Comparative study of eight oxytocin antagonists by simulated annealing

In this study, eight oxytocin (OT) antagonists were subjected to conformational analysis. By means of simulated annealing, 2,000 structures were generated to sample the conformational space. The preferred main-chain and side-chain dihedral angles, intramolecular H-bond interactions (types 1←4 and 1←3), and secondary-structure elements (β-turns) were determined in the series of compounds. The influence of the modified amino acid on these structural elements was established; the values determined will be used as filtration criterion in preliminary research.     

Predicted secondary structure of maltodextrin Phosphorylase fromEscherichia coli as deduced using Chou-Fasman model

Secondary structure of maltodextrin Phosphorylase fromEscherichia coli has been predicted using Chou-Fasman model. The enzyme protein contains 28% α-helix, 27% β-pleated sheets and 20% reverse β-turns. The secondary structure predicted 4 regions showing Rossman-fold super secondary structure. Two regions, one from residue 268–361 and the another from residue 606–684, having 4 consecutive strands of parallel β-pleated sheets and 3 joining α-helix, are predicted. Two regions, one from residue 379–434 and the another from residue 496–573, having 3 consecutive strands of parallel β-pleated sheets and two joining α-helix, are predicted.     

Development of cytochrome-c oxidase activity in rat heart

3′-azido-3′-deoxythymidine (AZT) is the first effective drug used clinically for the treatment of human immunodeficiency virus (HIV) infection. The drug interactions with DNA and protein are associated with its mechanism of action in vivo. This study was designed to examine the interaction of AZT with the Na,K-dependent adenosine triphosphatase (Na,K-ATPase) in H₂O and D₂O solutions at physiological pH using drug concentration of 0.1 μM to 1 mM and final protein concentration of 0.5 to 1 mg/mL. Ultraviolet absorption and Fourier transform infrared difference spectroscopy with its self-deconvolution second-derivative resolution enhancement, and curve-fitting procedures were used to characterize the drug-binding mode, the drug-binding constant, and the effects of drug interaction on the protein secondary structure Spectroscopic evidence showed that at low drug concentration (0.1 μM), AZT binds (H-bonding) mainly to the polypeptide C=O and C−N groups with two binding constants of K₁=5.3×10⁵ M ⁻¹ and K₂=9.8×10³ M ⁻¹. As drug content increased, AZT-lipid complex prevailed. At a high drug concentration (1 mM), drug binding resulted in minor protein secondary structural changes from that of the α-helix 19.8%; β-pleated 25.6%; turn 9.1%; β-antiparallel 7.5% and random 38%, in the free Na,K-ATPase to that of the α-helix 19%; β-pleated 21.1%; turn 10.1%; β-antiparallel 8.8% and random 41%, in the AZT-ATPase complexes.     

Protein secondary structure from circular dichroism spectra

Circular dichroism spectra of proteins are extremely sensitive to secondary structure. Nevertheless, circular dichroism spectra should not be analyzed for protein secondary structure unless they are measured to at least 184 nm. Even if all the various types ofβ-turns are lumped together, there are at least 5 different types of secondary structure in a protein (α-helix, antiparallelβ-sheet, parallelβ-sheet,β-turn, and other structures not included in the first 4 categories). It is not possible to solve for these 5 parameters unless there are 5 equations. Singular value decomposition can be used to show that circular dichroism spectra of proteins measured to 200 nm contain only 2 pieces of information, while spectra measured to 190 nm contain about 4. Adding the constraint that the sum of secondary structures must equal 1 provides another piece of information, but even with this constraint, spectra measured to 190 nm simply do not analyze well for the 5 unknowns in secondary structure. Spectra measured to 184 nm do contain 5 pieces of information and we have used such spectra successfully to analyze a variety of proteins for their component secondary structures.     

