Characterization of a type of β‐bend ribbon spiral generated by the repeating (Xaa–Yaa–Aib–Pro) motif: The solution structure of harzianin HC IX,a 14‐residue peptaibol forming voltage‐dependent ion channels

The three‐dimensional solution structure of harzianin HC IX, a peptaibol antibiotic isolated from the fungus Trichoderma harzianum, was determined using CD, homonuclear, and heteronuclear two‐dimensional nmr spectroscopy combined with molecular modeling. This 14‐residue peptide, Ac Aib¹ Asn² Leu³ Aib⁴ Pro⁵ Ala⁶ Ile⁷ Aib⁸ Pro⁹ Iva¹⁰ Leu¹¹ Aib¹² Pro¹³ Leuol¹⁴ (Aib, α‐aminoisobutyric acid; Iva, isovaline; Leuol, leucinol), is a main representative of a short‐sequence peptaibol class characterized by an acetylated N‐terminus, a C‐terminal amino alcohol, and the presence of three Aib‐L ‐Pro motifs at positions 4–5, 8–9, and 12–13, separated by two dipeptide units. In spite of a lower number of residues, compared to the 18/20‐residue peptaibols such as alamethicin, harzianin HC IX exhibits remarkable membrane‐perturbing properties. It interacts with phospholipid bilayers, increasing their permeability and forming voltage‐gated ion channels through a mechanism slightly differing from that proposed for alamethicin. Sequence‐specific ¹H‐ and ¹³C‐nmr assignments and conformational nmr parameters (³J_NHCαH coupling constants, quantitative nuclear Overhauser enhancement data, temperature coefficients of amide and carbonyl groups, NH–ND exchange rates) were obtained in methanol solution. Sixty structures were calculated based on 98 interproton distance restraints and 6 Φ dihedral angle restraints, using high temperature restrained molecular dynamics and energy minimization. Thirty‐seven out of the sixty generated structures were consistent with the nmr data and were convergent. The peptide backbone consists in a ribbon of overlapping β‐turns twisted into a continuous spiral from Asn² to Leuol¹⁴ and forming a 26 Å long helix‐like structure. This structure is slightly amphipathic, with the three Aib–Pro motifs aligned on the less hydrophobic face of the spiral where the Asn² side chain is also present, while the more hydrophobic bulky side chains of leucines, isoleucine, isovaline, and leucinol are located on the concave side. The repetitive (Xaa–Yaa–Aib–Pro) tetrapeptide subunit, making up the peptide sequence, is characterized by four sets of (Φ,Ψ) torsional angles, with the following mean values: Φ_i = −90°, Ψ_i = −27°; Φ_i+1 = −98°, Ψ_i+1 = −17°; Φ_i+2 = −49°, Ψ_i+2 = −50°; Φ_i+3 = −78°, Ψ_i+3 = +3°. We term this particular structure, specifically occurring in the case of (Xaa–Yaa–Aib–Pro)_n sequences, the (Xaa–Yaa–Aib–Pro)‐β‐bend ribbon spiral. It is stabilized by 4 → 1 intramolecular hydrogen bonds and differs from both the canonical 3₁₀‐helix made of a succession of type III β‐turns and from the β‐bend ribbon spiral that has been described in the case of (Aib–Pro)n peptide segments. © 1999 John Wiley & Sons, Inc. Biopoly 50: 71–85, 1999     

Solution structure of human calcitonin in membrane-mimetic environment: The role of the amphipathic helix

The 32 amino acid hormone human calcitonin was studied at pH 3.7 and 7.4 by multidimensional NMR spectroscopy in sodium dodecyl sulfate micelles at 310K. The secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), ³J_HNα coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 265 interproton distances derived from NOESY experiments, hydrogen-bond constraints obtained from amide exchange, and coupling constants were used. From the initial random conformations, 30 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In micelles, at both pHs, the hormone assumes an amphipathic α-helix from Leu9 to Phe16, followed by a type-I β-turn between residues Phe16 and Phe19. From His20 onward the molecule is extended and no interaction with the helix was observed. The relevance of the amphipathic helix for the structure–activity relationship, the possible mechanisms of interaction with the receptor, as well as the formation of fibrillar aggregates, is discussed. Proteins 32:314–323, 1998. © 1998 Wiley-Liss, Inc.     

