The 10th and 11th residues of short length N‐terminal PTH(1–12) analogue are important for its optimum potency

Abstract: The 10th and 11th residues of parathyroid hormone PTH(1–12) analogues were substituted to study the structure and function of PTH analogues. The substitution of Ala¹⁰ of [Ala^3,10,12(Leu⁷/Phe⁷)Arg¹¹]rPTH(1–12)NH₂ with Glu¹⁰ and/or the Arg¹¹ with Ile¹¹ markedly decreased cAMP generating activity. Data from circular dichroism (CD) and the nuclear magnetic resonance (NMR) structural analysis of [Ala^3,10,12(Leu⁷/Phe⁷)Arg¹¹]rPTH(1–12)NH₂ revealed tight α‐helical structures, while the Glu¹⁰ and/or Ile¹¹ substituted analogues showed unstable α‐helical structures. We conclude that 10th and 11th residues are important for stabilizing its helical conformation and that destabilization of the α‐helical structure, induced by substituting the above residues, remarkably affect its biological potency.     

Design,Synthesis, Structural Characterization by IR, 1H, 13C, 15N, 2D‐NMR,X‐Ray Diffraction and Evaluation of a New Class of Phenylaminoacetic Acid Benzylidene Hydrazines as pfENR Inhibitors

Recent studies have revealed that plasmodial enoyl‐ACP reductase (pfENR, FabI), one of the crucial enzymes in the plasmodial type II fatty acid synthesis II (FAS II) pathway, is a promising target for liver stage malaria infections. Hence, pfENR inhibitors have the potential to be used as causal malarial prophylactic agents. In this study, we report the design, synthesis, structural characterization and evaluation of a new class of pfENR inhibitors. The search for inhibitors began with a virtual screen of the iResearch database by molecular docking. Hits obtained from the virtual screen were ranked according to their Glide score. One hit was selected as a lead and modified to improve its binding to pfENR; from this, a series of phenylamino acetic acid benzylidene hydrazides were designed and synthesized. These molecules were thoroughly characterized by IR, ¹H, ¹³C, ¹⁵N, 2D‐NMR (COSY, NOESY, ¹H‐¹³C, ¹H‐¹⁵N HSQC and HMBC), and X‐ray diffraction. NMR studies revealed the existence of conformational/configurational isomers around the amide and imine functionalities. The major species in DMSO solution is the E, E form, which is in dynamic equilibrium with the Z, E isomer. In the solid state, the molecule has a completely extended conformation and forms helical structures that are stabilized by strong hydrogen bond interactions, forming a helical structure stabilized by N‐H…O interactions, a feature unique to this class of compounds. Furthermore, detailed investigation of the NMR spectra indicated the presence of a minor impurity in most compounds. The structure of this impurity was deduced as an imidazoline‐4‐one derivative based on ¹H‐¹³C and ¹H‐¹⁵H HMBC spectra and was confirmed from the NOESY spectra. The molecules were screened for in vitro activity against recombinant pfENR enzyme by a spectrophotometric assay. Four molecules, viz. 17, 7, 10, and 12 were found to be active at 7, 8, 10, and 12 μm concentration, respectively, showing promising pfENR inhibitory potential. A classification model was derived based on a binary QSAR approach termed recursive partitioning (RP) to highlight structural characteristics that could be tuned to improve activity.     

Bioactive N‐terminal undecapeptides derived from parathyroid hormone: the role of α‐helicity*

