High-field NMR and circular dichroism solvent-dependent conformational studies of the bradykinin C-terminal tetrapeptide Ser-Pro-Phe-Arg

The conformational properties of the tetrapeptide Ser1-Pro2-Phe3-Arg4, the C-terminal fragment of the nonapeptide hormone bradykinin, have been studied by circular dichroism and two-dimensional NMR techniques. Measurements of coupling constants, NH temperature dependence rates and nuclear Overhauser effects (performed with rotating frame nuclear Overhauser spectroscopy, ROESY) in H2O and CD3OH/D2O (80/20, v/v) reveal different conformations in the corresponding solvent. In aqueous solution the molecule exists in a random conformation or as an average of several conformations in rapid exchange. In CD3OH/D2O, however, the conformation is well-defined. The backbone of the peptide is extended, and the side-chains of Phe3 and Arg4 exhibit unusual rigidity for a peptide of this size. Evidently, the secondary structure is stabilized by a charge interaction between the guanidino group of Arg4 and the terminal carboxyl group, since experiments at various pH's show clearly that the definition of conformation decreases strongly upon protonation of the carboxyl function. A NH3+(Ser1)-COO-(Arg4) salt bridge, as well as any form of turn stabilized by hydrogen bonds can be ruled out with certainty.     

Conformational analysis of the type II and type III collagen α-1 chain N-telopeptides by 1H-NMR spectroscopy and restrained molecular mechanics calculations

The type II and type III collagen α-1 chain N-telopeptides are a nonadecamer with the sequence pEMAGGFDEKAGGAQLGVMQ-NH₂ and a tetradecamer with the sequence pEYEAYDVKSGVAGG-NH₂, respectively. Their conformations have been studied in CD₃OH/H₂O (60/40) solution by means of two-dimensional proton nmr spectroscopy. Based on double quantum filtered correlation spectroscopy, total correlation spectroscopy, rotating frame nuclear Overhauser enhancement (ROE) spectroscopy, and nuclear Over-hauser enhancement (NOE) spectroscopy experiments, all resonances were assigned and the conformational properties were analyzed in terms of vicinal NH-H_α coupling constants, sequential and medium-range NOEs (ROEs), and amide proton temperature coefficients. The NOE distance constraints as well as dihedral constraints based on the vicinal NH-H_α coupling constants were used as input parameters for restrained molecular mechanics, consisting of restrained molecular dynamics and restrained energy minimization calculations. The type II N-telopeptide's conformation is dominated by a fused βγ-turn between Phe⁶ and Ala¹⁰, stabilized by three hydrogen bonds and a salt bridge between the side-chain end groups of Glu⁸ and Lys⁹. The first 5 amino acids are extended with a much higher degree of conformational freedom. The 2 Gly residues following the turns were found to be highly flexible (hinge-like), leaving the spatial position of the second half of the molecule relative to the fused βγ-turn undefined. In the type III telopeptide, a series of sequential NH(i)-NH(i + 1) ROEs were observed between the amino acids Tyr² and Ser⁹, indicating that a fraction of the conformational space is helical. However, the absence of medium-range ROEs and the lack of regularity of the effects associated with α-helices suggest the presence of a nascent rather than a complete helix. © 1993 John Wiley & Sons, Inc.     

Conformational analysis of an EP24.15 inhibitor by NMR and molecular modelling

Summary The enzyme thimet oligopeptidase (EC3.4.24.15, EP24.15) is responsible for the hydrolysis of a number of neuropeptides. Despite much research examining its substrate specificity, little is known about the conformational requirements of its active site. We have used 1D¹H and 2D TOCSY NMR experiments to assign the proton resonances of the EP24.15 inhibitor,N-[1-(R, S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP), and 2D ROESY NMR to investigate whether cFP exhibits any conformational preferences in CD₃OD and in aqueous CD₃OD. Molecular modelling of charged cFP in the gaseous phase generated a number of conformations that were consistent with the NMR data obtained in CD₃OD. Analogous modelling on the uncharged cFP did not result in conformations consistent with any of the NMR data, but did suggest that, under non-polar conditions, cFP could adopt a hairpin conformation which would allow simultaneous coordination of the two carboxyl groups of cFP to the zinc ion in the active site of EP24.15.     

