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Results for “Spectroscopy, Fourier Transform Infrared”

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PubMed2004

Phosphorylation modulates the alpha-helical structure and polymerization of a peptide from the third tau microtubule-binding repeat.

Paired helical filaments (PHFs) isolated from patients with Alzheimer's disease (AD) mainly consist of the microtubule-associated protein tau in a hyperphosphorylated form. It has been found that PHFs are the first example of pathological protein aggregation associated with formation of alpha-helices [Biochemistry (2002) 41, 7150-5]. In an effort to investigate the interplay between phosphorylation and the putative role of short regions of alpha-helix in the polymerization of tau, we have focused on the region of tau encompassing residues 317 to 335. This region is able to form protein fibrils in vitro and has two serines that are often found phosphorylated in PHFs. Using trifluoroethanol as an indicator of the alpha-helix, we find that the stability of the alpha-helix conformation is enhanced by phosphorylation. Circular dichroism data show that the phosphorylated peptide in water presents a content in alpha-helix similar to the unphosphorylated peptide at 40% of trifluoroethanol. Phosphorylation also stimulates the effect of juglone in promoting the in vitro polymerization. Furthermore, Fourier transformed infrared spectroscopy of samples of phosphorylated peptide polymerized with juglone renders a spectrum with maxima at approximately 1665 and approximately 1675 cm(-1), which are suggestive of a mixture of turns and alpha-helix conformations. Our results provide a direct mechanistic connection between phosphorylation and polymerization in tau. The connection between phosphorylation and polymerization appears to involve formation of alpha-helix structure.

Amino Acid Sequence↗

PubMed1998

Intramembrane molecular dipoles affect the membrane insertion and folding of a model amphiphilic peptide.

The relationship between the dipole potential and the interaction of the mitochondrial amphipathic signal sequence known as p25 with model membranes has been studied using 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octyl-amino)-6-naphthyl]viny l] pyridinium betaine (di-8-ANEPPS) as a fluorescent probe. The dipole potential of phosphatidylcholine membranes was modified by incorporating into the bilayer the sterols phloretin and 6-ketocholestanol (KC), which decrease and increase the dipole potential, respectively. The results derived from the application of a dual-wavelength ratiometric fluorescence method for following the variation of the membrane dipole potential have shown that when p25 inserts into the lipidic bilayer, a decrease in the dipole potential takes place. The magnitude of this decrease depends on the initial value of the dipole potential, i.e., before interaction with the peptide. Thus, when KC was incorporated into the bilayer, the decrease caused by the membrane insertion of p25 was larger than that caused in PC membranes. Alternatively, in the presence of phloretin, the decrease in the potential caused by the peptide insertion was smaller. Complementary studies involving attenuated total reflectance-Fourier transform infrared spectroscopy of the peptide membrane interactions have shown that modification of the dipole potential affects the conformation of the peptide during the course of its interaction with the membrane. The presence of KC induces a higher amount of helicoidal structure. The presence of phloretin, however, does not appear to affect the secondary structure of the peptide. The differences observed in the dipole potential decreases caused by the presence of the peptide with the PC membranes and phloretin-PC membranes, therefore, must involve differences in the tertiary and, perhaps, quaternary conformations of p25.

Animals↗

PubMed2006

Two-dimensional ordered beta-sheet lipopeptide monolayers.

A series of amphiphilic lipopeptides, ALPs, consisting of an alternating hydrophilic and hydrophobic amino acid residue sequence coupled to a phospholipid tail, was designed to form supramolecular assemblies composed of beta-sheet monolayers decorated by lipid tails at the air-water interface. A straightforward synthetic approach based on solid-phase synthesis, followed by an efficient purification protocol was used to prepare the lipid-peptide conjugates. Structural insight into the organization of monolayers was provided by surface pressure versus area isotherms, circular dichroism, Fourier transform infrared spectroscopy, and Brewster angle microscopy. In situ grazing-incidence X-ray diffraction (GIXD) revealed that lipopeptides six to eight amino acids in length form a new type of 2D self-organized monolayers that exhibit beta-sheet ribbons segregated by lipid tails. The conclusions drawn from the experimental findings were supported by a representative model based on molecular dynamics simulations of amphiphilic lipopeptides at the vacuum-water interface.

