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Helical Content (helical + content)
Selected AbstractsImportant region in the ,-spectrin C -terminus for spectrin tetramer formationEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 2 2002Bing-Hao Luo Abstract: Many hereditary hemolytic anemias are due to spectrin mutations at the C -terminal region of ,-spectrin (the ,C region) that destabilize spectrin tetramer formation. However, little is known about the ,C region of spectrin. We have prepared four recombinant ,-peptides of different lengths from human erythrocyte spectrin, all starting at position 1898 of the C -terminal region, but terminating at position 2070, 2071, 2072 or 2073. Native polyacrylamide gel electrophoresis showed that the two peptides terminating at positions 2070 and 2071 did not associate with an N -terminal region ,-peptide (Sp,1,156) in the micromolar range. However, the peptides that terminated at positions 2072 and 2073 associated with the ,-peptide. Circular dichroism results showed that the unassociated helices in both ,- and ,-peptides became associated, presumably to form a helical bundle, for those ,-peptides that formed an ,, complex, but not for those ,-peptides that did not form an ,, complex. In addition, upon association, an increase in the ,-helical content was observed. These results showed that the ,-peptides ending prior to residue 2072 (Thr) would not associate with ,-peptide, and that no helical bundling of the partial domains was observed. Thus, we suggest that the C -terminal segment of ,-spectrin, starting from residue 2073 (Thr), is not critical to spectrin tetramer formation. However, the C -terminal region ending with residue 2072 is important for its association with ,-spectrin in forming tetramers. [source] Interaction of ostreolysin, a cytolytic protein from the edible mushroom Pleurotus ostreatus, with lipid membranes and modulation by lysophospholipidsFEBS JOURNAL, Issue 6 2003Kristina Sep Ostreolysin is a 16-kDa cytolytic protein specifically expressed in primordia and fruiting bodies of the edible mushroom Pleurotus ostreatus. To understand its interaction with lipid membranes, we compared its effects on mammalian cells, on vesicles prepared with either pure lipids or total lipid extracts, and on dispersions of lysophospholipids or fatty acids. At nanomolar concentrations, the protein lysed human, bovine and sheep erythrocytes by a colloid-osmotic mechanism, compatible with the formation of pores of 4 nm diameter, and was cytotoxic to mammalian tumor cells. A search for lipid inhibitors of hemolysis revealed a strong effect of lysophospholipids and fatty acids, occurring below their critical micellar concentration. This effect was distinct from the capacity of ostreolysin to bind to and permeabilize lipid membranes. In fact, permeabilization of vesicles occurred only when they were prepared with lipids extracted from erythrocytes, and not with lipids extracted from P. ostreatus or pure lipid mixtures, even if lysophospholipids or fatty acids were included. Interaction with lipid vesicles, and their permeabilization, correlated with an increase in the intrinsic fluorescence and ,-helical content of the protein, and with aggregation, which were not detected with lysophospholipids. It appears that either an unknown lipid acceptor or a specific lipid complex is required for binding, aggregation and pore formation. The inhibitory effect of lysophospholipids may reflect a regulatory role for these components on the physiological action of ostreolysin and related proteins during fruiting. [source] Structural consequences of site-directed mutagenesis in flexible protein domainsFEBS JOURNAL, Issue 8 200156)S mutant of RhoGDI, NMR characterization of the L(5 The guanine dissociation inhibitor RhoGDI consists of a folded C-terminal domain and a highly flexible N-terminal region, both of which are essential for biological activity, that is, inhibition of GDP dissociation from Rho GTPases, and regulation of their partitioning between membrane and cytosol. It was shown previously that the double mutation L55S/L56S in the flexible region of RhoGDI drastically decreases its affinity for Rac1. In the present work we study the effect of this double mutation on the conformational and dynamic properties of RhoGDI, and describe the weak interaction of the mutant with Rac1 using chemical shift mapping. We show that the helical content of the region 45,56 of RhoGDI is greatly reduced upon mutation, thus increasing the entropic penalty for the immobilization of the helix, and contributing to the loss of binding. In contrast to wild-type RhoGDI, no interaction with Rac1 could be identified for amino-acid residues of the flexible domain of the mutant RhoGDI and only very weak binding was observed for the folded domain of the mutant. The origins of the effect of the L55S/L56S mutation on the binding constant (decreased by at least three orders of magnitude relative to wild-type) are discussed with particular