2-Alkyl-2-carboxyazetidines as γ-turn inducers: incorporation into neurotrophin fragments

Conveniently substituted 2-alkyl-2-carboxyazetidine amino acids have been incorporated into NGF and NT3 tetrapeptide sequences to investigate their utility as reverse turn inducers (γ- vs. β-turns). Despite the presence of an Asp residue at i position, highly preferred in β-turns, molecular modeling and NMR studies indicated that the azetidine-containing peptides mainly stabilized γ-turn conformations.     

Studies on solution NMR structure of brazzein

Brazzein is a sweet-tasting protein isolated from the fruit of West African plantPentadiplandra brazzeana Baillon. It is the smallest and the most water-soluble sweet protein discovered so far and is highly thermostable. The proton NMR study of brazzein at 600 MHz (pH 3.5, 300 K) is presented. The complete sequence specific assignments of the individual backbone and sidechain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of brazzein contains one α-helix (residues 21–29), one short 3₁₀-helix (residues 14–17), two strands of antiparallel β-sheet (residues 34–39, 44–50) and probably a third strand (residues 5–7) near the N-terminus. A comparative analysis found that brazzein shares a so-called ‘cysteine-stabilized alpha-beta’ (CSαβ) motif with scorpion neurotoxins, insect defensins and plant γ - thionins. The significance of this multi-function motif, the possible active sites and the structural basis of themostability were discussed.     

Extracellular production of <Emphasis Type="Italic">Streptomyces exfoliatus</Emphasis> poly(3-hydroxybutyrate) depolymerase in <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. T104: determination of optimal biocatalyst conditions

The phaZ _Sex gene encoding poly(3-hydroxybutyrate) depolymerase from Streptomyces exfoliatus has been successfully cloned and expressed in Rhodococcus sp. T104 for the first time. Likewise, the recombinant enzyme was efficiently produced as an extracellular active form and purified to homogeneity by two hydrophobic chromatographic steps. MALDI-TOF analysis showed that the native enzyme is a monomer. Circular dichroism studies have revealed a secondary structure showing 25.6% α-helix, 21.4% β-sheet, 17.1% β-turns, and 35.2% random coil, with a midpoint transition temperature (T _m) of 55.8 °C. Magnesium and calcium ions enhanced the enzyme activity, whereas manganese inhibited it. EDTA moderately decreased the activity, and the enzyme was completely deactivated at 3 M NaCl. Chemical modification studies indicated the presence of the catalytic triad serine–histidine–carboxylic acid in the active site. High-performance liquid chromatography (HPLC)–mass spectrometry (MS) analysis of PHB products of enzymatic hydrolysis showed monomers and dimers of 3-hydroxybutyric acid, demonstrating that PHB depolymerase is an exo-hydrolase. Addition of methyl-β-cyclodextrin simultaneously increased the activity as well as preserved the enzyme during lyophilization. Finally, thermoinactivation studies showed that the enzyme is highly stable at 40 °C. All these features support the potential industrial application of this recombinant enzyme in the production of (R)-3-hydroxyalkanoic acid derivatives as well as in the degradation of bioplastics.     

Structural and functional importance of theβ-turn in proteins. Studies on proline-containing peptides

We report here two sets of results on proline-containing linear peptides, one of which brings out the role of theβ-turn conformation in the structure of nascent collagen while the other points to the functional importance of the β-turn in calcium-binding proteins. Based on the data on peptides containing the -Pro-Gly-sequence, we had proposed and experimentally verified that theβ-turn conformation in these peptides is a structural requirement for the enzymic hydroxylation of the proline residues in the nascent (unhydroxylated) procollagen molecule. Our recent data, presented here, on the conformation of peptides containing both the -Pro-Gly- and -Gly-Pro-sequences reveal that while theβ-turn in the substrate molecule is required at the catalytic site of prolyl hydroxylase, the polyproline-II structure is necessary for effective binding at the active site of the enzyme. Thus, peptides containing either theβ-turn or the polyproline-II structure alone are found to act only as inhibitors while those with the polyproline-II followed byβ-turn serve as substrates of the enzyme. In another study, we have synthesized the two linear peptides: Boc-Pro-D-Ala-Ala-NHCH₃ and Boc-Pro-Gly-Ala-NHCH₃ each of which adopts, in solution, a structure with two consecutiveβ-turns, as judged from circular dichroism, infrared and nuclear magnetic resonance data. Drastic spectral changes are seen in these peptides on binding to Ca²⁺. Both the peptides show a distinct specificity to Ca²⁺ over Mg²⁺, Na⁺ and Li₊. A conformational change in the peptides occurs on Ca²⁺ binding which brings together the carbonyl groups to coordinate with the metal ion. These results imply a functional role for theβ-turn in Ca²⁺ — binding proteins.     

