Helix Bundle Structure Research Articles

Self-association and Domains of Interactions of an Amphipathic Helix Peptide Inhibitor of HIV-1 Integrase Assessed by Analytical Ultracentrifugation and NMR Experiments in Trifluoroethanol/H2O Mixtures

EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) is a better inhibitor of human immunodeficiency virus, type 1, integrase than its parent Lys-159, reproducing the enzyme segment 147-175 with a nonpolar-polar/charged residue periodicity defined by four helical heptads (abcdefg) prone to collapse into a coiled-coil. Circular dichroism, nuclear magnetic resonance, sedimentation equilibrium, and chemical cross-linking were used to analyze EAA26 in various trifluoroethanol/H(2)O mixtures. In pure water the helix content is weak but increases regularly up to 50-60% trifluoroethanol. In contrast the multimerization follows a bell-shaped curve with monomers in pure water, tetramers at 10% trifluoroethanol, and dimers at 40% trifluoroethanol. All suggest that interhelical interactions between apolar side chains are required for the coiled-coil formation of EAA26 and subsist at medium trifluoroethanol concentration. The N(H) temperature coefficients measured by nuclear magnetic resonance show that at low trifluoroethanol concentration the amide groups buried in the hydrophobic interior of four alpha-helix bundles are weakly accessible to trifluoroethanol and are only weakly subject to its hydrogen bond strengthening effect. The increased accessibility of trifluoroethanol to buried amide groups at higher trifluoroethanol concentration entails the reduction of the hydrophobic interactions and the conversion of helix tetramers into helix dimers, the latter displaying a smaller hydrophobic interface. The better inhibitory activity of EAA26 compared with Lys-159 could arise from its better propensity to form a helix bundle structure with the biologically important helical part of the 147-175 segment in integrase.

Amoebapore homologs of Caenorhabditis elegans

It is shown that the Caenorhabditis elegans genome contains several distantly related members of the gene family of saposin-like proteins. The putative products of genes T07C4.4, T08A9.7A, T08A9.7B, T08A9.8, T08A9.9, T08A9.10 are similar to the amoebapores of Entamoeba histolytica, granulysin of cytotoxic T lymphocytes and a putative amoebapore-related protein of the liver fluke Fasciola hepatica inasmuch as they consist of only a single saposin-like domain and a secretory signal peptide. The saposin-like domain of protein T07C4.4, which is most closely related to NK-lysin and granulysin, has been expressed in Escherichia coli and the recombinant protein was shown to have a circular dichroism spectrum consistent with the helix bundle structure characteristic of saposin-like domains. Recombinant T07C4.4 protein was found to have antibacterial activity, suggesting that these amoebapore homologs may play a role in antibacterial mechanisms of C. elegans.

Structural information content and Lyapunov exponent calculation in protein unfolding

A lattice model with Monte Carlo dynamics is used to carry out computer simulations of protein dynamics on a four α-helix bundle. The interaction energies in the model can be set so that either the helix bundle structure remains relatively stable or changed so that it unfolds. The computer model produces output that simulates experimental measurements relating to the structure. We show how this output can be used with analytical techniques of nonlinear dynamics to obtain important information about the complex underlying protein dynamics. Time-delay reconstruction plots of structural parameters of unfolding bundles resemble strange attractors in a space of dimension 3–4. We calculate Lyapunov exponents for these unfolding runs and find positive Lyapunov exponents implying chaotic dynamics. For stable runs the Lyapunov exponents are close to zero. We use these Lyapunov exponents to calculate the rate of loss of structural information during the unfolding process and show how the approach may be useful for investigating the folding dynamics of proteins. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36: 2761–2769, 1998

Incorporation of hydrophobic helix-bundle peptides into lipid bilayer membranes facilitated by a peptide-umbrella structure

Hydrophobic helical peptides, which are covered by hydrophilic peptides, are transferred into a phospholipid bilayer to form helix-bundle structures as a consequence of their peptide-umbrella structure.