Two-dimensional 1H-NMR investigation of the water conformation of the atrial natriuretic factor (ANF 101-126)

The complete sequential assignment of the ¹H-nmr resonance frequencies of the active fragment of the rat atrial natriuretic factor (ANF 101–126) has been performed. Two-dimensional nmr techniques have been employed, including phase-sensitive nuclear Overhauser spectroscopy (NOESY), relayed coherence transfer spectroscopy (RELAY), and J-correlated spectroscopy (COSY). Experiments were performed both in D₂0 and H₂0 solutions at different pH values. With few exceptions, resonance frequencies were practically pH independent. NOESY spectra were recorded using both 300- and 500-ms mixing times, and no long-range connectivities were observed, leading to the conclusion that ANF 101–126 has no defined secondary nor tertiary structure in water in the pH range used (2.73–5.21).     

Lysine as helix C-capping residue in a synthetic peptide

The structure of the synthetic peptide CH₃CO (Leu-Ser-Leu-Leu-Leu-Ser-Leu)₃Lys-NH₂ in trifluoroethanol/water 60/40 (volume ratio) was characterized by two-dimensional nmr spectroscopy. The peptide, closely related to the amphiphilic helix models designed by W. F. DeGrado and co-workers to mimic protein ion channels [(1988) Science, Vol. 240, p. 1177–1181], folds into a regular helix spanning residues 1–20. Evidence for a helix C-terminal capping conformation, involving the terminal lysine residue, was observed from Overhauser effects and checked for consistency by restrained molecular dynamics simulations. The side-chain amino group of Lys22 forms a hydrogen bond with the carbonyl of Leu18, and the distorted helical geometry of the terminal dipeptide allows the inclusion of a water bridge between the backbone NH of the Lys22 residue and the carbonyls of Leu19 and Ser20. © 1997 John Wiley & Sons, Inc.     

Conformational studies of parathyroid hormone (PTH)/PTH‐related protein (PTHrP) point‐mutated hybrids

The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1–34)PTH/PTHrP segmental hybrid peptides, the N‐terminal 1–14 segment of PTHrP is incompatible with the C‐terminal 15–34 region of PTH leading to substantial reduction in potency. The sites of incompatibility were identified as positions 5 in PTH and 19 in PTHrP. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two point‐mutated PTH/PTHrP 1–34 hybrids in which the arginine residues at positions 19 and 21 of the native sequence of PTHrP have been replaced by valine (hybrid V²¹) and glutamic acid (hybrid E¹⁹), respectively, taken from the PTH sequence. Hybrid V²¹ exhibits both high receptor affinity and biological potency, while hybrid E¹⁹ binds weakly and is poorly active. The conformational properties of the two hybrids were studied in aqueous solution containing dodecylphosphocholine (DPC) micelles and in water/2,2,2‐trifluoroethanol (TFE) mixtures. Upon addition of TFE or DPC micelles to the aqueous solution, both hybrids undergo a coil‐helix transition. The maximum helix content in 1 : 1 water/TFE, obtained by CD data for both hybrids, is ∼ 80%. In the presence of DPC micelles, the maximum helix content is ∼ 40%. The conformational properties of the two hybrids in the micellar system were further investigated by combined 2D‐nmr, distance geometry (DG), and molecular dynamics (MD) calculations. The common structural motif, consisting of two helical segments located at N‐ and C‐termini, was observed in both hybrids. However, the biologically potent hybrid V²¹ exhibits two flexible sites, centered at residues 12 and 19 and connecting helical segments, while the flexibility sites in the weakly active hybrid E¹⁹ are located at position 11 and in the sequence 20–26. Our findings support the hypothesis that the presence and location of flexibility points between helical segments are essential for enabling the active analogs to fold into the bioactive conformation upon interaction with the receptor. © 1999 John Wiley & Sons, Inc. Biopoly 50: 525–535, 1999     

