magic-angle-spinning NMR of the drug resistant S31N M2 proton transporter from influenza A

We report chemical shift assignments of the drug-resistant S31N mutant of M2(18-60) determined using 3D magic-angle-spinning (MAS) NMR spectra acquired with a (15)N-(13)C ZF-TEDOR transfer followed by (13)C-(13)C mixing by RFDR. The MAS spectra reveal two sets of resonances, indicating that the tetramer assembles as a dimer of dimers, similar to the wild-type channel. Helicies from the two sets of chemical shifts are shown to be in close proximity at residue H37, and the assignments reveal a difference in the helix torsion angles, as predicted by TALOS+, for the key resistance residue N31. In contrast to wild-type M2(18-60), chemical shift changes are minimal upon addition of the inhibitor rimantadine, suggesting that the drug does not bind to S31N M2.     

Solid-state NMR spectroscopy method for determination of the backbone torsion angle psi in peptides with isolated uniformly labeled residues

We demonstrate a solid-state nuclear magnetic resonance technique, with the acronym ROCSA-LG, for the determination of backbone torsion angles psi in peptides with multiple, but isolated, uniformly labeled residues. The method correlates the 13C' chemical shift anisotropy and the 13Calpha-1Halpha heteronuclear dipolar tensors within a single uniformly labeled residue in a two-dimensional (2D) experiment. The technique requires the measurement of only five 2D spectra and is compatible with high-speed magic-angle spinning. Experimental results are presented for the 17-residue alpha-helical peptide MB(i+4)EK and for amyloid fibrils formed by the 15-residue peptide Abeta11-25.     

Synthesis and spectroscopy of N3P3X5OCH=CH2 (X = Cl, F, OCH3, OCH2CF3, N(CH3)2) and N3P3X4(OCH=CH2)2 (X = Cl, N(CH3)2). Correlations of ultraviolet photoelectron spectroscopy and nuclear magnetic resonance data to electronic and geometrical structure

The syntheses of the vinyloxycyclotriphosphazene derivatives N3P3X5OCH=CH2 (X = OMe, OCH2CF3) and the N3P3(NMe2)4(OCH=CH2)2 isomeric mixture along with improved preparations of N3P3X5OCH=CH2 (X = F, NMe2) are reported. The interactions between the vinyloxy function and the cyclophosphazene in these and the previously reported N3P3Cl5 (OCH=CH2) and N3P3F6-n(OCH=CH2)n (n = 1-4) have been examined by ultraviolet photoelectron spectroscopy (UPS) and NMR spectroscopy. The UPS data for the chloro and fluoro derivatives show a strong electron-withdrawing effect of the phosphazene on the olefin that is mediated with decreasing halogen substitution. The 1H and 13C NMR data for N3P3X5OCH=CH2 (X = F, Cl, OMe, OCH2CF3, NMe2) show significant changes as a function of the phosphazene substituent. There is a linear correlation between the beta-carbon chemical shift on the vinyloxy unit and the phosphorus chemical shift at the vinyloxyphosphorus centers. The chemical shifts of the different phosphorus centers on each ring are also related in a linear fashion. These relationships may be understood in terms of the relative electron donor-acceptor abilities of the substituents on the phosphazene ring. The 1H NMR spectra of the N3P3(NMe2)4(OCH-CH2)2 isomeric mixture allow for assignment of the relative amounts of cis and trans isomers. A model for the observed cis preference in the formation of N3P3Cl4(OCH=CH)2 is presented.     

Freezing point depression of water in phospholipid membranes: a solid-state NMR study