Air↗

PubMed1998

Specific inhibition of in vitro formation of protease-resistant prion protein by synthetic peptides.

The transmissible spongiform encephalopathies are characterized by the conversion of the protease-sensitive prion protein (PrPsen) into a protease-resistant isoform (PrPres) associated with the neuropathogenic process in vivo. Recently, PrPres has been shown to be capable of directly inducing the conversion of PrPsen to PrPres in a cell-free in vitro system. In the present experiments, various PrP peptides were studied for their ability to enhance or inhibit this cell-free conversion reaction. None of the synthetic peptides was able to confer protease-resistance to the labeled PrPsen molecules on their own. On the contrary, peptides from the central part of the hamster PrP sequence from 106 to 141 could completely inhibit the conversion induced by preformed PrPres. The presence of residues 119 and 120 from the highly hydrophobic sequence AGAAAAGA (position 113 to 120) was crucial for an efficient inhibitory effect. Fourier transform infrared spectroscopy analysis indicated that inhibitory peptides formed high beta-sheet aggregates under the conditions of the conversion reaction, but this was also true of certain peptides that were not inhibitory. Thus, the potential to form beta-sheeted aggregates may be necessary, but not sufficient, for peptides to act as inhibitors of PrPres formation. Clearly, the amino acid sequence of the peptide is also important for inhibition. The sequence specificity of the inhibition is consistent with the idea that residues in the vicinity of positions 106-141 of PrPres and/or PrPsen are critically involved in the intermolecular interactions that lead to PrPres formation.

Amino Acid Sequence↗

PubMed2000

[Infrared spectrometry and urolithiasis. Report of 80 cases].

Urolithiasis is a frequent disorder that is characterized by its recurrence following treatment and which can affect between 3-20% of the population, with an incidence that differs from country to country. The aim of the present study was to determine the composition of the calculi, and the remain characteristics of this pathology in Morocco. A series of 80 calculi was therefore analyzed by Fourier transform infrared spectroscopy. The findings showed that calcium monohydrate oxalate was the main lithiasic component, indicating that hyperoxaluria plays a major role in the formation of the calculi. The component identified were as follows: calcium oxalate (58.75%), calcium phosphate and magnesium phosphate (17.5%), uric acid (15%), and urate (8.75%). In 91.25% of cases, the calculi were of mixed composition. Regular patient follow-up is advocated and subjects should be informed of the risk factor involved, as urolithiasic recurrence was observed in 10% of the cases in this series.

Adolescent↗

PubMed2001

Peptide mimics of SNARE transmembrane segments drive membrane fusion depending on their conformational plasticity.

SNARE proteins are essential for different types of intracellular membrane fusion. Whereas interaction between their cytoplasmic domains is held responsible for establishing membrane proximity, the role of the transmembrane segments in the fusion process is currently not clear. Here, we used an in vitro approach based on lipid mixing and electron microscopy to examine a potential fusogenic activity of the transmembrane segments. We show that the presence of synthetic peptides representing the transmembrane segments of the presynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) synaptobrevin II (also referred to as VAMP II) or syntaxin 1A, but not of an unrelated control peptide, in liposomal membranes drives their fusion. Liposome aggregation by millimolar Ca(2+) concentrations strongly potentiated the effect of the peptides; this indicates that juxtaposition of the bilayers favours their fusion in the absence of the cytoplasmic SNARE domains. Peptide-driven fusion is reminiscent of natural membrane fusion, since it was suppressed by lysolipid and involved both bilayer leaflets. This suggests transient presence of a hemifusion intermediate followed by complete membrane merger. Structural studies of the peptides in lipid bilayers performed by Fourier transform infrared spectroscopy indicated mixtures of alpha-helical and beta-sheet conformations. In isotropic solution, circular dichroism spectroscopy showed the peptides to exist in a concentration-dependent equilibrium of alpha-helical and beta-sheet structures. Interestingly, the fusogenic activity decreased with increasing stability of the alpha-helical solution structure for a panel of variant peptides. Thus, structural plasticity of transmembrane segments may be important for SNARE protein function at a late step in membrane fusion.