Abstract: The N‐terminal 1–34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses in bone characteristic of the native intact PTH. Recent studies have demonstrated that N‐terminal fragments presenting the principal activating domain such as PTH(1–11) and PTH(1–14) with helicity‐enhancing substitutions yield potent analogues with PTH(1–34)‐like activity. To further investigate the role of α‐helicity on biological potency, we designed and synthesized by solid‐phase methodology the following hPTH(1–11) analogues substituted at positions 1 and/or 3 by the sterically hindered and helix‐promoting C^α‐tetrasubstituted α‐amino acids α‐amino isobutyric acid (Aib), 1‐aminocyclopentane‐1‐carboxylic acid (Ac₅c) and 1‐aminocyclohexane‐1‐carboxylic acid (Ac₆c): Ac₅c‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( I ); Aib‐V‐Ac₅c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( II ); Ac₆c‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( III ); Aib‐V‐Ac₆c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( IV ); Aib‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( V ); S‐V‐Aib‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( VI ), S‐V‐Ac₅c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( VII ); Ac₅c‐V‐S‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( VIII ); Ac₆c‐V‐S‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( IX ); Ac₅c‐V‐Ac₅c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( X ); Ac₆c‐V‐Ac₆c‐E‐I‐Q‐L‐M‐H‐Q‐R‐NH₂ ( XI ). All analogues were biologically evaluated and conformationally characterized in 2,2,2‐trifluoroethanol (TFE) solution by circular dichroism (CD). Analogues I – V , which cover the full range of biological activity observed in the present study, were further conformationally characterized in detail by nuclear magnetic resonance (NMR) and computer simulations studies. The results of ligand‐stimulated cAMP accumulation experiments indicated that analogues I and II are active, analogues III , VI and VII are very weakly active and analogues IV , V , VIII–XI are inactive. The most potent analogue, I exhibits biological activity 3500‐fold higher than that of the native PTH(1–11) and only 15‐fold weaker than that of the native sequence hPTH(1–34). Remarkably, the two most potent analogues, I and II , and the very weakly active analogues, VI and VII , exhibit similar helix contents. These results indicate that the presence of a stable N‐terminal helical sequence is an important but not sufficient condition for biological activity.     

Synthesis and structural identification of fluorine‐18 labeled parathyroid hormone

Parathyroid hormone (PTH) is an 84 amino acid peptide hormone that plays a key role in bone and mineral metabolism. The biological actions of PTH are mediated via the N‐terminal PTH(1–34) fragment, serving as the PTH receptor‐binding sequence, and which is therefore used clinically to treat conditions of low bone mass such as osteoporosis. In this study, PTH(1–34) was conjugated with non‐radioactive (stable F isotope) N‐succinimidyl 4‐fluorobenzoate (SFB) leading to three isomeric mono‐fluorobenzoated (FBz) PTH followed by Liquid chromatography‐Tandem mass spectrometry (LC‐MS/MS) assisted structural identification. Corresponding [¹⁸F]SFB‐labeled PTH derivatives were prepared respectively and the Lys¹³ site‐specific labeled [¹⁸F]FBz PTH was isolated by HPLC with radiochemical purity >99% and specific activity of 2.78 GBq/µmol, suitable for future application with in vivo pharmacokinetic/pharmacodynamic studies of PTH, using preclinical Positron Emission Tomography Computed Tomography (PET/CT) imaging.     

The cytoplasmic helix of cannabinoid receptor CB2, a conformational study by circular dichroism and 1H NMR spectroscopy in aqueous and membrane‐like environments

Abstract: The cytoplasmic helix domain (fourth cytoplasmic loop, helix 8) of numerous G protein‐coupled receptors (GPCRs) such as rhodopsin and the β‐adrenergic receptor exhibit unique structural and functional characteristics. Computer models also predict this structure for the cannabinoid CB2 receptor, another member of the GPCR superfamily. In our study, a peptide corresponding to helix 8 of the CB2 receptor was synthesized chemically and its secondary structure determined by circular dichroism (CD) and ¹H NMR spectroscopy. NMR and CD revealed an α‐helical structure in this region in both dodecylphosphocholine micelles and dimethylsulfoxide, in contrast to a random coil configuration found in aqueous solvent. This finding is in good agreement with other previous GPCR structural studies including X‐ray crystallography. By combining our finding with other studies, we further hypothesize that the amphipathic nature of helix 8 can play a significant role in the function and regulation of CB receptors as well as other GPCRs in general.     

Structural characterization of peptide fragments from hCD81–LEL

Abstract: The solution conformation of two peptides [ 1 : PSGSNIISNLFKED;  2 : GSSTLTALTTSVLKNNL] from human CD81 (hCD81) large extra‐cellular loop (LEL) with known importance in the hepatitis C virus glycoprotein E2 (HCV‐E2) binding interaction was characterized using circular dichroism spectroscopy. In addition, the solution structure of peptide 1 that contains a phenylalanine residue (F186 in hCD81) known to be critical in the binding interaction with HCV‐E2 was determined using 1D and 2D ¹H NMR spectroscopy. Both peptides are unstructured in water but begin forming significant helical conformation following the addition of 20% or more trifluoroethanol (v/v), a result consistent with their α‐helical conformation found in the native protein. The CD data recorded as a function of pH and NaCl concentration are consistent with stabilization of the helical structure from electrostatic forces for both peptides. Peptide 1 is able to block the binding interaction of recombinant HCV‐E2 (rHCV‐E2) to hCD81 expressed on Molt‐4 T cells at high concentrations (3.5 mm ), a low affinity that we attributed to the random coil structure in water.     