A Proton Magnetic Resonance and a Circular Dichroism Study of the Solvent Dependent Conformation of the Synthetic Tubulin Fragment Ac Tubulin,Alpha (430–441) Amide and its Interaction with Substance-P

Abstract

Proton magnetic resonance techniques were used to study the conformation of the synthetic tubulin fragment Ac-tubulin (430–441) amide in H₂0 and 80% CD₃OH/20% D₂0 solutions, using water suppression techniques. Proton assignments are based on two-dimensional COSY experiments combined with one-dimensional spin decoupling.

A comparison of the NH proton shifts between the two solvents, namely ?(CD₃OH/H₂0-H₂O) shows a small solvent effect for the Lys¹ to Val⁶ region of the molecule, whereas for Gly⁷ to Glu¹² the solvent effect is much larger. The smaller effects in the region of Lys¹ to Val⁶ may be due to some hydrogen bonding as these protons are shielded from the solvent These conclusions are in agreement with the circular dichroism results in 80% methano1/20% water where the a helix is present to the extent of 30%, whereas the peptide is completely unstructured in water with some aggregation.

The temperature dependence of the NH proton shifts was also carried out. In water these shifts are of the order of7-9 × 10^?3 ppm/K indicating that most of the protons are not involved in hydrogen bonding. In CD₃0H/H₂0, these values range from about 4–6 × 10^?3 ppm/K, which are compatible with the presence of hydrogen bonds.

Finally, binding studies were carried out between the tubulin peptide and the undecapeptide neurotransmitter substance P. The largest shifts are for the Tyr³ NH proton of the tubulin fragment, whereas for substance P it is for the Lys³, Gin⁵ and Leu¹⁰ NH protons, indicating a change in conformation of both peptides on interaction.     

Structural studies of a synthetic peptide derived from the carboxyl-terminal domain of RNA polymerase II

The conformation of the repeating heptapeptide unit of the carboxyl-terminal domain of RNA Polymerase II, Y¹S²P³T⁴S⁵P⁶S⁷ has been examined using nuclear magnetic resonance spectroscopy and circular dichroism. Nuclear Overhauser effects and CD spectra for the synthetic 56-residue peptide H₂N-(S²P³T⁴S⁵P⁶S⁷Y^l)₈-COOH in water indicate that the peptide is largely unordered. A small population of folded molecules is observed to contain β-turns located at Ser²-Pro³-Thr⁴-Ser⁵ (SPTS) and Ser⁵-Pro⁶-Ser⁷-Tyr¹ (SPSY). CD and NMR results in 90% TFE also indicate an equilibrium population of structures, but the fraction of turns is higher. Similarities of nuclear Overhauser effects in water and in 90% TFE suggest that the structures in TFE are biologically relevant. Based on these observations, the average structure of a single conformer of the heptapeptide repeat in 90% TFE was obtained by a distance geometry-simulated annealing method, using distance restraints extracted from nuclear Overhauser data. NMR spectra of the 56-mer show signals corresponding to only one repeat indicating that each repeat is in an identical environment. Thus it is possible to obtain an average structure of the heptapeptide repeat from NOE data on the 56-mer. Twenty-seven final structures were calculated and the root mean square deviations between the 27 structure and the mean coordinates was 1.52 Å for the backbone and 2.2 Å for all nonhydrogen atoms. The heptapeptide repeat consists of two overlapping β-turns which are potentially stabilized by hydrogen bonds. The hydroxyl side chains of Ser², Ser⁵, Thr⁴, and Ser⁷ all appear to be equally exposed for potential phosphorylation. The tyrosyl side chain of each repeat is folded inwards to the backbone and can potentially hydrogen bond to the carbonyl oxygen of the tyrosine in the preceding repeat. Iteration of the average structure of the heptapeptide repeat results in a model of the carboxyl-terminal domain with a regular but unusual secondary structure consisting of a series of staggered β-turns. © 1995 Wiley-Liss, Inc.     