Amino Acid Sequence↗

PubMed2004

Comparison of polypropylene and polyethylene terephthalate (Dacron) meshes for abdominal wall hernia repair: a chemical and morphological study.

For the first time, by scanning electron microscopy (SEM), polypropylene (PP) excised meshes (ethylene oxide sterilized) for abdominal wall hernia repair have been shown to be greatly damaged physically, independently of the implantation time, while the polyethylene terephthalate (PET), or Dacron, ones (gamma radiation sterilized), did not undergo alterations due to the sterilization process and were not damaged, even after long implantation periods. Fourier-Transform Infrared Spectroscopy (FTIR) study of PP and PET excised meshes, as well as of their extracts with cyclohexane, has shown the presence of species, such as squalene, palmitic and stearic acid, in some cases, cholesterol, transferred from the surrounding tissues to the polymer during the implantation period. In the case of PP meshes, these small organic molecules would reduce physical and mechanical properties of the material. A hypothesis is presented to account for the better behavior (not in the clinical sense) of PET meshes.

Adult↗

PubMed2003

Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin.

Surfactin, an acidic lipopeptide produced by various strains of Bacillus subtilis, behaves as a very powerful biosurfactant and possesses several other interesting biological activities. This work deals with the molecular mechanism of membrane permeabilization by incorporation of surfactin. The surfactin-induced vesicle contents leakage was monitored by following release of carboxyfluorescein entrapped into unilamellar vesicles made of palmitoyloleoylphosphatidylcholine (POPC). The effect of the addition of cholesterol, dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylethanolamine (POPE) was also checked. It was observed that surfactin was able to induce content leakage at concentrations far below the onset surfactin/lipid ratio for membrane solubilization to occur, which in our system was around 0.92. Electron microscopy showed that vesicles were present after addition of surfactin at a ratio below this value, whereas no vesicles could be observed at ratios above it. Cholesterol and POPE attenuated the membrane-perturbing effect of surfactin, whereas the effect of DPPC was to promote surfactin-induced leakage, indicating that bilayer sensitivity to surfactin increases with the lipid tendency to form lamellar phases, which is in agreement with our previous observation that surfactin destabilizes the inverted-hexagonal structure. Fourier-transform infrared spectroscopy (FTIR) was used to specifically follow the effect of surfactin on different parts of the phospholipid bilayer. The effect on the C=O stretching mode of vibration of POPC indicated a strong dehydration induced by surfactin. On the other hand, the C-H stretching bands showed that the lipopeptide interacts with the phospholipid acyl chains, resulting in considerable membrane fluidization. The reported effects could be useful to explain surfactin-induced 'pore' formation underlying the antibiotic and other important biological actions of this bacterial lipopeptide.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2001

A novel heterodimeric antimicrobial peptide from the tree-frog Phyllomedusa distincta.

We present here the purification and the analysis of the structural and functional properties of distinctin, a 5.4 kDa heterodimeric peptide with antimicrobial activity from the tree-frog Phyllomedusa distincta. This peptide was isolated from the crude extract of skin granular glands by different chromatographic steps. Its minimal inhibitory concentration was determined against pathogenic Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa strains. Amino acid sequencing and mass spectrometric investigations demonstrated that distinctin is constituted of two different polypeptide chains connected by an intermolecular disulphide bridge. Circular dichroism and Fourier-transformed infrared spectroscopy studies showed that this molecule adopts, in water, a structure containing a significant percentage of anti-parallel beta-sheet. A conformational variation was observed under experimental conditions mimicking a membrane-like environment. Database searches did not show sequence similarities with any known antimicrobial peptides. In the light of these results, we can consider distinctin as the first example of a new class of antimicrobial heterodimeric peptides from frog skin.