Structural and functional characterization of <Emphasis Type="Italic">Delphinus delphis</Emphasis> hemoglobin system

Structural analysis of the hemoglobin (Hb) system of Delphinus delphis revealed a high globin multiplicity: HPLC–electrospray ionization-mass spectrometry (ESI-MS) analysis evidenced three major β (β1 16,022 Da, β2 16,036 Da, β3 16,036 Da, labeled according to their progressive elution times) and two major α globins (α1 15,345 Da, α2 15,329 Da). ESI-tandem mass and nucleotide sequence analyses showed that β2 globin differs from β1 for the substitution Val126 → Leu, while β3 globin differs from β2 for the isobaric substitution Lys65 → Gln. The α2 globin differs from the α1 for the substitution Ser15 → Ala. Anion-exchange chromatography allowed the separation of two Hb fractions and HPLC–ESI-MS analysis revealed that the fraction with higher pI (HbI) contained β1, β2 and both the α globins, and the fraction with lower pI (HbII) contained β3 and both the α globins. Both D. delphis Hb fractions displayed a lower intrinsic oxygen affinity, a decreased effect of 2,3-BPG and a reduced cooperativity with respect to human HbA₀, with HbII showing the more pronounced differences. With respect to HbA₀, either the substitution Proβ5 → Gly or the Proβ5 → Ala is present in all the cetacean β globins sequenced so far, and it has been hypothesized that position 5 of β globins may have a role in the interaction with 2,3-BPG. Regarding the particularly lowered cooperativity of HbII, it is interesting to observe that the variant human HbA, characterized by the substitution Lysβ65 → Gln (HbJ-Cairo) has a decreased cooperativity with respect to HbA₀.     

Analysis of Chameleon Sequences by Energy Decomposition on a Pairwise Per-residue Basis

The conversion from α-helix to β-strand that has been widely observed in so-called chameleon sequences has received considerable attention since such a structural change may induce many amyloidogenic proteins to self-assemble into fibrils thus causing fatal diseases. Here we report a large scale-analysis of the energetics of secondary structural conversions in a collection of chameleon sequences retrieved from the Protein Data Bank. Major energetic contributions to the secondary structural conversion were analyzed by carrying out energy decomposition on a pairwise per-residue basis, i.e., (i,i), (i,i ± 1), (i,i ± 2), (i,i ± 3), (i,i ± 4) and > (i,i ± 4) intra-/inter-residual interactions. While the overall potential energy differences were subtle, individual residue-based interacting energy differences were observed to vary significantly depending on the specific type of secondary structural conversion. The average energy difference between α-helix and β-strand, <ΔE _α→β>, in the chameleon sequences varied significantly in (i,i), (i,i ± 1) and > (i,i ± 4) interactions. The major energetic factors in secondary structure conversions were electrostatic interactions and the polar term for solvation energy. In addition, residue-based average energy differences in α-helix → β-strand conversions were well-correlated to those in α-helix → random coil → β-strand conversions (R ² = 0.92). Assuming that three secondary structural elements can transform in either direction, this strong correlation indicates that the present energy decomposition method using database structures of chameleon sequences provides a reliable tool for the characterization of secondary structure fluctuations in amino acid sequences.     