Measurement of long-range2 13C-1H coupling constants of 95% uniformly 13C-labeled polysaccharide from streptococcus mitis J22

The coaggregation of Streptococcus mitis strain J22 in the early stages of dental plaque formation has been shown to result from interaction of cell wall polysaccharides with lectins on the surface of other oral bacterial species. This bacterium was grown in a medium containing ¹³C as the sole carbon source. We have isolated the lectin receptor polysaccharide from this strain with full enrichment in ¹³C and have determined a number of two-bond and three-bond ¹³C-¹H coupling constants from measurements of the offsets in two-dimensional homonuclear nmr spectra [exclusive correlated spectroscopy (E-COSY) method]. A scheme for reliable extraction of these coupling constants from homonuclear Hartmann-Hahn and nuclear Overhauser effect spectroscopy spectra is tested in model compounds. We interpret the three-bond coupling across the glycosidic linkage in terms of dihedral angles in order to provide conformational information to supplement molecular modeling and nuclear Overhauser effect data. We show that the E-COSY method works well even for coupling constants smaller than the nmr line width and that a number of the ³J_CH across the glycosidic linkage are in the range of 1–2 Hz, which is much smaller than many previously reported values. © 1994 John Wiley & Sons, Inc.     

Solution conformation study of substance P methyl ester and [Nle10]-neurokinin A (4–10) by nmr spectroscopy

High-resolution proton spectra at 500 MHz of two tachykinin peptides, substance P methyl ester (SPOMe) and [Nle¹⁰]-neurokinin A (4–10), have been obtained in dimethylsulfoxide (DMSO), and for SPOMe, also in 2, 2, 2-trifluoroethanol (TFE)/water mixtures. Complete chemical shift assignments for these peptides were made based on two-dimensional (2D) nmr techniques, correlated spectroscopy and total COSY. J coupling measurement and nuclear Overhauser effect spectroscopy (NOESY) were then used to determine the conformation of these peptides in the various solvents. Based on the J coupling, NOE correlations, and temperature coefficients of the NH resonances, it is concluded that these two peptides exist in DMSO at room temperature as a mixture of conformers that are primarily extended. For SPOMe in TFE/water with high TFE content, however, helical structures are found to be present, and they become quite clear at temperatures between 270 and 280 K. The variation of the ¹³C chemical shifts of the C_α (the secondary shift) with TFE contents corroborates this conclusion. The NOE and C_α shifts show that the main helical region for SPOMe lies between ⁴P and ⁹G. The C-terminus segment L? M? NH₂ is found to be quite flexible, which appears to be quite common for neurokinin-1 selective peptides. © 1993 John Wiley & Sons, Inc.     

1H and 15N NMR resonance assignments and solution secondary structure of oxidized Desulfovibrio desulfuricans flavodoxin

Summary Sequence-specific ¹H and ¹⁵N resonance assignments have been made for 137 of the 146 nonprolyl residues in oxidized Desulfovibrio desulfuricans [Essex 6] flavodoxin. Assignments were obtained by a concerted analysis of the heteronuclear three-dimensional ¹H-¹⁵N NOESY-HMQC and TOCSY-HMQC data sets, recorded on uniformly ¹⁵N-enriched protein at 300 K. Numerous side-chain resonances have been partially or fully assigned. Residues with overlapping ¹H^N chemical shifts were resolved by a three-dimensional ¹H-¹⁵N HMQC-NOESY-HMQC spectrum. Medium-and long-range NOEs, ³J_NH coupling constants, and ¹H^N exchange data indicate a secondary structure consisting of five parallel -strands and four -helices with a topology similar to that of Desulfovibrio vulgaris [Hidenborough] flavodoxin. Prolines at positions 106 and 134, which are not conserved in D. vulgaris flavodoxin, contort the two C-terminal -helices.Abbreviations CSI chemical shift index - DQF-COSY double-quantum-filtered correlation spectroscopy - DIPSI decoupling in the presence of scalar interactions - FMN flavin mononucleotide - GARP globally optimized alternating phase rectangular pulse - HMQC heteronuclear multiple-quantum coherence - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser effect - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy - TPPI time-proportional phase increments - TSP 3-(trimethylsilyl)propionic-2,2,3,3-d ₄ acid, sodium salt     