Special folding pathway to tetramer only through the micelle state of the corticotropin-releasing factor.

The ovine corticotropin-releasing factor (CRF), a peptide hormone of 41 residues stimulating the secretion of adrenocorticotropic hormone, was thermodynamically investigated. By means of size exclusion chromatography and/or ultrafiltration, the CRF solution could be separated into random coil monomers and highly alpha-helical tetramers, which seem to have amphipathic helix bundle structure. Circular dichroism measurements along with diluting or concentrating the CRF solution revealed that there exists the micelle state above the concentration of 0.1 mM, which would be the critical micelle concentration (cmc). The micelle state was also proved by binding ability for 8-anilino-1-naphthalenesulfonate and endothermic change by dilution across the cmc. The tetramer showed the cooperative thermal transition at about 55 degrees C in the buffer solution (pH 7.5), so that it would have native-protein-like folding. On the other hand, the micelle undergoes gradual change to dissociated state by heating, regardless of the similar alpha-helicity to the tetramer. Above the cmc the equilibrium between the tetramer and the micelle takes place as well as that between the monomer and the micelle. Whereas, the direct conversion between the tetramer and the monomer scarcely occurred below the cmc. The titration experiment with 2,2,2-trifluoroethanol (TFE) revealed that the cmc decreases with increasing the concentration of TFE. This tendency is the same as that of general surfactants. Most of experimental results can be well explained by this three-phase model involving the monomer, the tetramer, and the micelle. The lack of the equilibrium between the monomer and the tetramer indicates that the folding pathway of the tetramer is the transformation only through the micelle state and not from the monomer. This pathway resembles the collapse model among the phenomenological models for thermodynamic protein folding. By the mathematical consideration for the dissociation of micelle, we have demonstrated that the expected content of undegradable k-mer is 2/(k + 1), which agreed well with the observed tetramer content of CRF (40%).

Functionality of Polypeptide by Induction of Specific Tertiary Structure

Regular assemblies of peptide molecules to exhibit novel functionalities have been prepared in two different ways. One of them is a helix-bundle structure in which two or three hydrophobic α-helical peptides are connected together by a tie molecule. The helix-bundle structure formed an ion channel across lipid bilayer membrane at lower concentrations than a single-chain pepide. The other example is a two-dimensional assembly of amphiphilic peptide molecules carrying a chromophoric group. The assembly was an efficient photo-energy collector to generate photo-electron and to reduce cytochrome c.

Amino acid sequence of a new type of antifreeze protein, from the longhorn sculpin Myoxocephalus octodecimspinosis

A new type of fish antifreeze protein, designated here type IV, has been isolated from the longhorn sculpin, Myoxocephalus octodecimspinosis. Sequence analysis of the protein (LS-12) reveals that it contains 108 amino acids, is blocked at the N-terminus by a pyroglutamyl group and has a high (17%) content of glutamine; it is thus completely unrelated to the earlier described types I, II and III fish antifreeze proteins. Circular dichroism spectra and conformational analysis based on the sequence data indicate that LS-12 has a high helix content and probably folds as a four-helix bundle. LS-12 shows sequence similarity to certain plasma apolipoproteins known to have helix bundle structures, suggesting the possibility that LS-12 may have arisen by recruitment and mutation of a plasma apolipoprotein.

Phosphotransfer site of the chemotaxis-specific protein kinase CheA as revealed by NMR.