Solution structure of a cathelicidin‐derived antimicrobial peptide,CRAMP as determined by NMR spectroscopy

Abstract: CRAMP was identified from a cDNA clone derived from mouse femoral marrow cells as a member of cathelicidin‐derived antimicrobial peptides. This peptide shows potent antimicrobial activity against gram‐positive and gram‐negative bacteria but no hemolytic activity against human erythrocytes. CRAMP was known to cause rapid permeabilization of the inner membrane of Escherichia coli. In this study, the structure of CRAMP in TFE/H₂O (1 : 1, v/v) solution was determined by CD and NMR spectroscopy. CD spectra showed that CRAMP adopts a mainly α‐helical conformation in TFE/H₂O solution, DPC micelles, SDS micelles and liposomes, whereas it has a random structure in aqueous solution. The tertiary structure of CRAMP in TFE/H₂O (1 : 1, v/v), as determined by NMR spectroscopy, consists of two amphipathic α‐helices from Leu⁴ to Lys¹⁰ and from Gly¹⁶ to Leu³³. These two helices are connected by a flexible region from Gly¹¹ to Gly¹⁶. Previous analysis of series of fragments composed of various portion of CRAMP revealed that an 18‐residue fragment with the sequence from Gly¹⁶ to Leu³³ was found to retain antibacterial activity. Therefore, the amphipathic α‐helical region from Gly¹⁶ to Leu³³ of CRAMP plays important roles in spanning the lipid bilayers as well as its antibiotic activity. Based on this structure, novel antibiotic peptides having strong antibiotic activity, with no hemolytic effect will be developed.     

The conformational and biological analysis of a cyclic anti‐obesity peptide from the C‐terminal domain of human growth hormone

Abstract: The three‐dimensional solution structure of anti‐obesity drug (AOD), a 15‐residue, disulfide‐bonded, cyclic peptide, cyclo(6,13)‐H₂N‐Leu‐Arg‐Ile‐Val‐Gln‐Cys‐Arg‐Ser‐Val‐Glu‐Gly‐Ser‐Cys‐Gly‐Phe‐OH, derived from the C‐terminal domain of the human growth hormone (hGH) (residues 177–191) was determined using two‐dimensional ¹H NMR spectroscopy. AOD stimulates lipolysis and inhibits lipogenesis, in vitro, in rodent, porcine and human adipose tissues. These biological effects suggest that AOD is a potential therapeutic candidate for the treatment of obesity. Conformational studies of AOD were conducted in aqueous solution and in water/dimethylsulfoxide mixtures. In general, spectral quality was superior in the water/dimethylsulfoxide mixtures. The cyclic region of AOD in water/dimethylsulfoxide adopts type I β‐turns at residues Ser⁸‐Val⁹‐Glu¹⁰‐Gly¹¹ and Ser¹²‐Cys¹³‐Gly¹⁴‐Phe¹⁵, each preceded by loop‐like structures. Comparison of the conformation of this peptide with residues 177–191 in the native hGH protein X‐ray crystal structure indicates that the synthetic peptide retains some structural similarity to the intact protein. This study provides evidence that the C‐terminal region of hGH is a specific functional domain of the multifunctional hGH protein.     

Relationship between the tertiary structures of mastoparan B and its analogs and their lytic activities studied by NMR spectroscopy

Abstract: Mastoparan B (MP‐B), an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis, is an amphiphilic α‐helical peptide. MP‐B possesses a variety of biological activities, such as mast cells degradation histamine release, erythrocyte lysis and inhibition of the growth of Gram‐positive and Gram‐negative bacteria. In order to study the relationship between the structure and the biological activity of MP‐B, we used four analogs by replacing amino acids with alanine. Tertiary structures of MP‐B and its analogs in 2,2,2‐trifluoroethanol (TFE)‐containing aqueous solution have been determined by NMR spectroscopy and molecular modeling. The results indicate that [Ala⁴]MP‐B and [Ala¹²]MP‐B with higher hydrophobicity adopt a higher content of amphiphilic helical structures, and have better antimicrobial and hemolytic activities than MP‐B. However, [Ala³]MP‐B and [Ala⁹]MP‐B with lower hydrophobicity have disordered structures. [Ala³]MP‐B and [Ala⁹]MP‐B have low antimicrobial activity and much less hemolytic activity relative to MP‐B. It is likely that tryptophan residue in MP‐B and appropriate hydrophobicity of MP‐B to induce α‐helical structure is essential for the antibacterial and hemolytic activity of MP‐B. This study can aid understanding of the structure–activity relationship of MP‐B and to design peptides to possess lytic activity.     