13C-labeled oligodeoxyribonucleotides: A solution study of a CCAAT-containing sequence at the nuclear factor I recognition site of human adenovirus

The solution behavior of the single-stranded CCAAT-containing octamer 1 , d(AGCCAATA), that comprises part of the nuclear factor I (NF-I) recognition site at the origin of replication of human adenovirus has been studied by nmr spectroscopy at 500 and 600 MHz. Proton resonance assignments for 1 were aided by selective ¹³C enrichment at C1′ of A₁ or A₅. High-resolution ¹³C-¹H heteronuclear multiple-bond coherence spectra of the ¹³C-labeled oligomers permitted the selective detection of furanosyl ring protons within each labeled residue due to short- and long-range ¹³C-¹H couplings to the enriched C1′. The resulting assignments provided firm starting points in the interpretation of double quantum filtered correlated spectra, yielding information supplemented by total correlated spectroscopy (TOCSY) and rotating frame nuclear Overhauser effect spectroscopic data to completely assign the ¹H-nmr spectrum of 1 and extract ³J_HH values for furanose con-formational analysis. Several ¹³C-¹H spin-coupling constants within the ¹³C-enriched A₁ or A₅ residues were measured from cross-peak shifts in TOCSY spectra, and their signs determined by inspection of the relative orientations of these shifts. ¹H-²-H and ¹³C-¹H spin-couplings both indicate a preference (> 75%) for south (C2′-endo) conformations by the furanosyl rings of 1 . © 1994 John Wiley & Sons, Inc.     

Conformational analysis of β-methyl-para-nitrophenylalanine stereoisomers of cyclo[D-Pen2, D-Pen5]enkephalin by NMR spectroscopy and conformational energy calculations

Solution conformations of β-methyl-para-nitrophenylalanine⁴ analogues of the potent δ-opioid peptide cyclo[D-Pen², D-Pen⁵]enkephalin (DPDPE) were studied by combined use of nmr and conformational energy calculations. Nuclear Overhauser effect connectivities and ³J_HNC^α_H coupling constants measured for the (2S, 3S)-, (2S, 3R)-, and (2R, 3R)-stereoisomers of[β-Me-p-NO₂Phe⁴]DPDPE in DMSO were compared with low energy conformers obtained by energy minimization in the Empirical Conformational Energy Program for Peptides #2 force field. The conformers that satisfied all available nmr data were selected as probable solution conformations of these peptides. Side-chain rotamer populations, established using homonuclear (³J_H^α_H^β) and heteronuclear (³J_H^αC^γ) coupling constants and ¹³C chemical shifts, show that the β-methyl substituent eliminates one of the three staggered rotamers of the torsion angle x¹ for each stereoisomer of the β-Me-p-NO₂Phe⁴. Similar solution conformations were suggested for the L-Phe⁴-containing (2S, 3S)- and (2S, 3R)-stereoisomers. Despite some local differences, solution conformations of L- and D-Phe⁴-containing analogues have a common shape of the peptide backbone and allow similar orientations of the main δ-opioid pharmacophores. This type of structure differs from several models of the solution conformations of DPDPE, and from the model of biologically active conformations of DPDPE suggested earlier. The latter model is allowed for the potent (2S, 3S)- and (2S, 3R)-stereoisomers of [β-Me-p-NO₂Phe⁴] DPDPE, but it is forbidden for the less active (2R, 3R)- and (2R, 3S)-stereoisomers. It was concluded that the biologically active stereoisomers of [β-Me-p-No₂Phe⁴] DPDPE in the δ-receptor-bound state may assume a conformation different from their favorable conformations in DMSO. © 1996 John Wiley & Sons, Inc.     