Amino Acid Sequence↗

PubMed1998

Human complement factor I: its expression by insect cells and its biochemical and structural characterisation.

Factor I is a five-domain plasma serine protease which is essential for the regulation of the complement system. In order to express this, the factor I coding sequence was cloned into a recombinant baculovirus system, which was used to infect Trichoplusia ni cells. Using the native factor I leader sequence, recombinant factor I (rFI) was secreted into the culture medium. Purified rFI was recognised by polyclonal antisera and by the factor I-specific monoclonal antibody MRC-OX21. SDS PAGE showed that rFI was processed into two chains with molecular weights of 48,000 and 36,000. Amino acid sequence analysis showed that the N-terminal sequences of the rFI chains were the same as those of serum-derived factor I (sFI), confirming that processing was correct. Since both molecular weights were less than those observed for sFI, this is attributed to the replacement of complex-type oligosaccharides by high mannose ones in rFI. C3(NH,) cleavage assays showed that rFI had 55% the activity of sFI. Circular dichroism and Fourier transform infrared spectroscopy showed that the protein folding of rFI and sFI were very similar. Both had a secondary structure low in alpha-helix and high in beta-sheet, as expected from crystal structure and multiple sequence alignment analyses. It is inferred that the reduced activity of rFI is attributable to its changed glycosylation. The availability of rFI and structures for the domains in factor I makes possible new approaches to determine the molecular basis of its interactions with factor H and C3b.

Animals↗

PubMed2003

High pressure induces the formation of aggregation-prone states of proteins under reducing conditions.

The pressure stability of ribonuclease A and bovine pancreatic trypsin inhibitor has been investigated with Fourier transform infrared spectroscopy in the presence of the disulfide bond reducing agent 2-mercaptoethanol. The secondary structure of the reduced proteins at high pressure (1 GPa) is not significantly different from the pressure-induced conformation of the native form. Upon decompression under reducing conditions, amorphous aggregates are formed. Such aggregates are not formed upon decompression of the native proteins. Our data demonstrate that high pressure populates, and thus allows the potential characterization of highly aggregation-prone conformations. The relevance of these findings with regard to fibril formation is discussed and the possible role of conformational fluctuations of intermediates on the aggregation pathway is emphasized.

Animals↗

PubMed2003

In vitro effect of cholesterol on calcifying activity of vesicles isolated from rabbit aortas.

It has been shown that vesicles play a key role in the onset mechanism of aortic calcification related to cholesterol-induced atherosclerosis. This study using a rabbit model was conducted to determine whether cholesterol exerts a direct effect on vesicle's calcifiability. Inclusion of cholesterol in calcifying media stimulated ATP-initiated deposition of calcium in a dose-dependent manner by vesicles isolated from normal aortas using crude collagenase digestion. By contrast, cholesterol did not significantly affect ATP-promoted calcification if vesicles were isolated from atherosclerotic aortas. To determine whether high cholesterol levels in atherosclerotic vesicle preparations may have already maximized calcifying activity and therefore account for lack of the vesicle's response to the sterol, Fourier transform infrared spectroscopy (FT-IR) was used to compare the cholesterol contents in control and atherosclerotic vesicles. The spectral patterns revealed higher levels of cholesterol in vesicle preparations from atherosclerotic aortas than those from normal aortas. Removal of extra-vesicular cholesterol micelles from atherosclerotic vesicles by a relatively low centrifugal force sensitized the vesicles to cholesterol stimulation causing a 2-fold increase in calcifying activity. Of various oxidized forms of cholesterol tested, 7-keto and 6-keto cholesterol enhanced the activity by 2-fold. Altogether, these observations suggest that cholesterol and especially its oxidized forms may induce aortic calcification by directly enhancing the vesicle's ability to calcify.

Animals↗

PubMed2005

Electrolytic deposition of calcium etidronate drug coating on titanium substrate.