Conformational study of an Aib‐rich peptide in DMSO by NMR

Abstract: The strong propensity of 2‐amino‐2‐methyl propanoic acid (Aib)‐rich peptides to form stable helical structures is well documented. NMR analysis of the short peptide Z‐(Aib)₅‐L ‐Leu‐(Aib)₂‐OMe indicates the presence of a well‐characterized 3₁₀‐helix even in dimethylsulfoxide (DMSO), a solvent known to disrupt helical structures. The structure remains stable at least up to 348 K. Stereospecific assignment of the diastereotopic methyls of Aib was achieved, with the assumption of a specific helical screw sense. The methyl more eclipsed with respect to the CO vector resonates at a higher field in the carbon dimension. Molecular dynamics simulations successfully predict the ³J_CHNH coupling constant of Leu⁶ and most of the H‐bonding pattern. Discrepancies were found for Aib³ and Aib⁷ amide protons which can be explained by a higher sensitivity of the simulations to the helix fraying at the end of the peptide and by the presence of extended conformations for Leu⁶ during most of the simulations.     

Conformational behaviour of a synthetic peptide of the C-terminus of villin that interacts with actin: an NMR,CD and simulated annealing study

The solution structure of a synthetic 22-amino acid peptide (P1) corresponding to the extreme C-terminal end and one of the F-actin binding sites of villin has been determined by ¹H NMR and CD spectroscopy. The structure of this peptide was compared to that of a peptide in which lysine to glutamic acid substitutions were introduced at positions 17 and 19 (P11), abolishing F-actin binding. Both peptides are largely unstructured in aqueous solution. Changes observed in the NMR and CD spectra of both peptides are consistent with α-helix formation in trifluoroethanol/water mixtures. A set of 189 interproton distances derived from nuclear Overhauser enhancement (NOE) measurements, 17 φ-angle constraints obtained from ³J_nhα coupling constants, as well as about 10 N···O distance restraints deduced from amide proton exchange kinetics with deuterium, were used for the structure determination. The three-dimensional structure of P1 and P11 is characterized by two helical regions, one extending from residues 2 to 5 and a second covering residues 7 to 17. The central fragment, ranging from Leu-7 to Leu-15, is more stable. The C-terminal residues are less structured, particularly within peptide P11. The significance of these structural results is discussed in relation to the biological activity of villin. © Munksgaard 1995.     

Conformational studies of a potent Leu11, D-Trp12-containing lactam-bridged parathyroid hormone-related protein-derived antagonist

Human parathyroid hormone-related protein (PTHrP) is expressed in various tissues where it acts as an endocrine/paracrine factor involved in cellular growth, differentiation and development of fetal skeleton. As for parathyroid hormone (PTH), which is the hormone responsible for regulation of extracellular calcium homeostasis, the N-terminal 1-34 fragment can reproduce the full spectrum of calciotropic activities inherent in full-length PTH. Truncation of six amino acid residues from the N-terminus of both hormone sequences generates 7-34 fragments which act as weak antagonists. Although PTH(7-34) is a pure antagonist, PTHrP(7-34) acts as partial agonist against the receptor shared by both hormones, the PTH/PTHrP receptor. In the current study, we analyzed the conformation of [Leu¹¹,d -Trp¹²,Lys²⁶,Asp³⁰]PTHrP(7-34)NH₂ (hybrid-lactam) in a 1:1 mixture of H₂O/TFE-d₃ at pH 4 by circular dichroism, nuclear magnetic resonance and distance geometry calculations. This weak antagonist (K_b= 650 nm ) combines two modifications: Leu¹¹,d -Trp¹² (K_b= 5.1 nm ), reported to eliminate partial agonism and enhance potency, and Lys²⁶-Asp³⁰ lactamization (K_b= 31 nm ), aimed to stabilize the helical structure of the principal binding domain attributed to residues 25-34. The helical content in 30% trifluoroethanol is 88%, i.e., higher than the corresponding linear analog, and comprises the d -Trp¹²-Thr³³ segment. This hybrid lactam contains a rigid helical segment spanning the 14-18 sequence followed by a hinge motif around Arg^19-20, but the sequence 14-18 forms a stable helix. In all potent lactam-containing, PTHrP-derived agonists and antagonists studied so far, the dominant structural motif consists of two helical domains at the two ends of the sequence and of two hinge regions centered around Gly¹²-Lys¹³ and Arg¹⁹. The weakly active agonists and antagonists do not exhibit the “hinge” around position 19. These findings suggest that the presence and location of discrete hinge regions that connect the N- and C-terminal helices are essential for generating the bioactive conformation of ligands for the PTH/ PTHrP receptor.     