Investigation of the thermal unfolding of secondary and tertiary structure in E. coli tRNAfMet by high-resolution nmr

The high-resolution (300 MHz) proton nmr spectrum of E. coli tRNA^fMet has been examined in 0.17M NaCl, with and without Mg²⁺, and at various temperatures. In light of recent studies of other E. coli tRNA and fragments of tRNA^fMet, some low field (11–15 ppm) resonances previously assigned to secondary structure base pairs are reassigned to a tertiary structure A₁₄–S⁴U₈ base pair and a protected uridine residue in the anticodon loop. These two resonances and other low field resonances which are assigned to secondary structure base pairs are used to monitor the thermal unfolding of the molecule. In the absence of Mg²⁺ the tertiary structure base pair is present only to ～45°C, but in the presence of Mg²⁺ it remains until at least 70°C. Analysis of the temperature dependence of other low field resonances indicates that the melting of the dihydrouridine stem occurs more or less simultaneously with the loss of tertiary structure. The observation of the resonance from the A₁₄–S⁴U₈ base pair proves that tertiary structure is present in this molecule below 40°C, even in the absence of Mg²⁺.     

Conformational preferences of Leu-enkephalin in reverse micelles as membrane-mimicking environment

Enkephalin represents one of the bioactive peptide molecules most extensively investigated both in solution and in the crystal state. Depending upon the environment chosen for such studies, three main conformational states were identified for this flexible, linear pentapeptide—i.e., an extended conformation, a single-bend, and a double-bend structure. Since CD and Fourier transform ir (FTIR) spectra of Leu-enkephalin solubilized in negatively charged reverse micelles of bis (2-ethylhexyl)sulfosuccinate sodium salt/isooctane/water were supportive of a restricted conformational space of the aromatic side chains and of a bended type fold, we have analyzed by nmr the conformational preferences of Leu-enkephalin in reverse micelles using a synthetic [¹³C, ¹⁵N]-backbone-labeled sample. The overall conformation derived from nuclear Overhauser effect spectroscopy (NOESY) and ¹⁵N-filtered rotating frame NOESY (ROESY) spectra and by distance geometry calculations is a double-bend fold of the backbone that is comparable to one of the known x-ray structures. Thereby the tyrosine side chain is inserted into the hydrophobic core of the reverse micelles in a restrained conformational space as well evidenced by NOEs between the aromatic ring protons and the surfactant. The proximity of the aromatic rings of tyrosine and phenylalanine indicate a preferred structure consistent with the postulated conformation of the opioid peptide in the δ-receptor-bound state. These results confirm the interesting and promising properties of reverse micelles as membrane mimetica. © 1997 John Wiley & Sons, Inc. Biopoly 41: 591–606, 1997     

Structure of human salivary histatin 5 in aqueous and nonaqueous solutions

The solution structure of human salivary histatin 5 (D-S-H-A-K-R-H-H-G-Y-K-R-K-F-H-E-K-H-H-S-H-R-G-Y) was examined in water (pH 3.8) and dimethyl sulfoxide solutions using 500 MHz homo- and heteronuclear two-dimensional (2D) nmr. The resonance assignment of peptide backbone and side-chain protons was accomplished by 2D total correlated spectroscopy and nuclear Overhauser effect (NOE) spectroscopy. The high J values (≥7.4 Hz), absence of any characteristic NH-NH(i, i + 1) or C^αH-C^βH(i, i + 3) NOE connectivities, high dδ/dT values (≥0.004 ppm K⁻¹) and the fast ¹H/²H amide exchange suggest that histatin 5 molecules remain unstructured in aqueous solution at pH 3.8. In contrast, histatin 5 prefers largely α-helical conformation in dimethyl sulfoxide solution as evident from the J values (≤6.4 Hz), slow ¹H/²H exchange, low dδ/dT values (≤0.003 ppm K⁻¹) observed for amide resonances of residues 6–24, and the characteristic NH-NH(i, i + 1) and C^αH-C^βH(i, i +3) NOE connectivities. All backbone amide ¹⁵N-¹H connectivities fall within 6 ppm on the ¹⁵N scale in the 2D heteronuclear single quantum correlated spectrum, and the restrained structure calculations using DIANA suggest the prevalence of α-helical conformations stabilized by 19 (5 → 1) intramolecular backbone amide hydrogen bonds in polar aprotic medium such as dimethyl sulfoxide. The interside-chain hydrogen bonding and salt-bridge type interactions that normally stabilize the helical structure of linear peptides in aqueous solutions are not observed. Histatin 5, unlike other naturally occurring antimicrobial polypeptides such as magainins, defensins, and tachyplesins, does not adopt amphiphilic structure, precluding its insertion into microbial membranes and formation of ion channels across membranes. Electrostatic (ionic type) and hydrogen bonding interactions of the positively charged and polar residues with the head groups of microbial membranes or with a membrane-bound receptor could be the initial step involved in the mechanism of antimicrobial activity of histatins. © 1998 John Wiley & Sons, Inc. Biopoly 45: 51–67, 1998     