A 1H and 13C NMR Study on the Role of Salt-Bridges in the Formation of a Type I β-Turn in N-Acetyl-L-Asp-L-Glu-L-Lys-L-Ser-NH2

Abstract

The conformation of the tetrapeptide N-Acetyl-Asp⁷-Glu⁸-Lys⁹-Ser¹⁰-NH₂, a fragment of the type I collagen α-1 chain N-telopeptide, has been studied by ¹H and ¹³C NMR and circular dichroism spectroscopy. The spectroscopic evidence, based on two-dimensional, phase-sensitive NMR techniques such as COSY, ROESY, proton-carbon shift correlation and selective COLOC, indicates a strong dependence of the conformation on the experimental conditions. In CD₃OH/H₂O (60/40) at ca. neutral pH the tetrapeptide forms a β-turn, stabilized by a hydrogen bond between NH(S¹⁰) and CO(D⁷) and a strong salt-bridge between COO^?(E⁸) and NH₃ ⁺(K⁹). The β-turn is type I and appears to coexist with a non-hydrogen-bonded structure. The coexistence of these two conformers is proven by proton NMR data such as NH-NH ROEs, reduced NH-H_α(E⁸) coupling constant NH(E⁸) low-field shift and the temperature coefficient of NH(S¹⁰), whereas the conclusion regarding the salt-bridge is based on ¹³C results. In the same solvent, at a pH below the pKa of the carboxyl groups, no evidence for a conformation other than extended can be found. In aqueous solution at approximately neutral pH, evidence for the E⁸-K⁹ charge interaction is observed, but not for a hydrogen bond anywhere in the molecule.     

A CD and an nmr study of multiple bradykinin conformations in aqueous trifluoroethanol solutions

CD and nmr studies have been carried out on aqueous trifluoroethanol (TFE) solutions of bradykinin (BK) and a bradykinin antagonist. The CD results exhibit a striking effect of TFE on the spectra of BK, with sequence Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, and the BK antagonist, with sequence D -Arg-Arg-Pro-Hyp-Gly-Thi-D -Ser-D -Cpg-Cpg-Arg [where Hyp is 4-hydroxy-L -proline; Thi refers to β-(2-thienyl)-L -alanine and Cpg refers to α-cyclopentylglycine]. The effect of increasing concentration of TFE in water on the difference ellipticity at 222 nm was examined and showed that BK may be a mixture of at least two different conformers, one of which largely forms when the TFE concentration is increased beyond 80%. The linear extrapolation of 100% of the difference ellipticity of BK at low TFE concentrations yields a value in agreement with that shown by the BK antagonist, indicating that the conformation of BK at the lower TFE concentrations is similar to that of the BK antagonist. The conformational analysis was carried out using both one-dimensional and two-dimensional ¹H-nmr techniques. The total correlation spectroscopy (TOCSY) spectrum of BK in a 60/40% (v/v) TFE/H₂O solution at 10°C and a nuclear Overhauser effect spectroscopy (NOESY) spectrum that shows only sequential H_α(i) – NH(i + 1) or the H_α(i) – H_δδ′(i + 1) NOEs indicate that the majority of the molecules adopt an all-trans extended conformation. The TOCSY for BK in the 95/5% (v/v) TFE/H₂O solution shows that there are two major conformations in the solution with about equal population. The NOESY experiment shows two new important cross peaks for one conformation, namely Pro²(α)-Pro³ (α) and the Pro²(α)-Gly⁴(NH), indicating a cis Pro²-Pro³ bond and a type VI β-turn between residues Arg¹ and Gly⁴ involving cis proline at position 3, respectively. The low temperature coefficient of Gly⁴ for this conformation suggests the presence of an intramolecular hydrogen bond, therefore a type VIa β-turn is present. The other conformation is all trans and extended. The BK antafonist shows difference CD spectra in TFE solutions referred to H₂O that are superficially indicative of a β-bend. However, nmr speaks against this possibility, as only one set of peaks were observed in the TOCSY and NOESY experiments, indicating an all-trans extended confirmation over the range of TFE concentrations. The BK-antagonist CD data suggest that solvent perturbation of the CD of an extended confirmation perturbation of the optical activity of the thienyl moiety of the peptide since the CD spectrum of N-acetyl-β-thienyl-L -alanine N-methylamide is strongly perturbed by TFE. The present results again demonstrate the complementary relationship between CD and nmr. © 1994 John Wiley & Sons, Inc.     

