Glycopeptides related to beta-endorphin adopt helical amphipathic conformations in the presence of lipid bilayers

A series of glycosylated endorphin analogues designed to penetrate the blood-brain barrier (BBB) have been studied by circular dichroism and by 2D-NMR in the presence of water; TFE/water; SDS micelles; and in the presence of both neutral and anionic bicelles. In water, the glycopeptides showed only nascent helix behavior and random coil conformations. Chemical shift indices and nuclear Overhauser effects (NOE) confirmed helices in the presence of membrane mimics. NOE volumes provided distance constraints for molecular dynamics calculations used to provide detailed backbone conformations. In all cases, the glycopeptides were largely helical in the presence of membrane bilayer models (micelles or bicelles). Plasmon waveguide resonance (PWR) studies showed hen egg phosphatidyl choline (PC) bilayers produce amphipathic helices laying parallel to the membrane surface, with dissociation constants (K(D)) in the low nanomolar to micromolar concentration range. Two low-energy states are suggested for the glycosylated endorphin analogues, a flexible aqueous state and a restricted membrane bound state. Strong interactions between the glycopeptide amphipaths and membranes are crucial for penetration of the BBB via an endocytotic mechanism (transcytosis).     

Conformation of a diastereoisomeric pair of enkephalin analogues: A 400 MHz 1H NMR study

¹H NMR spectra recorded at 400 MHz of the enkephalin analogues Tyr-D -NIe-Gly-Phe-D (and L )-NIeS and the dipeptide Ac-Phe-D -NIeS as solutes in DMSO-d₆ are reported and assigned. A preferred conformation of the D ²L ⁵ peptide is proposed, based on evidence of an intramolecular hydrogen bond between Phe⁴ NH and Tyr¹ CO and NH/a-CH coupling constants. The possibility of conformational differences between the isomeric peptides accounting for differences in their ability to bind to δ-opiate receptors is discussed.     

Sequence and structural duality: designing peptides to adopt two stable conformations

To improve our understanding of conformational transitions in proteins, we are attempting the de novo design of peptides that switch structural state. Here, we describe coiled-coil peptides with sequence and structural duality; that is, features compatible with two different coiled-coil motifs superimposed within the same sequence. Specifically, we promoted a parallel leucine-zipper dimer under reducing conditions, and a monomeric helical hairpin in an intramolecularly disulfide bridged state. Using an iterative process, we engineered peptides that formed stable structures consistent with both targets under the different conditions. Finally, for one of the designs, we demonstrated a one-way switch from the helical hairpin to the coiled-coil dimer upon addition of disulfide-reducing agents.     

Self-assembled amphotericin B is probably surrounded by ergosterol: bimolecular interactions as evidenced by solid-state NMR and CD spectra

Amphotericin B (AmB) is thought to exert its pharmacological effects by forming a barrel-stave assembly with ergosterol in fungal membranes. To examine the interaction between AmB and ergosterol (Erg) or cholesterol (Cho), (13)C- and (19)F-labelled covalent conjugates were prepared as reported previously (N. Matsumori et al. Chem. Biol. 2004, 11, 673-679). The CD spectra of the conjugates in a membrane-bound form suggested that the distance between the heptaene moieties of the ergosterol conjugates AmB-C(2)-(6-F)Erg 2 and AmB-C(2)-Erg 3 is similar to that of AmB in ergosterol-containing membranes, but significantly larger than that of AmB in nonsterol or cholesterol-containing membranes. These observations suggest that, as is the case with ergosterol-containing membranes, the conjugated sterol moiety prevents the close contact between the heptaene moieties within the membrane that would reduce channel conductivity of the AmB assemblies. To further investigate this bimolecular interaction, we recorded the solid-state NMR spectra of conjugates 2 and AmB-C(2)-(6-F)Cho 4, which are composed of uniformly (13)C-labelled AmB and 6-fluorinated ergosterol or cholesterol; the conjugates were expected to facilitate the estimation of distances between the fluorine and carbon atoms. By using rotor-synchronous double resonance (rotational echo double resonance of X cluster; RDX) experiments, we deduced the distance between the fluorine atom and its nearest carbon atom in the heptaene moiety of 2 to be less than 8.6 A. This indicates that the B ring of ergosterol comes close to the AmB polyene moiety. A conformational search of the AmB-ergosterol conjugate by using distance constraints derived from the RDX results suggested that ergosterol molecules possibly surround the AmB assembly, which is in contrast with the conventional image in which ergosterol is inserted into AmB molecules.     