reference to the flexibility of this part of the protein. [source] Properties of 2,2,2-Trifluoroethanol/Water Mixtures: Acidity, Basicity, and DipolarityHELVETICA CHIMICA ACTA, Issue 2 2005Paz Sevilla, Sierra In this report, we focus our attention on the characterization of 2,2,2-trifluoroethanol(TFE)/H2O mixtures and describe their intrinsic parameters; i.e., solvent acidity (SA), solvent basicity (SB), and solvent dipolarity/polarizability (SPP), by the probe/homomorph-couple method for a range of mixtures from 0,100% (v/v) TFE. Variation of these parameters is not linear and has a singular and unpredictable behavior depending on the precise composition of the mixture. Based on these parameters, we describe the TFE-induced changes in some physical properties; i.e., viscosity (,), partial molar volume (V,), density (,), dielectric constant (,), vapor pressure (pv), and spectroscopic properties; i.e., NMR chemical shifts (,(1H)) of TFE Me group for all molar fractions studied. In addition, by means of CD studies, we report that formation of the secondary structure, as percentage of helical content, ,, of a polypeptide, poly(L -lysine), in several TFE/H2O mixtures is adequately described by these mixture parameters. SA, SB, and SPP of TFE/H2O mixtures provide an excellent tool for the interpretation of formation and stability of intramolecular H-bonds, and, thus, of secondary structures in polypeptides. [source] Structural characterization of a neurotoxic threonine-rich peptide corresponding to the human prion protein ,2-helical 180,195 segment, and comparison with full-length ,2-helix-derived peptides,JOURNAL OF PEPTIDE SCIENCE, Issue 10 2008Luisa Ronga Abstract The 173,195 segment corresponding to the helix 2 of the globular PrP domain is a good candidate to be one of the several ,spots' of intrinsic structural flexibility, which might induce local destabilization and concur to protein transformation, leading to aggregation-prone conformations. Here, we report CD and NMR studies on the ,2-helix-derived peptide of maximal length (hPrP[180,195]) that is able to exhibit a regular structure different from the prevalently random arrangement of other ,2-helix-derived peptides. This peptide, which has previously been shown to be affected by buffer composition via the ion charge density dependence typical of Hofmeister effects, corresponds to the C -terminal sequence of the PrPC full-length ,2-helix and includes the highly conserved threonine-rich 188,195 segment. At neutral pH, its conformation is dominated by ,-type contributions, which only very strong environmental modifications are able to modify. On TFE addition, an increase of ,-helical content can be observed, but a fully helical conformation is only obtained in neat TFE. However, linking of the 173,179 segment, as occurring in wild-type and mutant peptides corresponding to the full-length ,2-helix, perturbs these intrinsic structural propensities in a manner that depends on whether the environment is water or TFE. Overall, these results confirm that the 180,195 parental region in hPrPC makes a strong contribution to the chameleon conformational behavior of the segment corresponding to the full-length ,2-helix, and could play a role in determining structural rearrangements of the entire globular domain. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [source] Oligopeptide-mediated acceleration of amyloid fibril formation of amyloid ,(A,) and ,-synuclein fragment peptide (NAC)JOURNAL OF PEPTIDE SCIENCE, Issue 1 2004Dr Yoshihiro Kuroda Abstract The effects of oligopeptides on the secondary structures of A, and NAC, a fragment of ,-synuclein protein, were studied by circular dichroism (CD) spectra. The effects of oligopeptides on the amyloid fibril formation were also studied by fluorescence spectra due to thioflavine-T. The oligopeptides were composed of a fragment of A, or NAC and were interposed by acidic or basic amino acid residues. The peptide, Ac-ELVFFAKK-NH2, which involved a fragment Leu-Val-Phe-Phe-Ala at A,(17,21), had no effect on the secondary structures of A,(1,28) in 60% or 90% trifluoroethanol (TFE) solutions at both pH 3.2 and pH 7.2. However, it showed pronounced effects on the secondary structure of A,(1,28) at pH 5.4. The Ac-ELVFFAKK-NH2 reduced the ,-helical content, while it increased the ,-sheet content of A,(1,28). In phosphate buffer solutions at pH 7.0, Ac-ELVFFAKK-NH2 had little effect on the secondary structures of A,(1,28). However, it accelerated amyloid fibril formation when monitored by fluorescence spectra due to thioflavine-T. On the other hand, LPFFD, a peptide known as a ,-sheet breaker, caused neither an appreciable extent of change in the secondary structure nor amyloid fibril formation in the same buffer solution. The peptide, Ac-ETVK-NH2, which involved a fragment Thr-Val at NAC(21,22), had no effect on the secondary structure of NAC in 90% TFE and in isotonic phosphate buffer. However, Ac-ETVK-NH2 in water with small amounts of NaN3 and hexafluoroisopropanol greatly increased the ,-sheet content of NAC after standing the solution for more than 1 week. Interestingly, in this solution, Ac-ETVK-NH2, accelerated the fibril formation of NAC. It was concluded that an oligopeptide that involves a fragment of amyloidogenic proteins could be a trigger for the formation of amyloid plaques of the proteins even when it had little effect on the secondary structures of the proteins as monitored by CD spectra for a short incubation time. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd. [source] Effects of detergents on the secondary structures of prion protein peptides as studied by CD spectroscopyJOURNAL OF PEPTIDE SCIENCE, Issue 4 2003Dr Yoshihiro Kuroda Abstract Pathogenic prion proteins (PrPSc) are thought to be produced by ,-helical to ,-sheet conformational changes in the normal cellular prion proteins (PrPC) located solely in the caveolar compartments. In order to inquire into the possible conformational changes due to the influences of hydrophobic environments within caveolae, the secondary structures of prion protein peptides were studied in various kinds of detergents by CD spectra. The peptides studied were PrP(129,154) and PrP(192,213); the former is supposed to assume ,-sheets and the latter ,-helices, in PrPSc. The secondary structure analyses for the CD spectra revealed that in buffer solutions, both PrP(129,154) and PrP(192,213) mainly adopted random-coils (,60%), followed by ,-sheets (30%,40%). PrP(129,154) showed no changes in the secondary structures even in various kinds of detergents such as octyl-,- D -glucopyranoside (OG), octy-,- D -maltopyranoside (OM), sodium dodecyl sulfate (SDS), Zwittergent 3,14 (ZW) and dodecylphosphocholine (DPC). In contrast, PrP(192,213) changed its secondary structure depending on the concentration of the detergents. SDS, ZW, OG and OM increased the ,-helical content, and decreased the ,-sheet and random-coil contents. DPC also increased the ,-helical content, but to a lesser extent than did SDS, ZW, OG or OM. These results indicate that PrP(129,154) has a propensity to adopt predominantly ,-sheets. On the other hand, PrP(192,213) has a rather fickle propensity and varies its secondary structure depending on the environmental conditions. It is considered that the hydrophobic environments provided by these detergents may mimic those provided by gangliosides in caveolae, the head groups of which consist of oligosaccharide chains containing sialic acids. It is concluded that PrPC could be converted into a nascent PrPSc having a transient PrPSc like structure under the hydrophobic environments produced by gangliosides. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd. [source] Investigation of penetratin peptides Part 1.JOURNAL OF PEPTIDE SCIENCE, Issue 4 2002The environment dependent conformational properties of penetratin, two of its derivatives Abstract The homeodomain, the DNA-binding domain of Antennapedia homeoprotein, is composed of three ,-helices and one ,-turn between helices II and III. Its third helix from the N -terminal (helix III) can translocate through the cell membrane into the nucleus and can be used as an intracellular vehicle for the delivery of oligopeptides and oligonucleotides. To the best of our knowledge, this helix III, called penetratin, which consists of 16 amino acids, is internalized by cells in a specific, non-receptor-mediated manner. For a better understanding of the mechanism of the transfer, the structure of penetratin was examined in both extracellular matrix-mimetic and membrane-mimetic environments; 1H-NMR and CD spectroscopic measurements were performed in mixtures of TFE/water with different ratios. The molecular conformations of two analogue peptides [(6,14-Phe)-penetratin and a 12 amino acid penetratin derivative (peptide 3)] were also studied. An atomic level comprehensive analysis of penetratin and its two analogues was performed. In a membrane-mimetic solvent system (TFEd2/water = 9 : 1), on the basis of 553 distance restraints, the 4,12 region of penetratin exhibits a bent, irregular helical structure on NMR examination. Interactions between hydrophobic amino acid residues in conjunction with H-bonds stabilize the secondary structure of the molecule. Thus, both derivatives adopt a helix-like conformation. However, while (6,14-Phe)-penetratin displays both ,-helical and 310 -helical features, the structure of peptide 3 is predominantly a 310 -helix. Of the three peptides, surprisingly (6,14-Phe)-penetratin has the largest helical content. An increase in the polarity of the molecular environment gradually disintegrates these helix-like secondary structures. In a highly aqueous molecular system (TFEd2/water = 1 : 9), the fast exchange of multiple conformers leads to too few distance restraints being extracted, therefore the NMR structures can no longer be determined. The NMR data show that only short-range order can be traced in these peptides. Under these conditions, the molecules adopt nascent