A proton magnetic resonance study of two synthetic agonist–antagonist pairs of bradykinin analogues

The conformation of two agonist–antagonist pairs of bradykinin (Arg¹-Pro²-Pro³-Gly⁴-Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹) analogues were studied in CD₃OH/H₂O solution by ¹H-nmr techniques. The first agonist peptide studied, D -Arg⁰-Arg¹-Pro²-Hyp³-Gly⁴-Thi⁵-Ser⁶-Pro⁷-Thi⁸-Arg⁹, differs from the bradykinin sequence by the addition of D -Arg⁰, the replacement of the Phe moieties in positions 5 and 8 by Thi (Thi = β-(2-thienyl)-L -alanine), and Hyp³ (Hyp = L -4-hydroxy-L -proline) in position 3. In the corresponding antagonist sequence, Pro⁷ is replaced by D -Phe⁷. The second agonist–antagonist pair studied does not contain the D -Arg⁰ residue, which is present only to slow down the rate of metabolism. Based on complete resonance assignments from two-dimensional total correlation spectroscopy and rotating frame nuclear Overhauser effect spectroscopy spectra at 500 MHz, the peptides were analyzed in terms of intraresidue, sequential, and medium-range nuclear Overhauser effects, amide proton temperature coefficients, and vicinal coupling constants. Both agonist peptides show clear evidence for the existence of a type I β-turn comprising the C-terminal residues Ser⁶-Pro⁷-Thi⁸-Arg⁹ in fast conformational equilibrium with extended structures throughout. Although the conformational space is dominated by extended structures, the presence of the β-turn is spectroscopically clearly discernible. The two antagonist peptides, on the other hand, do not show evidence of turn formation but rather the presence of an extended conformation with some irregularities in the N-terminal region of the peptide. While the existence of a turn at the C-terminal end of bradykinin and its analogues with agonist activity has been predicted by empirical calculations and measurements in very apolar solvents, this study, for the first time, provides evidence based on physical data in a polar solvent environment that the turn is present, that it is type I and that it is essential for agonist activity. In the particular solvent used in these studies, the Pro⁷ to D -Phe⁷ substitution precluded the formation of the turn for the C-terminal residues of the antagonist. © 1993 John Wiley & Sons, Inc.     

Conformational analysis of an EP24.15 inhibitor by NMR and molecular modelling

The enzyme thimet oligopeptidase (EC3.4.24.15, EP24.15) is responsible for the hydrolysis of a number of neuropeptides. Despite much research examining its substrate specificity, little is known about the conformational requirements of its active site. We have used 1D ¹H and 2D TOCSY NMR experiments to assign the proton resonances of the EP24.15 inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP), and 2D ROESY NMR to investigate whether cFP exhibits any conformational preferences in CD₃OD and in aqueous CD₃OD. Molecular modelling of charged cFP in the gaseous phase generated a number of conformations that were consistent with the NMR data obtained in CD₃OD. Analogous modelling on the uncharged cFP did not result in conformations consistent with any of the NMR data, but did suggest that, under non-polar conditions, cFP could adopt a hairpin conformation which would allow simultaneous coordination of the two carboxyl groups of cFP to the zinc ion in the active site of EP24.15.     