Peptide models II. Intramolecular interactions and stable conformations of glycine,alanine, and valine peptide analogues

Summary The stable conformations of N and C protected amino acids of the type: HCONH-CHR-CONH₂ of glycine,l-alanine andl-valine have been obtained by fully optimizedab-initio computations with a 3–21G basis set. An original procedure has been devised to extract the side-chain/backbone interaction energy and the backbone and side-chain distortion energies. This enables us to analyze the stabilization/destabilization effects introduced by the side-chain in terms of electrostatic, induction and steric hindrance contributions.Dedicated to Dr. A. Pullman     

Ring conformations and intermolecular interactions in two fused dibenzoazocines

5‐Acetyl‐2‐chloro‐8,11‐dimethyl‐5,6,11,12‐tetrahydrodibenzo[b,f]azocine, C₁₉H₂₀ClNO, (I), crystallizes as a single fully ordered isomer, but 14‐acetyl‐8,11‐dimethyl‐7,8,13,14‐tetrahydrobenzo[f]naphtho[1,2‐b]azocine–14‐acetyl‐8,9‐dimethyl‐7,8,13,14‐tetrahydrobenzo[f]naphtho[1,2‐b]azocine (74/26), C₂₃H₂₃NO, (II), exhibits threefold whole‐molecule disorder involving both configurational and structural isomers. In (I) and in the predominant form of (II), the azocine rings adopt very similar conformations, forming boat‐shaped rings having approximate twofold rotational symmetry. There are no direction‐specific intermolecular interactions in the crystal structure of (I), but the molecules of (II) are weakly linked into chains by an aromatic π–π stacking interaction. The compounds were made under green conditions using an acid‐catalysed cyclization process having very high atom utilization.     

Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity

We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation.     

The NMR spectra and conformations of cyclic compounds–III: The conformations of some pinane derivatives

The 220 MHz ¹H spectra of isoverbanone, nopinone and verbanone are reported. The spectra of the first two are completely assigned but that of verbanone only partially. The coupling constants obtained provide information about the conformation of these molecules. The isoverbanone molecule is almost Y shaped, but that of nopinone is between a Y shape and a half-chair conformation with the six membered ring bent away form the gem dimethyl groups. These conformations are consistent with the known steric interactions in these molecules.     

Aromatic C-H...O interactions in a series of bindone analogues. NMR and quantum mechanical study

The aromatic C-H...O hydrogen bonding within the series of the structurally relative indenone derivatives has been studied. The presence of the hydrogen bonds is corroborated by the large low-field chemical shifts of the protons involved in the hydrogen bond observed experimentally and reproduced by quantum mechanical calculations. Further confirmation is provided by analysis of the orbital overlap coefficients, (13)C NMR chemical shifts, and one-bond spin-spin coupling constants J((13)C-(1)H). The relationship between molecular geometry and (1)H NMR chemical shifts of involved protons has a complex nature, but the C-H...O distance is the principal factor.     

Azolium-linked cyclophanes: a comprehensive examination of conformations by 1H NMR spectroscopy and structural studies

The synthesis and characterization of a series of azolium-linked cyclophanes are reported. The cyclophanes consist of two azolium groups (17 examples) or three imidazolium groups (1 example) linked to two benzenoid units (benzene, naphthalene, p-xylene, mesitylene, 1,2,3,4- and 1,2,4,5-tetramethylbenzene, 2,6-pyridine, and p-tert-butylphenol) via methylene groups. Cyclophanes containing ortho-, meta-, and para-substitution patterns in the benzenoid units were examined. The conformations of the cyclophanes were examined in solution by variable-temperature NMR studies and in the solid state by crystallographic studies. The p-cyclophanes and mesitylene-based m- and o/m-cyclophanes are rigid on the NMR time scale, as indicated by sharp (1)H NMR spectra at all accessible temperatures. The non-mesitylene-based m-cyclophanes and the o-cyclophanes are fluxional on the NMR time scale at high temperatures, but in most cases, specific conformations can be "frozen out" at low temperatures. Many structures deduced from solution studies were consistent with those in the solid state.     

Synthesis and hybridization properties of l-oligodeoxynucleotide analogues fixed in a low anti glycosyl conformation

We have synthesized l-type enantiomers (cU and cA) of nucleoside analogues, whose glycosyl bonds are fixed in a low anti conformation (ap glycosyl conformation, [small chi][approximate] 180[degree]), and incorporated them into oligonucleotides to evaluate the hybridization ability with natural DNA and RNA sequences. Although the incorporation of the modified nucleosides into oligonucleotides decreased the hybridization ability with unmodified complementary DNA sequences, the fully-substituted 12mers (cU(12) and cA(12)) still retained the hybridization ability with the complementary unmodified DNA 12mers, regardless of their unnatural l-chirality. In contrast, cU(12) and cA(12) showed different hybridization behavior with complementary unmodified RNA 12mers. cU(12) forms a more stable duplex with rA(12) than the corresponding natural 12mer (dT(12)), whereas cA(12) cannot hybridize with rU(12). Based on the model structure of cU(12)-rA(12), we discuss these experimental results.     