Bacterial chemotaxis involves autophosphorylation of a histidine kinase and transfer of the phosphoryl group to response regulators to control flagellar rotation and receptor adaptation. The phosphotransfer domain, CheA1-134, of the chemotaxis-specific histidine autokinase CheA from Escherichia coli contains the site of phosphorylation, His48, and two other histidine residues, His26 and His67. Two-dimensional 1H-15N NMR techniques were applied to characterize the protonation states of these histidine residues and to evaluate the structural changes in the domain that occur upon phosphorylation of His48. The pKa of His48 was determined to be 7.8 (in 50 mM NaPO4 buffer at 30 degrees C). At high pH, its imidazole ring exists primarily as the normally unfavored N delta 1H tautomer, suggesting hydrogen bond formation to the ring nitrogen atom(s) to stabilize this state. The pKa values and predominant tautomeric states of the imidazole rings of His26 (pKa approximately 7.1, N epsilon 2H tautomer) and His67 (pKa approximately 6.5, N delta 1H tautomer) were also determined. His48 of CheA1-134 and CheA1-233 was phosphorylated by full-length CheA. The phosphorylation site was confirmed to be the N epsilon 2 position in the imidazole ring. Phosphorylation of His48 only results in small changes in the amide 1H and 15N chemical shifts of a few residues from helices B and C, suggesting that only very small changes in structure are associated with phosphorylation of the phosphotransfer domain of CheA. These residues occupy a small surface area of the helix bundle and form the active site of the protein. At the active site, in addition to His48, residues Gly52, His67, and Glu70 are conserved in the CheA homologous phosphotransfer domains from 10 different organisms. Sequence comparison of these CheA homologs suggest that the phosphotransfer domains likely fold in a similar helix-bundle structure and the structural features at the active site are well-conserved.

Crystal Structure of L-2-Haloacid Dehalogenase from Pseudomonas sp. YL: AN α/β HYDROLASE STRUCTURE THAT IS DIFFERENT FROM THE α/β HYDROLASE FOLD

L-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. The crystal structure of the homodimeric enzyme from Pseudomonas sp. YL has been determined by a multiple isomorphous replacement method and refined at 2.5 A resolution to a crystallographic R-factor of 19.5%. The subunit consists of two structurally distinct domains: the core domain and the subdomain. The core domain has an alpha/beta structure formed by a six-stranded parallel beta-sheet flanked by five alpha-helices. The subdomain inserted into the core domain has a four helix bundle structure providing the greater part of the interface for dimer formation. There is an active site cavity between the domains. An experimentally identified nucleophilic residue, Asp-10, is located on a loop following the amino-terminal beta-strand in the core domain, and other functional residues, Thr-14, Arg-41, Ser-118, Lys-151, Tyr-157, Ser-175, Asn-177, and Asp-180, detected by a site-directed mutagenesis experiment, are arranged around the nucleophile in the active site. Although the enzyme is an alpha/beta-type hydrolase, it does not belong to the alpha/beta hydrolase fold family, from the viewpoint of the topological feature and the position of the nucleophile.

Supramolecular systems composed of α-helical peptides

Hydrophobic helical peptides having alternating hydrophobic amino acid and Aib in the sequence were synthesized to construct supramolecular systems. Three types of supramolecular systems were constructed by the peptides and the derivatives in different environments. First, the dispersion of TFA · H-(Ala-Aib) 8-OBzl in water was studied by dynamic light scattering, which suggests the formation of a vesicular structure with an average diameter of 76 nm. We call the peptide assembly in water “peptosome”. Second, Boc-Ser(Ant)-(Ala-Aib) 8-OMe spanned the phospholipid bilayer membrane and formed a helix-bundle structure. The bundle structure was supported by ion-channel formation in the membrane. Third, Boc-(Ala-Aib) 8-OMe and Boc-(Leu-Aib) 8-OBzl formed a two-dimensional crystal at the air-water interface. Boc-(Ala-Aib) 12-OBzl also formed a monolayer in a solid state at the air-water interface, but the helix orientation was perpendicular to the interface, which presents a contrast to the parallel orientation of the former hexadecapeptides.

The Building of Antiparallel Four α-Helix Bundle Structure with a Pair of Pyrenylalanines on a Cyclic Pseudo-peptide Template

Abstract The 4α -helix bundle structure bearing a pair of pyrenylalanines as a fluorescence probe was built on a cyclic pseudo-peptide template and characterized by the pyrene excimer emission.