Omega amino acids in peptide design: incorporation into helices

Incorporation of easily available achiral ω-amino acid residues into an oligopeptide results in substitution of amide bonds by polymethylene units of an aliphatic chain, thereby providing a convenient strategy for constructing a peptidomimetic. The central Gly-Gly segment of the helical octapeptide Boc-Leu-Aib-Val-Gly-Gly-Leu-Aib-Val-Ome(1) has been replaced by δ-amino-valeric acid (δ-Ava) residue in the newly designed peptide Boc-Leu-Aib-Val-δ-Ava-Leu-Aib-Val-OMe(2). ¹H-nmr results clearly suggest that in the apolar solvent CDCl₃, the δ-Ava residue is accommodated into a folded helical conformation, stabilized by successive hydrogen bonds involving the NH groups of Val(3), δ-Ava(4), and Leu(5). The δ-Ava residue must adopt a gauche-gauche-trans-gauche-gauche conformation along the central polymethylene unit of the aliphatic segment, a feature seen in an energy-minimized model conformation based on nmr parameters. The absence of hydrogen bonding functionalities, however, limits the elongation of the helix. In fact, in CDCl₃, the folded conformation consists of an N-terminal helix spanning residues 1–4, followed by a Type II β-turn at residues 5 and 6, whereas in strongly solvating media like (CD₃)₂SO, the unfolding of the N-terminal helix results in β-turn conformations at Leu(1)-Aib(2). The Type II β-turn at the Leu(5)-Aib(6) segment remains intact even in (CD₃)₂SO. CD comparisons of peptides 1 and 2 reveal a “nonhelical” spectrum for 2 in 2,2,2-trifluoroethanol. © 1996 John Wiley & Sons, Inc.     

Assignment of the 270 MHz nuclear magnetic resonance spectrum of somatostatin in water

The proton nuclear magnetic resonance spectrum of the 14 residue peptide hormone somatostatin in D₂O at 270 MHz has been assigned by comparing the spectra of synthetic analogs with those of the native peptide. Extensive difference double resonance studies of all somatostatins and pH titrations confirmed all assignments. ³J_NHCH values and conventional NMR hydrogen bonding studies confirm the existence of preferred secondary conformations but not with a predominant conformation possessing a β-turn in either of the sequences 7–8–9–10 or 8–9–10–11. More extensive data treatment is needed before the actual conformation(s) of somatostatin is elucidated, but several NMR criteria for conformations are proposed.     

Conformational Analysis of the Type II and Type III Collagen α-1 Chain C-Telopeptides by 1H NMR and Circular Dichroism Spectroscopy

Abstract

The type II and type III collagen α-1 chain C-telopeptides are a 27 mer with the sequence NAc- GPGIDMSAFAGLGPREKGPDPLQYMRA and a 22mer, NAc-GGGVASLGAGEKGPVG- YGYEYR, respectively. Their conformations have been studied in CD₃OH/H₂O (80/20) solution by means of two-dimensional proton NMR and CD spectroscopy. Based on TOCSY and NOESY experiments, all resonances were assigned and the conformational properties were analyzed in terms of vicinal NH-H_α coupling constants, sequential and medium range NOEs and amide proton temperature coefficients.

The conformation of the type II C-telopeptide is essentially extended. Evidence from CD spectroscopy suggests that a very minor proportion of the peptide might be helical (ca. 8%), but the NMR data show no evidence for a non-linear structure. The observation of reduced amide proton temperature dependence coefficients in certain sections of the molecule can, in view of the absence of any other supporting evidence, only be interpreted in terms of local shielding from solvent for sterical reasons (large hydrophobic side-chains).