Use of graph theory for secondary structure recognition and sequential assignment in heteronuclear (13C, 15N) NMR spectra: Application to HU protein from Bacillus stearothermophilus

A computer-assisted procedure, based upon a branch of mathematics known as graph theory, has been developed to recognize secondary structure elements in proteins from their corresponding nuclear Overhauser effect spectroscopy (NOESY)-type spectra and to carry out their sequential assignment. In the method, NOE connectivity templates characteristic of regular secondary structures are identified in the spectra. Resonance assignment is then achieved by connecting these NOE patterns of secondary structure together, and thereby matching connected spin systems to specific parts of the primary sequence. The range of NOE-graph templates of secondary structure motifs, incorporating α-helices and β-strand motifs, has been examined for reliability and extent of secondary structure identification in a data base composed of the high resolution structures of 20 proteins. The analysis identified several robust NOE-graph templates and supports the implementation of an ordered search strategy. The method, known as SERENDIPITY, has been applied to the analysis of nuclear Overhauser effect data from a three-dimensional time-shared nuclear Overhauser effect spectroscopy (¹³C, ¹⁵N) heteronuclear single quantum correlation spectrum of the (α + β) type protein HU from Bacillus stearothermophilus. The arrangement of the elucidated elements of secondary structure is very similar to that of the x-ray and nmr structures of HU. In addition, our analysis revealed a pattern of interstrand nuclear Overhauser effect in the β-arm region (residues 53–76) of HU, which suggest irregularities, not reported in the x-ray structure, in both strands of the β-arm at Ala57 and Pro72, respectively. At these residues, both strands of the β-arm appear to flip inside out before continuing as a regular antiparallel β-sheet. © 1996 John Wiley & Sons, Inc.     

The temperature dependence and thermodynamic functions of partitioning of substance P peptides in dodecylphosphocholine micelles

The temperature dependence of the partition of a neuropeptide, Substance P (SP), and its [Tyr⁸] analogue in a widely used membrane mimic, dodecylphosphocholine micelles, was studied by using a pulsed field gradient nmr diffusion technique. The partition coefficient was found to decrease when the temperature is increased, indicating a favorable (negative) enthalpy change upon partitioning of the peptides. Thermodynamic functions of the partitioning were determined. The enthalpy of partition ΔH_part, was found to be in the −2.5 to −3.0 kcal/mol range, which is between 2 and 3 times higher than the entropic term −TΔS_part. The free energy of partitioning is consistent with a model in which the SP peptides interact with the micelles mainly through the hydrophobic side chains of the residues Phe⁷, Phe⁸ (or Tyr⁸), Leu¹⁰, and Met¹¹, and without the insertion of a major portion of the peptide into the hydrophobic core of the micelles. © 1998 John Wiley & Sons, Inc. Biopoly 45: 395–403, 1998     

3H-uridine labelling patterns and chromosomal polymorphism inDrosophila subobscura: J and U chromosomes