(αMe)Aun: a highly lipophilic,chiral, Cα‐tetrasubstituted α‐amino acid. Incorporation into model peptides and preferred conformation

Abstract: Using a chemo‐enzymatic approach we prepared the highly lipophilic, chiral, C^α‐methylated α‐amino acid (αMe)Aun. Two series of terminally protected model peptides containing either d ‐(αMe)Aun in combination with Aib or l ‐(αMe)Aun in combination with Gly were synthesized using solution methods and fully characterized. A detailed solution conformational analysis, based on FT‐IR absorption, ¹H NMR and CD techniques, allowed us to determine the preferred conformation of this amino acid and the relationship between chirality at its α‐carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that d ‐(αMe)Aun favors the formation of the left‐handed 3₁₀‐helical conformation.     

Didanosine Polymorphism in a Supercritical Antisolvent Process

Solid‐state properties of active ingredients are crucial in pharmaceutical development owing to their significant clinical and economical implications. In the present work we investigated the solid‐state properties and the solubility in water of didanosine, DDI, re‐crystallized from a dimethylsulfoxide solution using supercritical CO₂ as an antisolvent (SAS process) for comparison with the commercially available drug product. We also applied modern solid‐state NMR (SS NMR) techniques, namely 2D ¹H DQ CRAMPS (Combined Rotation And Multiple Pulse Spectroscopy) and ¹H–¹³C on‐ and off‐resonance CP (cross polarization) FSLG‐HETCOR experiments, known for providing reliable information about ¹H–¹H and ¹H–¹³C intra‐ and intermolecular proximities, in order to address polymorphism issues arising from the crystallization of a new form in the supercritical process. A new polymorph of didanosine was obtained from the supercritical antisolvent process and characterized by means of 1D and 2D multinuclear (¹H, ¹³C, ¹⁵N) SS NMR. The particle size of the new crystal phase was reduced by varying the antisolvent density through a pressure increase. The structural differences between the commercial product and the SAS re‐crystallized DDI are highlighted by X‐ray diffractometry and well described by solid‐state NMR. The carbon C6 ¹³C chemical shift suggests that both commercial and re‐crystallized didanosine samples are in the enol form. The analysis of homo‐ and heteronuclear proximities obtained by means of 2D NMR experiments shows that commercial and SAS re‐crystallized DDI possess very similar molecular conformation and hydrogen bond network, but different packing. The new polymorph proved to be a metastable form at ambient conditions, showing higher solubility in water and lower stability to mechanical stress. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1855–1870, 2010     

Structural features of linear,homo‐Aib‐based peptides in solution: a spectroscopic and molecular mechanics investigation

Abstract: In continuation of our studies on the determination of the structural features of functionalized peptides in solution by combining time‐resolved fluorescence data and molecular mechanics results, the conformational properties of a series of linear, homo‐Aib peptides in methanol (a structure‐supporting solvent) were investigated. These compounds have the general formula P(Aib)_nN, where Aib is α‐aminoisobutyric acid, N is naphthalene and P is the monomethylated protoporphyrin IX, the two latter chromophores being covalently attached to the peptide C‐ and N‐termini, respectively, while n = 3, 6 and 9. According to ¹H NMR and IR spectra, the peptides investigated largely populate a 3₁₀‐helical structure in CDCl₃, which is also a structure‐supporting solvent. Both steady‐state and time‐resolved fluorescence measurements show a strong quenching of the N emission that parallels an increase of the P fluorescence intensity, suggesting the occurrence of long‐range energy transfer from ¹N* to ground‐state P. Comparison of quenching efficiencies and lifetime pre‐exponents with those obtained theoretically from the deepest energy minimum conformers is very satisfactory. The computed structures, built up by partially taking into account the solvent medium, exhibit a rigid, highly compact arrangement, owing to both the 3₁₀‐helix conformation of the backbone chain and the very few peptide‐to‐chromophore covalent linkages. As a result, only one or two stable conformations for each peptide were theoretically found, in full agreement with the time‐resolved fluorescence data. Orientational effects between the probes must be taken into account for a correct interpretation of the fluorescence decay results, which implies that interconversion among conformational substates of the N linkages is slower than 10 ns, corresponding to the upper limit of the energy transfer
characteristic time.     