The conformation of the type III C-telopeptide is mostly extended except for a β-turn ranging from Gly⁸ to Glu¹¹, which is stabilized by a hydrogen-bond between NH of Glu¹¹ and the carbonyl group of Gly⁸. The low temperature coefficient of NH(Glu¹¹) and, in particular, the observation of a medium range NOE between H_α (A⁹) and NH(E¹¹) corroborate the existence of a β-turn in this region. Although spectral overlap prevents a precise conclusion with regard to the type of β-turn present, there is some evidence that it might be type II.     

Osteopontin Is Cleaved at Multiple Sites Close to Its Integrin-binding Motifs in Milk and Is a Novel Substrate for Plasmin and Cathepsin D

Osteopontin (OPN) is a highly modified integrin-binding protein present in most tissues and body fluids where it has been implicated in numerous biological processes. A significant regulation of OPN function is mediated through phosphorylation and proteolytic processing. Proteolytic cleavage by thrombin and matrix metalloproteinases close to the integrin-binding Arg-Gly-Asp sequence modulates the function of OPN and its integrin binding properties. In this study, seven N-terminal OPN fragments originating from proteolytic cleavage have been characterized from human milk. Identification of the cleavage sites revealed that all fragments contained the Arg–Gly–Asp¹⁴⁵ sequence and were generated by cleavage of the Leu¹⁵¹–Arg¹⁵², Arg¹⁵²–Ser¹⁵³, Ser¹⁵³–Lys¹⁵⁴, Lys¹⁵⁴–Ser¹⁵⁵, Ser¹⁵⁵–Lys¹⁵⁶, Lys¹⁵⁶–Lys¹⁵⁷, or Phe¹⁵⁸–Arg¹⁵⁹ peptide bonds. Six cleavages cannot be ascribed to thrombin or matrix metalloproteinase activity, whereas the cleavage at Arg¹⁵²–Ser¹⁵³ matches thrombin specificity for OPN. The principal protease in milk, plasmin, hydrolyzed the same peptide bond as thrombin, but its main cleavage site was identified to be Lys¹⁵⁴–Ser¹⁵⁵. Another endogenous milk protease, cathepsin D, cleaved the Leu¹⁵¹–Arg¹⁵² bond. OPN fragments corresponding to plasmin activity were also identified in urine showing that plasmin cleavage of OPN is not restricted to milk. Plasmin, but not cathepsin D, cleavage of OPN increased cell adhesion mediated by the α_Vβ₃- or α₅β₁-integrins. Similar cellular adhesion was mediated by plasmin and thrombin-cleaved OPN showing that plasmin can be a potent regulator of OPN activity. These data show that OPN is highly susceptible to cleavage near its integrin-binding motifs, and the protein is a novel substrate for plasmin and cathepsin D.     

Solid state and solution conformations of a helical peptide with a central gly-gly segment

The influence of amino acids with contrasting conformational tendencies on the stereochemistry of oligopeptides has been investigated using an octapeptide Boc-Leu-Aib-Val-Gly-Gly-Leu-Aib-Val-OMe, which contains two helix-promoting Aib residues and a central helix-destabilizing Gly-Gly segment. Single crystal x-ray diffraction studies reveal that a 3 ₁₀-helix is formed up to the penultimate Aib residue, at which point there is a helix reversal in the backbone, reminiscent of a C-terminal 6 → I hydrogen bond. The curious feature in the crystal is the solvation of the possible 6 → 1 bond by a CH₃OH molecule, where the OH is inserted between O(3) and N(8) and participates in hydrogen bonds with both. The cell parameters are as follows: space group P2₁2₁2₁, a = 10.649(4) Å, b = 15.694(5) Å, c = 30.181(8) Å, R = 6.7% for 3427 data (| F₀| > 3σF) observed to 0.9 Å. Nuclear magnetic resonance studies in CDCl₃ using NH group solvent accessibility and nuclear Overhauser effects as probes are consistent with a 3 ₁₀-helical conformation. In contrast, in (CD₃)₂SO, unfolding of the central segment results in a multiple β-turn structure, with β-turn conformations populated at residues 1–2, 3–4, and 6–7. CD studies in methanol-2,2,2-trifluoroethanol (TFE) mixtures also provide evidence for a solvent-dependent structural transition. Helical conformations are populated in TFE, while type II β-turn structures are favored in methanol. © 1996 John Wiley & Sons, Inc.     