Lipid-water interaction plays an important role in the properties of lipid bilayers, cryoprotectants, and membrane-associated peptides and proteins. The temperature at which water bound to lipid bilayers freezes is lower than that of free water. Here, we report a solid-state NMR investigation on the freezing point depression of water in phospholipid bilayers in the presence and absence of cholesterol. Deuterium NMR spectra at different temperatures ranging from -75 to + 10 degrees C were obtained from fully (2)H2O-hydrated POPC (1-palmitoyl-2-oleoylphosphatidylcholine) multilamellar vesicles (MLVs), prepared with and without cholesterol, to determine the freezing temperature of water and the effect of cholesterol on the freezing temperature of water in POPC bilayers. Our 2H NMR experiments reveal the motional behavior of unfrozen water molecules in POPC bilayers even at temperatures significantly below 0 degrees C and show that the presence of cholesterol further lowered the freezing temperature of water in POPC bilayers. These results suggest that in the presence of cholesterol the fluidity and dynamics of lipid bilayers can be retained even at very low temperatures as exist in the liquid crystalline phase of the lipid. Therefore, bilayer samples prepared with a cryoprotectant like cholesterol should enable the performance of multidimensional solid-state NMR experiments to investigate the structure, dynamics, and topology of membrane proteins at a very low temperature with enhanced sample stability and possibly a better sensitivity. Phosphorus-31 NMR data suggest that lipid bilayers can be aligned at low temperatures, while 15N NMR experiments demonstrate that such aligned samples can be used to enhance the signal-to-noise ratio of is 15N chemical shift spectra of a 37-residue human antimicrobial peptide, LL-37.     

Simultaneous detection and deconvolution of congested NMR spectra containing three isotopically labeled species

We present a procedure for isolating subspectra corresponding to individual protein or peptide components in a ternary mixture or complex. Each of the three-component species is labeled differently: species A uniformly with 15N, species B uniformly with 15N and 13C, and species C uniformly with 15N but selectively with 13C' or 13Calpha. By using the dual carbon label selective HSQC (DCLS-HSQC) pulse sequence and exploiting differences in 1J 15N-13C coupling patterns to filter selected 15N resonances from detection during a constant time period, a subspectrum from each species can be generated from three spectra acquired from a single sample. Many important biological pathways involve dynamic interactions among members of multicomponent protein assemblies, and this approach offers a powerful way to monitor such processes.     

Use of 13C as an indirect tag in 15N specifically labeled nucleosides. Syntheses of [8-13C-1,7,NH2-15N3]adenosine, -guanosine, and their deoxy analogues

We have previously reported the use of a 13C tag at the C2 of 15N-multilabeled purine nucleosides to distinguish the adjacent-labeled 15N atoms from those in an untagged nucleoside. We now introduce the use of an indirect tag at the C8 of 15N7-labeled purine nucleosides. This tag allows unambiguous differentiation between a pair of 15N7-labeled purines in which only one is 13C8 labeled. Although the very small C8-N7 coupling (<1 Hz) precludes its direct detection in 1D 15N spectra, 2D 1H-15N NMR experiments display the large C8-H8 coupling (>200 Hz) because H8 is coupled to both N7 and C8. The 13C8 atom is introduced by means of a ring closure of the exocyclic amino groups of a pyrimidinone using [13C]sodium ethyl xanthate. Here, we present methods for the syntheses of [8-13C-1,7,NH2-15N3]adenosine, -guanosine, and their deoxy analogues.     

Short polyglutamine peptide forms a high-affinity binding site for thioflavin-T at the N-terminus

Thioflavin-T is one of the most important amyloid specific dyes and has been used for more than 50 years; however, the molecular mechanism of staining is still not understood. Chemically synthesized short polyglutamine peptides (Q(n), n = 5-10) were subjected to the thioflavin-T (ThT) staining assay. It was found that the minimum Q(n) peptide that stained positive to ThT was Q(6). Two types of ThT-binding sites, a high-affinity site (k(d1) = 0.1-0.17 μM) and a low-affinity site (k(d2) = 5.7-7.4 μM), were observed in short polyQs (n = 6-9). (13)C{(2)H}REDOR NMR experiments were carried out to extract the local structure of ThT binding sites in Q(8) peptide aggregates by observing the intermolecular dipolar coupling between [3-Me-d(3)]ThT and natural abundance Q(8) or residue-specific [1,2-(13)C(2)] labeled Q(8)s. (13)C{(2)H}REDOR difference spectra of the [3-Me-d(3)]ThT/natural abundance Q(8) (1/9) complex indicated that all of the five carbons of the glutamine residue participated in the formation of ThT-binding sites. (13)C{(2)H}DQF-REDOR experiments of [3-Me-d(3)]ThT/residue-specific [1,2-(13)C(2)] labeled Q(8) (1/50) complexes demonstrated that the N-terminal glutamine residue had direct contact with the ThT molecule at the high-affinity ThT-binding sites.     