Molecular dynamics simulations of the folding of poly(alanine) peptides

The secondary structures and the shapes of long-chain polyalanine (PA) molecules were investigated by all-atom molecular dynamics simulations using a modified Amber force field. Homopolymers of polyaminoacids such as PA are convenient models to study the mechanism of protein folding. It was found that the conformational structures of PA peptides are highly sensitive to the chain length. In the absence of solvent, straight α-helices dominate in short (n ∼ 20) peptides at room temperature. A shape transition occurs at a chain length n of 40–45; the compact helix-turn-helix structure (the double-leg hairpin) becomes favored over a straight α-helix. For n = 60, double-leg and the triple-leg hairpins are the only structures present in PA molecules. An exploration of a chain organization in a cubic cavity revealed a clear predisposition of PA molecules for additional breaks in α-helices and the formation of multifolded hairpins. Furthermore, under confinement the hairpin structure becomes much looser, the antiparallel positions of helical stems are disturbed, and a sizeable proportion of the helical stems are transformed from α-helices into 3₁₀-helices.     

Spectroscopic Detection of Pseudo-Turns in Homodetic Cyclic Penta- and Hexapeptides Comprising β-Homoproline

β-Amino acids with side chains at C² and/or at C³ are of growing interest in drug design, as they may induce astonishing and unusual peptide conformations. Therefore it is of eminent importance to gather information on the consequences of β-amino acid incorporation on the three-dimensional structure of a peptide. This paper describes the synthesis and conformational analysis of cyclic penta- and hexapeptides comprising either (S)-Pro or (S)-β-Hpro. The conformational influence of the β-homoproline building block was analyzed by the combined application of CD, FT-IR and NMR. While the CD spectra of the proline containing peptides indicate the presence of inverse γ-turns and βII-turns, the CD spectra of the β-homoamino acid analogs are dominated by an unprecedented negative band near 205 nm associated with a pseudo-β-turn (Ψβ) or pseudo-γ-turn (Ψγ). These results were confirmed by FT-IR spectroscopy, which also indicates the formation of two internal hydrogen bonds in the cyclic peptides containing the β-homoproline. The conformations of the β-homoproline containing pentapeptides were additionally determined by NMR in combination with MD simulations in two different solvents. The conformation in trifluoroethanol (TFE) is characterized by a bifurcated hydrogen bond stabilizing a pseudo-γ-turn with β-homoproline in the central position, nested with a pseudo-β-turn with β-homoproline in the i+1 position. The combined CD/FT-IR studies clearly show that the replacement of proline by β-homoproline gives rise to a more flexible peptide backbone, and CD spectroscopy hints towards the presence of pseudo-β- or pseudo-γ-turns.     

Domain Evolution in the α-Amylase Family

The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their domain B, a distinct domain that protrudes from the regular catalytic (β/α)₈-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase, cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable similarity with (β/α)₈-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family. Received: 4 December 1996 / Accepted: 13 March 1997     

Prediction of beta-turns at over 80% accuracy based on an ensemble of predicted secondary structures and multiple alignments

Background  

β-turn is a secondary protein structure type that plays significant role in protein folding, stability, and molecular recognition. To date, several methods for prediction of β-turns from protein sequences were developed, but they are characterized by relatively poor prediction quality. The novelty of the proposed sequence-based β-turn predictor stems from the usage of a window based information extracted from four predicted three-state secondary structures, which together with a selected set of position specific scoring matrix (PSSM) values serve as an input to the support vector machine (SVM) predictor.     