Characterization of the interaction of NADH with proton pumping E. coli transhydrogenase reconstituted in the absence and in the presence of bacteriorhodopsin

(1) Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli was purified in a reconstitutively active form employing affinity chromatography on immobilized palmitoyl-Coenzyme A. Reconstituted transhydrogenase showed an active proton pumping and a stimulation of the rate of reduction of 3-acetylpyridine-NAD + by NADPH by uncouplers. Reconstitution in the absence of a thiol-reducing agent, e.g. dithiothreitol, abolished proton pumping without affecting catalytic activity, giving a decoupled transhydrogenase. (2) Co-reconstitution of transhydrogenase with bacteriorhodopsin gave vesicles which catalyzed a 5–10 fold increased rate of reduction of thio-NADP + by NADH in the light. The K m for NADH, but not that for thio-NADP +, decreased markedly in the light, indicating an effect of the electrochemical proton potential on the affinity of the enzyme for NADH. Inhibition by substrate derivatives in the absence or presence of light supported this conclusion. Replacement of NADH with 2'-deoxy-NADH gave a strongly sigmoidal concentration dependence, indicating an allosteric change induced by binding to the NAD(H)-site. (3) Reduction of 3-acetylpyridine-NAD + by NADH in the presence of NADPH, previously demonstrated to be catalyzed by both reconstitued bovine transhydrogenase and detergent-dispersed E. coli transhydrogenase, occurred at a pH below 6.5. This reaction did not pump protons. Proton pumping by 3-acetylpyridine-NAD + plus NADPH occurred at a pH above 5.5. The two reactions were thus close to mutually exclusive, with a cross point at pH 5.8. Assuming a helix bundle structure of the membrane domain of transhydrogenase, a model is proposed involving histidine 91 of the β subunit which previously was shown to be essential by site-directed mutagenesis. According to the model the extent of protonation of this histidine determines whether proton pumping or the NADH-3-acetylpyridine-NAD + reaction takes place.

Binding Properties of de Novo Designed Proteins.

By means of "de novo design" approach several artificial proteins are developing. The construction of helix bundle structures and the characteristics of their hydrophobic binding are discussed.

2·9 Å resolution structure of the N-terminal domain of a variant surface glycoprotein from Trypanosoma brucei

The variant surface glycoprotein (VSG) of Trypanosoma brucei forms a coat on the surface of the parasite; by the expression of a series of antigenically distinct VSGs in the surface coat the parasite escapes the host immune response. The 2.9 A resolution crystal structure of the N-terminal domain of one variant, MITat 1.2, has been determined. The structure was solved using data collected from two crystal forms. Initially a partial model was built into an electron density map based on multiple isomorphous replacement phases and improved by phase combination methods. Subsequently this model was used to obtain the molecular replacement solution for a second crystal form, providing starting phases which were refined using 2-fold non-crystallographic symmetry averaging. The current model includes 362 residues and has been refined using X-PLOR to an R value of 0.22 for data between 7 and 2.9 A. The molecule is a dimer, approximately 100 A long, having an asymmetrical cross section with maximum dimensions of approximately 40 A x 60 A. Two long, approximately 70 A, alpha-helices from each monomer pack together to form, with several other helices, a core helix bundle that extends nearly the full length of the molecule. The "top" of the protein, which in the surface coat may be exposed to the external environment, is formed from the ends of the two long helices, a short three-stranded beta-sheet, and a strand having irregular conformation that packs above these secondary structure elements. Two conserved disulfide bridges are in this part of the molecule. Several elements of the MITat 1.2 sequence, which contribute to the formation of the helix bundle structure, have been identified. These elements can be found in the sequences of several different VSGs, suggesting that to some extent the VSG structure is conserved in those variants.