Lamellar structure in poly(ala-gly) determined by solid-state NMR and statistical mechanical calculations

Lamellar structure of poly(Ala-Gly) or (AG)n in the solid was examined using 13C solid-state NMR and statistical mechanical approaches. Two doubly labeled versions, [1-13C]Gly14[1-13C]Ala15- and [1-13C]Gly18[1-13C]Ala19 of (AG)15 were examined by two-dimensional (2D) 13C spin diffusion NMR in the solid state. In addition five doubly labeled [15N,13C]-versions of the same peptide, (AG) 15 and 15 versions labeled [3-13C] in each of the successive Ala residues were utilized for REDOR and 13C CP/MAS NMR measurements, respectively. The observed spin diffusion NMR spectra were consistent with a structure containing a combination of distorted beta-turns with a large distribution of the torsion angles and antiparallel beta-sheets. The relative proportion of the distorted beta-turn form was evaluated by examination of 13C CP/MAS NMR spectra of [3-13C]Ala-(AG)15. In addition, REDOR determinations showed five kinds of atomic distances between doubly labeled 13C and 15N nuclei which were also interpreted in terms of a combination of beta-sheets and beta-turns. Our statistical mechanical analysis is in excellent agreement with our Ala Cbeta 13C CP/MAS NMR data strongly suggesting that (AG)15 has a lamellar structure.     

CN-HMBC: a powerful NMR technique for the simultaneous detection of long-range 1H,13C and 1H,15N connectivities

[structure: see text] A new one-shot NMR experiment (CN-HMBC) is proposed for the simultaneous acquisition of 2D 1H,13C and 1H,15N HMBC spectra. Important sensitivity enhancements (up to 41% simultaneously for both 13C and 15N) or time savings (about 50%) can be achieved when compared to the separate acquisition of individual HMBC spectra. The experiment is highly recommended for the complete structural analysis and simultaneous chemical shift assignments of protonated and nonprotonated 13C and 15N resonances in nitrogen-containing organic compounds.     

Structural investigation of Titan tholins by solution-state 1H, 13C, and 15N NMR: one-dimensional and decoupling experiments

Titan, the largest moon of Saturn, is enveloped in a reddish brown organic haze. Titan haze is presumed to be formed from methane and nitrogen (CH(4) and N(2)) in Titan's upper atmosphere through energetic photochemistry and particle bombardment. Though Titan haze has been directly investigated using methods including the Cassini mission, its formation mechanism and the contributing chemical structures and prebiotic potential are still not well developed. We report here the structural investigation of the (13)C and (15)N labeled, simulated Titan haze aerosol (tholin) by solution-state NMR. The one-dimensional (1)H, (13)C, and (15)N NMR spectra and decoupling experiments indicate that the tholin sample contains amine, nitrile, imine, and N-heteroaromatic compounds of tremendous import in understanding complex organic chemistry in anaerobic, extraterrestrial environments.     

Solid-state NMR study of amyloid nanocrystals and fibrils formed by the peptide GNNQQNY from yeast prion protein Sup35p

Sup35p is a prion protein found in yeast that contains a prion-forming domain characterized by a repetitive sequence rich in Gln, Asn, Tyr, and Gly amino acid residues. The peptide GNNQQNY7-13 is one of the shortest segments of this domain found to form amyloid fibrils, in a fashion similar to the protein itself. Upon dissolution in water, GNNQQNY displays a concentration-dependent polymorphism, forming monoclinic and orthorhombic crystals at low concentrations and amyloid fibrils at higher concentrations. We prepared nanocrystals of both space groups as well as fibril samples that reproducibly contain three (coexisting) structural forms and examined the specimens with magic angle spinning (MAS) solid-state nuclear magnetic resonance. 13C and 15N MAS spectra of both nanocrystals and fibrils reveal narrow resonances indicative of a high level of microscopic sample homogeneity that permitted resonance assignments of all five species. We observed variations in chemical shift among the three dominant forms of the fibrils which were indicated by the presence of three distinct, self-consistent sets of correlated NMR signals. Similarly, the monoclinic and orthorhombic crystals exhibit chemical shifts that differ from one another and from the fibrils. Collectively, the chemical shift data suggest that the peptide assumes five conformations in the crystals and fibrils that differ from one another in subtle but distinct ways. This includes variations in the mobility of the aromatic Tyr ring. The data also suggest that various structures assumed by the peptide may be correlated to the "steric zipper" observed in the monoclinic crystals.