Stabilizing effects of 2-methylalanine residues on β-turns and α-helices

¹³C-, ¹H-nmr, CD, and x-ray crystallography revealed β-turns of type III for Boc-Gly-L-Ala-Aib-OMe, Boc-L-Ala-Aib-L-Ala-OMe; the 3₁₀-helix for Boc-Aib-L-Ala-Aib-L-Ala-Aib-OMe; and antiparallel arranged α-helices for Boc-L-Ala-Aib-Ala-Aib-Ala-Glu(OBzl)-Ala-Aib-Ala-Aib-Ala-OMe. An N-terminal rigid α-helical segment is found in the polypeptide antibiotics alamethicin, suzukacillin, and trichotoxin. The α-helix dipole is essential for their voltage-dependent pore formation in lipid bilayer membranes, which is explained by a flip-flop gating mechanism based on dipole–dipole interactions of parallel and antiparallel arranged α-helices within oligomeric structures.     

α-Helix to β-sheet transition within the LeuiLysj (i = 2j) series of lytic amphipathic peptides by decreasing their size

Summary De novo designed extremely simplified amphipathic basic Leu_iLys_j (i=2j) peptides of 8, 9 and 15 residues were synthesized to clarify the mechanism of action of natural cytotoxic and hemolytic small proteins or peptides. They proved to have strong hemolytic activity towards human erythrocytes which increases with peptide length. These peptides are highly surface active and form stable peptidic films at the air/water interface. The sensitive and efficient FTIR modulated polarization technique (PMIRRAS) allows one to obtain in situ structural and orientational information about the peptides at the interface. A transition of secondary structure is observed: the shorter peptides (8 and 9 residues) adopt β-sheet structures while the longer one (15 residues) is folded into an α-helix. In both cases, the peptides lie with the axis parallel to the interface. Their insertion into a dimyristoylphosphatidylcholine monolayer can be followed from the increase in the surface and/or pressure of the films. In the mixed films, the peptides adopt the same structure and orientation as observed at the air/water interface. Therefore, among the same series of peptides, a transition from β-sheet to α-helix occurs when the length increases (roughly>10 aa), but despite this drastic change both types of structures result in strongly hemolytic peptides.     

Effects of side-chain orientation on the <Superscript>13</Superscript>C chemical shifts of antiparallel β-sheet model peptides

The dependence of the ¹³C chemical shift on side-chain orientation was investigated at the density functional level for a two-strand antiparallel β-sheet model peptide represented by the amino acid sequence Ac-(Ala)₃-X-(Ala)₁₂-NH₂ where X represents any of the 17 naturally occurring amino acids, i.e., not including alanine, glycine and proline. The dihedral angles adopted for the backbone were taken from, and fixed at, observed experimental values of an antiparallel β-sheet. We carried out a cluster analysis of the ensembles of conformations generated by considering the side-chain dihedral angles for each residue X as variables, and use them to compute the ¹³C chemical shifts at the density functional theory level. It is shown that the adoption of the locally-dense basis set approach for the quantum chemical calculations enabled us to reduce the length of the chemical-shift calculations while maintaining good accuracy of the results. For the 17 naturally occurring amino acids in an antiparallel β-sheet, there is (i) good agreement between computed and observed ¹³C^α and ¹³C^β chemical shifts, with correlation coefficients of 0.95 and 0.99, respectively; (ii) significant variability of the computed ¹³C^α and ¹³C^β chemical shifts as a function of χ¹ for all amino acid residues except Ser; and (iii) a smaller, although significant, dependence of the computed ¹³C^α chemical shifts on χ^ξ (with ξ ≥ 2) compared to χ¹ for eleven out of seventeen residues. Our results suggest that predicted ¹³C^α and ¹³C^β chemical shifts, based only on backbone (φ,ψ) dihedral angles from high-resolution X-ray structure data or from NMR-derived models, may differ significantly from those observed in solution if the dihedral-angle preferences for the side chains are not taken into account. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Interaction of detergents with bovine lens α-crystallin: evidence for an oligomeric structure based on amphiphilic interactions