(αMe)Nva: stereoselective syntheses and preferred conformations of selected model peptides

Abstract: Using different stereoselective chemical and chemoenzymatic approaches we synthesized the chiral, C^α‐methylated α‐amino acid l ‐(αMe)Nva with a short, linear side‐chain. A set of terminally protected model peptides to the pentamer level containing either (αMe)Nva or Nva in combination with Ala and/or Aib was prepared using solution methods and characterized fully. Two (αMe)Nva peptides were also synthesized using side‐chain hydrogenation of the corresponding C^α‐methyl, C^α‐allylglycine (Mag) peptides. A detailed solution and crystal‐state conformational analysis based on FT‐IR absorption, ¹H NMR and X‐ray diffraction techniques allowed us to define that: (i) (αMe)Nva is an effective β‐turn and 3₁₀‐helix former; and (ii) the relationship between (αMe)Nva chirality and the screw sense of the turn/helix formed is that typical of protein amino acids, i.e. l ‐(αMe)Nva induces the preferential formation of right‐handed folded structures. In more general terms, this study reinforced previous conclusions that peptides based on α‐amino acids with a C^α‐methyl substituent and a C^α‐linear alkyl substituent are characterized by a strong tendency to fold into turn and helical structures.     

Tryptophan‐containing peptide helices: interactions involving the indole side chain*

Abstract: Two designed peptide sequences containing Trp residues at positions i and i + 5 (Boc‐Leu‐Trp‐Val‐Ala‐Aib‐Leu‐Trp‐Val‐OMe, 1 ) as well as i and i + 6 (Boc‐Leu‐Trp‐Val‐Aib‐Ala‐Aib‐Leu‐Trp‐Val‐OMe, 2 ) containing one and two centrally positioned Aib residues, respectively, for helix nucleation, have been shown to form stable helices in chloroform solutions. Structures derived from nuclear magnetic resonance (NMR) data reveal six and seven intramolecularly hydrogen‐bonded NH groups in peptides 1 and 2 , respectively. The helical conformation of octapeptide 1 has also been established in the solid state by X‐ray diffraction. The crystal structure reveals an interesting packing motif in which helical columns are stabilized by side chain–backbone hydrogen bonding involving the indole N?1H of Trp(2) as donor, and an acceptor C=O group from Leu(6) of a neighboring molecule. Helical columns also associate laterally, and strong interactions are observed between the Trp(2) and Trp(7) residues on neighboring molecules. The edge‐to‐face aromatic interactions between the indoles suggest a potential C‐H…π interaction involving the Cζ3H of Trp(2). Concentration dependence of NMR chemical shifts provides evidence for peptide association in solution involving the Trp(2) N?1H protons, presumably in a manner similar to that observed in the crystal.     

Solution structure of the phosphorylated sites of ribosomal protein S6 by 1H NMR spectroscopy

An increase in the rate of protein synthesis is found to be accompanied by phosphorylation of the 40S ribosomal protein S6. Treatment of S6 by cyanogen bromide produced three fragments, and one of the fragments of S6, which is a C-terminal portion of S6 (M_r? 4000), contains all phosphorylation sites of S6. The C-terminal fragment of S6 contains seven serines. S6 kinase phosphorylates S6 specifically, i.e. five serines in the C-terminal of S6 are phosphorylated. The three-dimensional structure of S6 peptide was studied in 50% trifluoroethanol/50% H₂O solution by ¹H NMR with combined use of distance geometry and restrained molecular dynamics calculations. NMR results indicated that it takes an α-helix between Glu5 and Arg21 and a distorted helical structure for the following three residues, but no rigid structure was present from Ser25 through the C-terminus and for the N-terminal region (Lys1-Lys4). The specificity of the phosphorylation of the peptide is discussed from a structural aspect. © Munksgaard 1996.