Wear debris-induced osteolysis is the major cause of aseptic loosening and failure of hip implants. One of the promising therapeutic interventions to improve the longevity of hip implants is to administrate bisphosphonate drug to inhibit osteoclastic bone resorption. This study aimed at developing new techniques of directly combining bisphosphonate with implants to achieve local delivery and controlled release of the drug. Instead of using soluble sodium salt, we proposed to apply sparingly soluble calcium salt of bisphosphonate as a potential long-term antiosteolysis coating on hip implants. Calcium salt of etidronate, a member of the bisphosphonate family of potent osteoclast inhibitors, was used in this pilot study. By adopting the electrolytic deposition (ELD) technique, which was developed for ceramic coatings including calcium phosphates, we demonstrated that a thin layer of calcium bisphosphonate could be deposited onto titanium surface. The drug coating is amorphous as characterized with X-ray diffraction, and has globular morphology under the scanning electron microscope. Electrospray-ionization mass-spectrometry (ESI-MS) and Fourier-transformed infrared spectroscopy confirmed that the molecular structure of the etidronate (m/z 205, H3L-, the single dissociated form of parent etidronic acid, denoted as H4L) was preserved after the ELD process. In vitro release into a "physiological" buffer solution confirmed that the etidronate concentration was limited by its low solubility. The etidronate concentration was 8 x 10(-5) M at day 1 and kept relatively stable at approximately 6 x 10(-5) M from day 2 to day 8. The deposition mechanisms of the drug coating and its potential efficacy as an antiosteolytic release source were discussed.

Alloys↗

PubMed2006

Characterization of physiochemical and biological properties of an insulin/lauryl sulfate complex formed by hydrophobic ion pairing.

An insulin/lauryl sulfate complex was prepared by hydrophobic ion pairing (HIP). The physiochemical and biological properties of the HIP complex were characterized using octanol/water partition measurement, isothermal titration calorimetry (ITC), ultraviolet-circular dichroism (UV-CD) and Fourier transform infrared spectroscopy (FTIR). Sodium dodecyl sulfate (SDS) bound to the insulin in a stoichiometric manner. The formed complex exhibited lipophilicity, and its insulin retained its native structure integrity. The in vivo bioactivity of the complex insulin was evaluated in rats by monitoring the plasma glucose level after intravenous (i.v.) injection, and the glucose level was compared with that for free insulin. The pharmacodynamic study result in rats showed that the complex insulin had in vivo bioactivity comparable to free insulin.

1-Octanol↗

PubMed2000

Identification of impurities affecting commercial ethylene glycol UV transmittance.

As a raw material for making polyesters, ethylene glycol has a special specification of UV transmittance. At present, ethylene glycol produced by some plants still has low UV transmittance rendering it unsuitable for use in polyester production. In this paper, a method was developed for the identification of the impurities that cause commercial ethylene glycol to have low UV transmittance, using solid-phase extraction (SPE) and some analytical techniques such as high-performance liquid chromatography, gas chromatography-mass spectrometry and gas chromatography-Fourier transform infrared spectroscopy. The major UV-absorbing impurities were identified as some alkyl homologues of 2-hydroxycyclopent-2-en-1-one, including 2-hydroxy-3,5-dimethylcyclopent-2-en-1-one, 2-hydroxy-3-methylcyclopent-2-en-1-one, 2-hydroxy-3,4-dimethylcyclopent-2-en-1-one, 2-hydroxy-3-ethyl-4-methylcyclopent-2-en-1-one and 2-hydroxy-3-ethylcyclopent-2-en-1-one. Their concentrations were estimated to be less than 2 microg ml(-1). It is believed that with the above results, ethylene glycol-producing plants might make process improvements to remove these impurities more effectively and more easily.

Chromatography, High Pressure Liquid↗

PubMed2004

Temperature driven morphological changes of chemically precipitated hydroxyapatite nanoparticles.