The³H-uridine labelling patterns in J and U polytene chromosomes ofDrosophila subobscura were determined. The analysis was carried out in two developmental stages and in two strains proceeding from the same geographical origin whose genotypes were: J_st/J_st; U₁₊₂/U₁₊₂ and J₁/J₁; U₁₊₂₊₈/U₁₊₂₊₈ respectively. It was observed that the labelling pattern coincided very approximately with the puffing pattern in the same stages and chromosomal arrangements. Comparison of the³H-Uridine incorporation patterns between chromosomal arrangements showed light quantitative differences. These results are discussed in relation to the inversion effect.     

Terminal effect of chiral residue on helical screw sense in achiral peptides

To understand the terminal effect of chiral residue for determining a helical screw sense, we adopted five kinds of peptides I – V containing N‐ and/or C‐terminal chiral Leu residue(s): Boc–L ‐Leu–(Aib–ΔPhe)₂–Aib–OMe ( I ), Boc–(Aib–ΔPhe)₂–L ‐Leu–OMe ( II ), Boc–L ‐Leu–(Aib–ΔPhe)₂–L ‐Leu–OMe ( III ), Boc–D ‐Leu–(Aib–ΔPhe)₂–L ‐Leu–OMe ( IV ), and Boc–D ‐Leu–(Aib–ΔPhe)₂–Aib–OMe ( V ). The segment –(Aib–ΔPhe)₂– was used for a backbone composed of two “enantiomeric” (left‐/right‐handed) helices. Actually, this could be confirmed by ¹H‐nmr [nuclear Overhauser effect (NOE) and solvent accessibility of NH resonances] and CD spectroscopy on Boc–(Aib–ΔPhe)₂–Aib–OMe, which took a left‐/right‐handed 3₁₀‐helix. Peptides I – V were also found to take 3₁₀‐type helical conformations in CDCl₃, from difference NOE measurement and solvent accessibility of NH resonances. Chloroform, acetonitrile, methanol, and tetrahydrofuran were used for CD measurement. The CD spectra of peptides I – III in all solvents showed marked exciton couplets with a positive peak at longer wavelengths, indicating that their main chains prefer a left‐handed screw sense over a right‐handed one. Peptide V in all solvents showed exciton couplets with a negative peak at longer wavelengths, indicating it prefers a right‐handed screw sense. Peptide IV in chloroform showed a nonsplit type CD pattern having only a small negative signal around 280 nm, meaning that left‐ and right‐handed helices should exist with almost the same content. In the other solvents, peptide IV showed exciton couplets with a negative peak at longer wavelengths, corresponding to a right‐handed screw sense. From conformational energy calculation and the above ¹H‐nmr studies, an N‐ or C‐terminal L ‐Leu residue in the lowest energy left‐handed 3₁₀‐helical conformation was found to take an irregular conformation that deviates from a left‐handed helix. The positional effect of the L ‐residue on helical screw sense was discussed based on CD data of peptides I – V and of Boc–(L ‐Leu–ΔPhe)_n–L ‐Leu–OMe (n = 2 and 3). © 1999 John Wiley & Sons, Inc. Biopoly 49: 551–564, 1999     

Quantitative two-dimensional nmr study of dermenkephalin,a highly potent and selective δ-opioid peptide