helix-like structures. On the other hand, CD spectra could be recorded at any TFE/water ratio and the conformational interconversion could therefore be monitored as a function of the polarity of the molecular environment. The CD data were analysed comprehensively by the quantitative deconvolution method (CCA+). All three penetratin peptides display helical conformational features in a low dielectric medium, with significant differences as a function of their amino acid composition. However, these conformational features are gradually lost during the shift from an apolar to a polar molecular environment. Copyright © 2002 European Peptide Society and John Wiley & Sons, Ltd. [source] Urea Unfolding of Opsin in Phospholipid Bicelles,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2009Craig McKibbin Opsin is the unstable apo-protein of the light-activated G protein-coupled receptor rhodopsin. We investigated the stability of bovine opsin, solubilized in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/detergent bicelles, against urea-induced unfolding. A single irreversible protein unfolding transition was observed from changes in intrinsic tryptophan fluorescence and far-UV circular dichroism. This unfolding transition correlated with loss of protein activity. Changes in tertiary structure, as indicated by fluorescence measurements, were concomitant with an approximate 50% reduction in ,-helical content of opsin, indicating that global unfolding had been induced by urea. The urea concentration at the midpoint of unfolding was dependent on the lipid/detergent environment, occurring at approximately 1.2 m urea in DMPC/1,2-dihexanoyl-sn-glycero-3-phosphocholine bicelles, while being significantly stabilized to approximately 3.5 m urea in DMPC/3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate bicelles. These findings demonstrate that interactions with the surrounding lipids and detergent are highly influential in the unfolding of membrane protein structure. The urea/bicelle system offers the possibility for a more detailed understanding of the structural changes that take place upon irreversible unfolding of opsin. [source] The role of ,-, 310 -, and ,-helix in helix,coil transitionsPROTEIN SCIENCE, Issue 6 2003Roger Armen Abstract The conformational equilibrium between 310 - and ,-helical structure has been studied via high-resolution NMR spectroscopy by Millhauser and coworkers using the MW peptide Ac-AMAAKAWAAKA AAARA-NH2. Their 750-MHz nuclear Overhauser effect spectroscopy (NOESY) spectra were interpreted to reflect appreciable populations of 310 -helix throughout the peptide, with the greatest contribution at the N and C termini. The presence of simultaneous ,N(i,i + 2) and ,N(i,i + 4) NOE cross-peaks was proposed to represent conformational averaging between 310 - and ,-helical structures. In this study, we describe 25-nsec molecular dynamics simulations of the MW peptide at 298 K, using both an 8 Å and a 10 Å force-shifted nonbonded cutoff. The ensemble averages of both simulations are in reasonable agreement with the experimental helical content from circular dichroism (CD), the 3JHN, coupling constants, and the 57 observed NOEs. Analysis of the structures from both simulations revealed very little formation of contiguous i , i + 3 hydrogen bonds (310 -helix); however, there was a large population of bifurcated i , i + 3 and i , i + 4 ,-helical hydrogen bonds. In addition, both simulations contained considerable populations of ,-helix (i , i + 5 hydrogen bonds). Individual turns formed over residues 1,9, which we predict contribute to the intensities of the experimentally observed ,N(i,i + 2) NOEs. Here we show how sampling of both folded and unfolded structures can provide a structural framework for deconvolution of the conformational contributions to experimental ensemble averages. [source] A helix-turn motif in the C-terminal domain of histone H1PROTEIN SCIENCE, Issue 4 2000Roger Vila Abstract The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKT KKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone Hl° (residues 99,121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i, i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the C, protons, span from Pro100 to Val 116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the ,-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) ,-turn conformation, a ,-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs. [source] Structure of the C-terminal domain of nsp4 from feline coronavirusACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009Ioannis Manolaridis Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26,31,kb) encodes 15,16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication,transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (,100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8,Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P43. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly ,-helical content displaying a unique fold that could be engaged in protein,protein interactions. [source] ,-Zein secondary structure in solution by circular dichroismBIOPOLYMERS, Issue 3 2008Tatiana C. Bicudo Abstract The proline-rich N-terminal domain of ,-zein has been reported in relevant processes, which include its ability to cross the cell membranes. Evidences indicate that synthetic hexapeptide (PPPVHL), naturally found in N-terminal portion of ,-zein, can adopt the polyproline II (PPII) conformation in aqueous solution. The secondary structure of ,-zein in maize protein bodies had been analyzed by solid state Fourier transform infrared and nuclear magnetic resonance spectroscopies. However, it was not possible to measure PPII content in physiological environment since the ,-sheet and PPII signals overlap in both solid state techniques. Here, the secondary structure of ,-zein has been analyzed by circular dichroism in SDS aqueous solution with and without ditiothreitol (DTT), and in 60% of 2-propanol and water with DTT. The results show that ,-zein has high helical content in all solutions. The PPII conformation was present at about 7% only in water/DTT solution. © 2007 Wiley Periodicals, Inc. Biopolymers 89: 175,178, 2008. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley. com [source] Does the anesthetic 2,2,2-trifluoroethanol interact with bovine serum albumin by direct binding or by solvent-mediated effects?BIOPOLYMERS, Issue 2 2005A calorimetric, spectroscopic investigation Abstract Thermal unfolding of bovine serum albumin (BSA) has been studied in the presence of 2,2,2-trifluoroethanol (TFE) using high-sensitivity microdifferential scanning calorimetry. Quantitative thermodynamic parameters accompanying the thermal transitions have been evaluated. TFE is observed to be a stabilizer or a destabilizer of the folded state of BSA depending on the pH. CD spectroscopy revealed an increase in the ,-helical content of BSA and a decrease in the tertiary structure in the presence of increasing molalities of TFE. Isothermal titration calorimetric results do not indicate appreciable binding of the TFE molecules to BSA. TFE quenches the steady-state tryptophan fluorescence of BSA only at higher molalities and there is no effect on the tryptophan fluorescence at lower molalities. The calorimetric and spectroscopic results obtained in this work suggest that solvent-mediated effects dominate the interaction of TFE with BSA and the binding component may be very weak. Since the binding component is very weak, one of the possibilities of anesthetic action of TFE molecules on the actual targets may be through perturbation of the structural and dynamic properties of the lipid bilayer so that the function of crucial but unspecified membrane proteins is affected. © 2005 Wiley Periodicals, Inc. Biopolymers 78: 78,86, 2005 [source] The Interaction of Highly Helical Structural Mutants with the NOP Receptor Discloses the Role of the Address Domain of Nociceptin/Orphanin FQCHEMISTRY - A EUROPEAN JOURNAL, Issue 7 2005Teodorico Tancredi Dr. Abstract Nociceptin is a heptadecapeptide whose sequence is similar to that of Dynorphin A, sharing a message domain characterized by two glycines and two aromatic residues, and a highly basic C-terminal address domain but, in spite of these similarities, displays no opioid activity. Establishing the relative importance of the message and address domains of nociceptin has so far been hampered by its extreme conformational flexibility. Here we show that mutants of this peptide, designed to increase the helical content in the address domain, can be employed to explain the mode of interaction with the NOP receptor. Nociceptin analogues in which Ala residues are substituted with aminoisobutyric acid (Aib) show a substantial increment of activity in their interaction with the NOP receptor. The increment of biological activity was attributed to the well-documented ability of Aib to induce helicity. Here we have verified this working hypothesis by a conformational investigation extended to new analogues in which the role of Aib is taken up by Leu. The NMR conformational analysis confirms that all Ala/Aib peptides as well as [Leu7,11]-N/OFQ-amide and [Leu11,15]-N/OFQ-amide mutants (N/OFQ=nociceptin/orphanin FQ) have comparable helix content in helix-promoting media. We show that the helical address domain of nociceptin can place key basic residues at an optimal distance from complementary acidic groups of the EL2 loop of the receptor. Our structural data are used to rationalize pharmacological data which show that although [Leu11,15]-N/OFQ-amide has an activity comparable to those of Ala/Aib peptides, [Leu7,11]-N/OFQ-amide is less active than N/OFQ-amide. We hypothesize that bulky residues cannot be hosted in or near the hinge region (Thr5 -Gly6 -Ala7) without severe steric clash with the receptor. This hypothesis is also consistent with previous data on this hinge region obtained by systematic substitution of Thr, Gly, and Ala with Pro. [source] | ||||||||||||||||||||||