We have studied the quaternary structure of α-crystallin in the presence of increasing concentrations of amphiphilic and neutral detergents using gel filtration, light-scattering, boundary and equilibrium sedimentation. We observed a continuous reduction of the molar mass of the polymeric α-crystallin on increasing the concentration of sodium dodecyl sulphate from 0.1 mM to 5 mM, ending up with the monomeric peptides. Dodecyltrimethylammonium bromide also disrupts the oligomeric structure of α-crystallin but the interaction appears to be cooperative: in the sharp transition region (for a 1 mg/ml protein solution) from 3 to 8 mM of the detergent, only the native protein and a mixture of monomeric and dimeric peptide-DTAB complexes can be observed. Concomitant studies of the circular dichroism in the far UV revealed a substantial decrease of the β-sheet and increase of the α-helix secondary structure. The latter can be related to the presence of amphiphilic polypeptide sequences in the constituent αA and αB peptides. These studies reveal for the first time a direct relation between changes in the secondary structure of the αA and αB peptides and the formation of the oligomeric α-crystallin structure: the binding of the amphiphilic detergent reduces the β-sheet content, induces the formation of α-helix secondary structure and reduces the tendency of the peptide to form large aggregates. The different mechanisms for reducing the oligomeric size by anionic and cationic detergents with identical apolar parts stresses the importance of charge interactions. Our findings support some aspects of the micelle model of α-crystallin and can be related to its chaperone activity. Accepted: 18 October 1996     

Ca 2+-induced self-assembly in designed peptides with optimally spaced gamma-carboxyglutamic acid residues

We have previously elucidated a new paradigm for the metal ion-induced helix-helix assembly in the natural γ-carboxyglutamic acid (Gla)-containing class of conantokin (con) peptides, typified by con-G and a variant of con-T, con-T[K7Gla], independent of the hydrophobic effect. In these “metallo-zipper” structures, Gla residues spaced at i, i + 4, i + 7, i + 11 intervals, which is similar to the arrangement of a and d residues in typical heptads of coiled-coils, coordinate with Ca²⁺ and form specific antiparallel helical dimers. In order to evaluate the common role of Gla residues in peptide self-assembly, we extend herein the same Gla arrangement to designed peptides: NH₂-(γLSγEAK)₃-CONH₂ (peptide 1) and NH₂-γLSγEAKγLSγQANγLSγKAE-CONH₂ (peptide 2). Peptide 1 and peptide 2 exhibit no helicity alone, but undergo structural transitions to helical conformations in the presence of a variety of divalent cations. Sedimentation equilibrium ultracentrifugation analyses showed that peptide 1 and peptide 2 form helical dimers in the presence of Ca²⁺, but not Mg²⁺. Folding and thiol-disulfide rearrangement assays with Cys-containing peptide variants indicated that the helical dimers are mixtures of antiparallel and parallel dimers, which is different from the strict antiparallel strand orientations of con-G and con-T[K7γGla] dimers. These findings suggest that the Gla arrangement, i, i + 4, i + 7, i + 11, i + 14, plays a key role in helix formation, without a strict adherence to strand orientation of the helical dimer.     

Effects of hypericin on the structure and aggregation properties of β-amyloid peptides

We have determined the secondary structure of 1–40 β-amyloid peptides by Fourier-transform infrared spectroscopy (FTIR) and characterized the peptide photophysical properties before and after self-assembly by using intrinsic tyrosine steady-state and time-resolved fluorescence. All measurements were performed in the presence and absence of hypericin (Hyp), an exogenous natural polycyclic pigment that has been shown to inhibit fibril formation and has also been used as a fluorescent probe. We monitored the time course of the aggregation process measuring 405 nm light diffusion at 90° and used thioflavin T to reveal the presence of fibrils. FTIR quantitative analysis evidenced a prevalent random conformation at t = 0 with and without Hyp. Fibrils showed a predominant parallel β-sheet structure and a small percentage of α-helix. The results of fluorescence measurements showed that Hyp does significantly interact with peptides in β-sheet conformation. In conclusion, hypericin does hinder the formation of fibrils, but the percentages of parallel β-sheets were not significantly different from those found in samples not treated with Hyp.