A nonhelical,multiple β-turn conformation in a glycine-rich heptapeptide fragment of trichogin A IV containing a single central α-aminoisobutyric acid residue

The conformational properties of the protected seven-residue C-terminal fragment the lipopeptaibol antibiotic Trichogin A IV (Boc-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe) has been examined in CDCl₃ and (CD₃)₂SO by ¹H-nmr. Evidence for a multiple β-turn conformation [type I′ at Gly(1)-Gly(2), type II at Leu(3)-Aib(4), and a type I′ at Aib(4)-Gly(5)] suggests that Leu(3) has preferred an extended or semiextended conformation over a helical conformation in CDCl₃. This structure is thus in contrast to earlier observations of seven-residue peptides containing a single central Aib preferring helical conformations in both solution and crystalline slates. A structural transition to a frayed right-handed helix is absented in (CD₃)₂SO. These results suggest that nonhelical conformations may be important in Gly-rich peptides containing Aib. Further, the presence of amino acids with contradictory influences on backbone conformational freedom can lead to well-defined conformational transitions even in small peptides. © 1995 John Wiley & Sons, Inc.     

Reactions of Tp–Os nitrido complexes with the nucleophiles hydroxide and thiosulfate

The reaction between TpOs(N)Cl₂ (1) [Tp = hydrotris(1-pyrazolyl)borate] and aqueous (ⁿBu₄N)(OH) in THF-d₈ forms the nitrosyl complex TpOs(NO)Cl₂ (5) among other products, suggesting an initial hydroxide attack at the nitrido ligand. In contrast, the reaction of the acetate complex TpOs(N)(OAc)₂ (2) with NaOH in Me₂CO/H₂O yields the osmium bis-hydroxide complex TpOs(N)(OH)₂ (3), which has been structurally characterized by single-crystal X-ray diffraction. Acetate for hydroxide exchange could occur by ligand substitution or by nucleophilic attack at the carbonyl carbon of the acetate ligands (saponification). Reacting 2 with Na¹⁸OH in H₂¹⁸O/CD₃CN yields predominantly doubly ¹⁸O-labeled TpOs(N)(¹⁸OH)₂ (3-¹⁸O₂) and unlabeled acetate, by ESI/MS and ¹³C{¹H} NMR. This indicates that hydroxide reacts by substitution rather than by attack at the ligand. The reaction of 2 with the softer nucleophile thiosulfate occurs at the nitrido ligand, giving the thionitrosyl complex TpOs(NS)(OAc)₂ (4). Reacting 4 with NaOH in (CD₃)₂CO/D₂O also generates the bis-hydroxide complex 3.     

Raman and infrared spectroscopic study of gramicidin A conformations

Raman scattering and infrared spectroscopic techniques were used to study the vibrational spectrum and conformation of the membrane channel protein gramicidin A in the solid state, in organic solutions and, using Raman scattering only, in a phospholipid environment. The investigation also includes measurements on head- and tail-group-modifled gramicidin A and a potassium thiocyanate-gramicidin A complex. Tentative identification of the molecular vibrations is proposed on the basis of the data on model compounds. The existence of four distinct conformations of the gramicidin A chain is established: conformation I present in the solid state, and CH₃OH and CD₃OD solutions; conformation II present in films cast from CHCl₃ solution; conformation III present in (CH₃)₂SO and (CD₃)₂SO solutions at concentrations below 0.5 m gramicidin A; and conformation IV present in the potassium thiocyanate-gramicidin A complex. The data obtainable on a gramicidin A-phospholipid suspension indicate a gramicidin A conformation in this environment corresponding either to the conformation I or II. The details of the spectra in the amide I region are shown to be consistent with a β-parallel hydrogen-bonded π_LD helix for conformational I, in terms of the polypeptide vibrational calculations of Nevskaya and co-workers. Conformation II is found to be consistent with an antiparallel double-stranded π_LD helix, while conformations III and IV probably have π-helical structures with larger channel diameters. The data on head- and tail-modified gramicidin A molecules indicate that their conformations are only slightly different from that of gramicidin A in conformation I.     