Hydroxyapatite (HA) is synthesized by a wet chemical route using calcium hydroxide and ortho-phosphoric acid at various temperatures (40, 80, and 100 degrees C). X-ray diffraction of the precipitate particles revealed HA as the predominant phase (>99%) with a small amount of beta-tricalcium phosphate. Fourier transform infrared spectroscopy indicated the presence of carbonate substitution, which decreased with increasing temperature. Transmission electron microscopy observations revealed needle-shaped particles with a high aspect ratio at 40 degrees C, which changed to spheroidal when the precipitation temperature was increased to 100 degrees C. The changes in the morphology with temperature were analyzed taking into account the driving force for the HA precipitation and the supersaturation level of Ca2+ and PO4(3-) ions with respect to HA. The analysis indicated that the supersaturation level of the reactants, especially the concentration of Ca2+ ions, played a predominant role on the precipitate morphology for this classical acid-base reaction.

Chemical Precipitation↗

PubMed1998

A synthetic all D-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 recognizes the wild-type fusion peptide in the membrane and inhibits HIV-1 envelope glycoprotein-mediated cell fusion.

Recent studies demonstrated that a synthetic fusion peptide of HIV-1 self-associates in phospholipid membranes and inhibits HIV-1 envelope glycoprotein-mediated cell fusion, presumably by interacting with the N-terminal domain of gp41 and forming inactive heteroaggregates [Kliger, Y., Aharoni, A., Rapaport, D., Jones, P., Blumenthal, R. & Shai, Y. (1997) J. Biol. Chem. 272, 13496-13505]. Here, we show that a synthetic all D-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 (D-WT) of HIV-1 associates with its enantiomeric wild-type fusion (WT) peptide in the membrane and inhibits cell fusion mediated by the HIV-1 envelope glycoprotein. D-WT does not inhibit cell fusion mediated by the HIV-2 envelope glycoprotein. WT and D-WT are equally potent in inducing membrane fusion. D-WT peptide but not WT peptide is resistant to proteolytic digestion. Structural analysis showed that the CD spectra of D-WT in trifluoroethanol/water is a mirror image of that of WT, and attenuated total reflectance-fourier transform infrared spectroscopy revealed similar structures and orientation for the two enantiomers in the membrane. The results reveal that the chirality of the synthetic peptide corresponding to the HIV-1 gp41 N-terminal sequence does not play a role in liposome fusion and that the peptides' chirality is not necessarily required for peptide-peptide interaction within the membrane environment. Furthermore, studies along these lines may provide criteria to design protease-resistant therapeutic agents against HIV and other viruses.

Amino Acid Sequence↗

PubMed1997

Effect of nucleotides on the thermal stability and on the deuteration kinetics of the thermophilic F0F1 ATP synthase.

Differential scanning calorimetry has been used to characterize the influence of specific nucleotide binding on the thermal unfolding of the F0F1-type ATP synthase from the thermophilic Bacillus PS3 (TF0F1). The calorimetric trace shows an irreversible and kinetically controlled endothermic transition for TF0F1 in the absence of nucleotides. The thermal denaturation occurs at a transition temperature (t(m)) of 81.7 degrees C. The remarkable thermostability of this enzyme was decreased upon tight binding of Mg2+ x ATP to noncatalytic sites, whereas binding of Mg2+ x ADP increased the temperature at which thermal denaturation occurred. At high temperatures, an exothermic transition due to aggregation processes was also affected by nucleotide binding. With the aim to correlate these thermal effects with possible structural differences among the various forms of TF0F1, Fourier transform infrared spectroscopy was carried out. Hydrogen/deuterium exchange was clearly affected by specific nucleotide occupancy. As illustrated by the total extent of protons exchanged, our results demonstrate that more peptide groups are exposed to the medium in the presence of Mg2+ x ATP than in the presence of Mg2+ x ADP. Therefore, consistent with microcalorimetric data, binding of Mg2+ x ADP induces conformational changes which shield amide protons to more buried hydrogen-bonded structures, whereas binding of Mg2+ x ATP results in a more open or flexible structure.

Adenosine Diphosphate↗