C NMR spectra of cyclomercurated ferrocenylimines: Substituent effects and conformations in the ferrocenes

Three series of cyclomercurated ferrocenylimines (2-chloromercurio-ferrocenylimines) have been studied using ¹³C NMR spectroscopy. Good to excellent linear relationships have been found to exist between the chemical shifts of the carbon atoms in the ferrocenyl moiety and normal Hammett substituent constants σ_m and σ_p. The δ values of the iminyl carbon atoms show excellent linear correlations with the σ values. In three series of the ferrocenes, the sensitivity of the carbon atoms to the substituent effect is different, which is discussed in terms of the twist angle between the N-phenyl ring and the C---C=N---C plane. The relative sensitivity of the chemical shifts on different positions in ferrocenyl moiety to the substituent effect has also been presented.     

Virus-ligand interactions: identification and characterization of ligand binding by NMR spectroscopy

We demonstrate the detection and characterization of ligand binding to viruses via NMR. To illustrate the methodology, the interaction of an antiviral compound with human rhinovirus serotype 2 (HRV2) was investigated. Specific interaction of a capsid-binding inhibitor and native HRV2 was monitored utilizing saturation transfer difference (STD) NMR. STD NMR experiments at atomic resolution allowed those regions of the ligand that are involved in the interaction with the virus to be determined. The approach allows for (i) the fast and robust assessment of binding, (ii) the determination of the ligand binding epitope at atomic resolution without the necessity to crystallize virus-ligand complexes, and (iii) the reuse of the virus in subsequent assays. This methodology enables one to easily identify binding of drugs, peptides, and receptor or antibody fragments to the viral capsid.     

Structure and dynamics of surfactin studied by NMR in micellar media

The NMR structure of the cyclic lipopeptide surfactin from Bacillus subtilis was determined in sodium dodecyl sulfate (SDS) micellar solution. The two negatively charged side chains of surfactin form a polar head opposite to most hydrophobic side chains, accounting for its amphiphilic nature and its strong surfactant properties. Disorder was observed around the fatty acid chain, and 15N relaxation studies were performed to investigate whether it originates from a dynamic phenomenon. A very large exchange contribution to transverse relaxation rate R(2) was effectively observed in this region, indicating slow conformational exchange. Temperature variation and Carr-Purcell-Meiboom-Gill (CPMG) delay variation relaxation studies provided an estimation of the apparent activation energy around 35-43 kJ x mol(-1) and an exchange rate of about 200 ms(-1) for this conformational exchange. 15N relaxation parameters were also recorded in dodecylphosphocholine (DPC) micelles and DMSO. Similar chemical exchange around the fatty acid was found in DPC but not in DMSO, which demonstrates that this phenomenon only occurs in micellar media. Consequently, it may either reflect the disorder observed in our structures determined in SDS or originate from an interaction of the lipopeptide with the detergent, which would be qualitatively similar with an anionic (SDS) or a zwitterionic (DPC) detergent. These structural and dynamics results on surfactin are the first NMR characterization of a lipopeptide incorporated in micelles. Moreover, they provide a model of surfactin determined in a more biomimetic environment than an organic solvent, which could be useful for understanding the molecular mechanism of its biological activity.     

Characterization of peptide-pyrazole interactions in solution by low-temperature NMR studies

Complexation of the amino- and carboxyl-protected tripeptide Piv-L-Val-L-Val-L-Val-tBu with 3-methylpyrazole and 3-amino-5-methylpyrazole was studied by low-temperature NMR experiments in a freonic solvent. The peptide forms an extended beta-type structure at all temperatures and associates through hydrogen bonding with the two pyrazole-based beta-sheet ligands. A detailed structural characterization of the formed complexes by one- and two-dimensional NMR experiments under slow exchange conditions was made possible by employing very low temperatures. The tripeptide associates to stable antiparallel dimers that are symmetrically capped on both sides by two pyrazole receptors to form 2:2 complexes. Amide groups of two neighboring residues in an extended conformation are involved in cyclic hydrogen bonds to the pyrazole. Based on amide chemical shift changes, the relative strength of intermolecular hydrogen bonds can be assessed and correlated with the electronic effects of the substituents on the pyrazole.     