Conformational energy calculations of enzyme-substrate complexes of lysozyme. I. Energy minimization of monosaccharide and oligosaccharide inhibitors and substrates of lysozyme

Conformational energy calculations were performed on monosaccharide and oligosaccharide inhibitors and substrates of lysozyme to examine the preferred conformations of these molecules. A grid-search method was used to locate all of the low-energy conformational regions for N-acetyl-β-D -glycosamine (NAG), and energy minimization was then carried out in each of these regions. Three stable positions for the N-acetyl group have ben located, in two of which the plane of the amide unit is normal to the mean plane of the pyranosyl ring. Nine local energy minima were located for the —CH₂OH group. The positions of the two vicinal cis —OH groups are determined predominantly by interactions with either the —CH₂OH or the N-acetyl group. The most stable conformations of β-N-acetylmuramic acid (NAM) were determined from the study of the low-energy conformations of NAG. In the two stable orientations for the D -lactic acid side chain, the O—C—C′ plane (C′ being the carbon atom of the terminal carboxyl group) was found to be normal to the mean plane of the pyranosyl ring. The low-energy positions for the COOH group of NAM are determined mainly by interactions with neighboring groups. The conformational preferences of the α-anomers of NAG and NAM were also explored. The calculated conformation of the N-acetyl group for α-NAG was quite close to that determined by X-ray analysis. Two of the three lowest energy conformations of α-NAM are similar to the corresponding conformations of the β-anomer. A third low-energy structure, which has a hydrogen bond from the NH of the N-acetyl group to the C?O of the lactic acid group, corresponds very closely to the X-ray structure of this molecule. The preferred conformations of the disaccharides NAG–NAG, NAM–NAG and NAG–NAM were also investigated. Two preferred orientations of the reducing pyranosyl ring relative to the nonreducing ring were found for all of these disaccharides, both of which are close to the extended conformation. In one of these conformations, a hydrogen bond can form between the OH group attached to C₃ of the reducing sugar and the ring oxygen of the preceding residue. Each conformation can be stabilized further by a hydrogen bond between the CH₂OH (donor) of residue i + 1 and the C?O of residue i (acceptor). The interactions that determine conformations for all oligosaccharides containing both NAG and NAM are shown to be exclusively intraresidue and nearest neighbor interactions, so that it is possible to predict all stable conformations of oligosaccharides containing NAG and NAM in any sequence.     

The conformation of tetrafluorinated methyl galactoside anomers: crystallographic and NMR studies

The first single-crystal X-ray diffraction study of tetrafluorinated monosaccharide derivatives is presented. Both α- and β-methyl 2,3-dideoxy-2,2,3,3-tetrafluoro-d-galactopyranoside anomers adopt the ⁴C₁ conformation. The values for the C1–O1 and C1–O5 bond lengths and the O5–C1–O1–CH₃ dihedral angles are in line with what can be expected from the anomeric and exo-anomeric effects. The chair conformations are slightly distorted, presumably due to repulsion between 1,3-diaxial C–O and C–F bonds. The asymmetric unit of both compounds contains up to three independent molecules, which differ in the conformation of the hydroxymethyl group (including in one case a ‘forbidden’ gg rotamer). The molecular packing of the β-anomer shows a clear segregation between fluorinated and hydrophilic domains, while for the α-anomer the regions of fluorine segregation are broken by interleafing of OMe groups. There is one close OH?F contact, which is likely to arise from the crystal packing. NMR studies show that the two anomers also adopt a ⁴C₁ conformation in solution (D₂O, CDCl₃).     

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.