Dermenkephalin, H-Tyr-(D ) Met-Phe-His-Leu-Met-Asp-NH₂, a highly potent and selective δ-opioid peptide isolated from frog skin, was studied in DMSO-d₆ solution by two-dimensional nmr spectroscopy, including the determination of NH temperature coefficients, the evaluation of ³J coupling constants from phase-sensitive correlated spectroscopy (COSY) and the volumes of nuclear Overhauser effect (NOE) correlations. The two-dimensional NOE spectroscopy (NOESY) spectrum of dermenkephalin revealed sequential, medium-, and long-range effects. To put this information on a quantitative basis, special attention was devoted to J cross-peak suppression, quantification of the NOE volumes and analysis of the overlaps, normalization of the NOEs against diagonal peaks and H_ββ′ geminal interactions. Although most of the dihedral angles deduced from the ³J_Nα coupling constants together with several N_iα_i and α_iN_{i + 1} NOEs pointed to a partially extended peptide backbone, several N_i N_{i + 1} NOEs and β_i N_{i + 1} interactions argued in favor of a folded structure. Moreover, several long-range correlations of strong intensities were found that supported a close spatial proximity between the side chains of D -Met² and Met⁶, Tyr¹ and His⁴, Tyr¹ and Asp⁷, and His⁴ and the C-terminal amide group. In Phe, the g^? rotamer in the side chain is deduced from the ³J_αβ coupling constants and αβ and Nβ NOE correlations. Whereas the amide proton dependency was not indicative of stable hydrogen bonds, the nonuniform values of the temperature coefficient may reflect an equilibrium mixture of folded and extended conformers. The overall data should provide realistic starting models for energy minimization and modelization studies. © 1993 John Wiley & Sons, Inc.     

Nuclear magnetic resonance studies on oligo-deoxyribonucleotides containing thedam methylation site GATC. Adenine methylation of d(GGATCC) does not change the helix geometry

We have studied the conformation of two hexanucleotides d(GGATCC) and d(GGm⁶ATCC) using proton nuclear magnetic resonance. Nuclear Overhauser effect measurements show that d(GGATCC) assumes a normal right handed B helix. The single and double strand resonances are in fast exchange on a proton nuclear magnetic resonance time scale. For d(GGm⁶ATCC), up to the Tm separate resonances are observed for each state, indicating slow exchange, though above the Tm it becomes more rapid. The orientation of the adenosine methyl-amino group, preferentiallycis to N¹, hinders base pair formation.The connectivities of the resonances of the two states were established by saturation transfer experiments. At 0°C irradiation of the m⁶ A-T imino proton gives an nuclear Overhauser effect to AH² showing that base pairing is Watson-Crick. Intra and interresidue nuclear Overhauser effects starting from the 3′ terminus show that the helix is right handed and in the B-form.The results on the two oligomers demonstrate that adenosine methylation induces little or no change in the conformation of the helix, but reduces the Tm from 45° to 32°C and slows the opening and closing of the m₆A.T base pair by a factor of about 100.     

Stereochemical constraints in peptide design: Analysis of the influence of a disulfide bridge and an α-aminoisobutyryl residue on the conformation of a hexapeptide

The competing effects of a disulfide bridge and an α-aminoisobutyryl residue (Aib) in determining the conformation of a hexapeptide have been investigated, by comparing the cyclic disulfide (1) and the acylic peptide Boc-Cys(SBzl)-Val-Aib-Ala-Leu-Cys(SBzl)-NHMe ( 2 ). Previously published nmr and crystallographic studies [R. Kishore, S. Raghothama, and P. Balaram (1987) Biopolymers, Vol. 26, pp. 873–891; I. L. Karle, R. Kishore, S. Raghothama, & P. Balaram, (1988) Journal of the American Chemical Society Vol. 110, pp. 1958–1963] have established an antiparallel β-hairpin structure for 1 with a central Aib-Ala β-turn. A comparison of nmr data for 1 and 2 in chloroform and dimethylsulfoxide reveals that the acyclic peptide is conformationally labile. Evidence for a 3₁₀-helical conformation in CDCl₃ is obtained from sensitivity of NH chemical shifts to temperature and solvent perturbation and low J_HNCαH values. Studies in solvent mixtures establish a conformational transition on going from CDCl₃ to (CD₃)₂SO. The changes in NH nmr parameters, together with the observation of several interresidue C H-N_{i + 1}H nuclear Overhauser effects support a conformation having a central β-turn with extended arms in (CD₃)₂SO. A single Aib residue appears to stabilize a helix in apolar solvents, for the acyclic hexapeptide, while the disulfide bridge serves to lock the β-hairpin conformation. © 1993 John Wiley & Sons, Inc.