NMR studies of conformations and dynamic processes—III : Cyclophanes with unsaturated bridges

Three cyclophanes, each displaying a different type of dynamic process, have been studied by NMR methods. The barriers to these processes are attributed mainly to the decrease in π-electron overlap between the benzene rings and adjacent double bonds which occurs in the transition state for each process. In [5₂] paracyclophanetetraene, two successive flippings of the benzene rings interconvert the two hydrogens in the methylene groups (Scheme 1). In tetramethyl [2₄] paracyclophanetetraene, the passage of one methyl group through the central cavity of the molecule interconverts two conformations of similar, but not equal, free energy (Scheme 2). In [2₆] orthoparacyclophanehexaene, the orthosubstituted rings change sides by passing through the centre of the cyclophane (Scheme 3).     

Highly populated turn conformations in natively unfolded tau protein identified from residual dipolar couplings and molecular simulation

Tau, a natively unstructured protein that regulates the organization of neuronal microtubules, is also found in high concentrations in neurofibrillary tangles of Alzheimer's disease and other neurodegenerative disorders. The conformational transition between these vastly different healthy and pathological forms remains poorly understood. We have measured residual dipolar couplings (RDCs), J-couplings, and nuclear Overhauser enhancement (NOE) in construct K18 of tau, containing all four repeat domains R1-R4. NHN RDCs were compared with prediction on the basis of a statistical model describing the intrinsic conformational sampling of unfolded proteins in solution. While local variation and relative amplitude of RDCs agrees with propensity-based prediction for most of the protein, homologous sequences in each repeat domain (DLKN, DLSN, DLSK, and DKFD in repeats R1-R4) show strong disagreement characterized by inversion of the sign of the central couplings. Accelerated molecular dynamic simulations (AMD) in explicit solvent revealed strong tendencies to form turns, identified as type I beta-turns for repeats R1-R3. Incorporation of the backbone dihedral sampling resulting from AMD into the statistical coil model closely reproduces experimental RDC values. These localized sequence-dependent conformational tendencies interrupt the propensity to sample more extended conformations in adjacent strands and are remarkably resistant to local environmental factors, as demonstrated by the persistence of the RDC signature even under harsh denaturing conditions (8 M urea). The role that this specific conformational behavior may play in the transition to the pathological form is discussed.     

The NMR spectra and conformations of cyclic compounds: IX—conformational studies of bicyclo(3,1,0)hexane derivatives by 13C NMR

The ¹³C spectra of α-thujene ( 1 ), isothujone ( 2 ), (?)isothujol ( 3 ), (+)neoisothujol ( 4 ), sabinol ( 5 ), dihydroumbellulone ( 6 ) and umbellulone ( 7 ) and the alcohol acetates are recorded and assigned. The C-6 chemical shift may be used in conjunction with the steric shift mechanism as a conformational probe in these molecules. The results obtained indicate that isothujol, neoisothujol, isothujone and dihydroumbellulone adopt boat-like conformations whilst sabinol has a much flatter conformation. Conjugation of the isolated double bond of α-thujene and the carbonyl group of dihydroumbellulone with the cyclopropane ring has virtually no effect on the ¹³C shifts, but those of umbellulone itself are anomalous, indicating conjugation of the α,β unsaturated ketone system with the cyclopropyl ring.     

Hydrogen-bonded LC nanocomposites: characterisation of nanoparticle-LC interactions by solid-state NMR and FTIR spectroscopies

Although the molecular-level nanoparticle-liquid crystal (NP-LC) interactions are key for forming stable NP dispersions in LC, characterisation of these interactions is scarce in the literature. Recently, we have developed hydrogen-bonded LC nanocomposites based on mesogenic carboxylic acids and NPs functionalised with hydrogen-bond acceptor/donor groups. Here, we apply solid-state ¹H and ¹³C NMR and Fourier transform infrared spectroscopies to model systems consisting of mixtures of trans-4-n-butylcyclohexanecarboxylic acid (4-BCHA), 4-hexylbenzoic acid (6BA) and 4,4?-bipyridine. The binary mixture, 4-BCHA:6BA, was also studied. The results were compared to monolayer quantities of 6BA and 4-BCHA adsorbed on pyridine functionalised zirconia NPs (ZrO₂-n₃-pyridine) to understand the different effects of the functionalised NPs on the LC properties.     

The conformations of bicyclo[3.1.0]hexane derivatives by 1H and 13C NMR

The ¹H and ¹³C spectra of bicyclo[3.1.0]hexanes and thujanes have been recorded and assigned. Application of the Karplus equation has yielded dihedral angles, and a computer calculation of the angle of ring buckle as a function of the main dihedral angles has been carried out. The calculated angles of ring buckle agree well with known values in the bicyclo[3.1.0]hexanes, but for 1-methylbicyclo[3.1.0]hexanes and thujanes the results are not self consistent. It is suggested that the bridgehead substituent causes the boat to twist, although the twist can be reduced by an axial methyl substituent on C-4.