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At least 1,171 records · Page 65Linked to original sources
PubMed2005

In situ conformational analysis of fibrinogen adsorbed on Si surfaces.

Fibrinogen is a major plasma protein. Previous investigations of structural changes of fibrinogen due to adsorption are mostly based on indirect evidence after its desorption, whereas our measurements were performed on fibrinogen in its adsorbed state. Specific enzyme-linked immunosorption experiments showed that the amount of adsorbed fibrinogen increased as the surface became more hydrophobic. Atomic force microscopy (AFM) investigations revealed the trinodular shape of fibrinogen molecules adsorbed on hydrophilic surfaces, whereas all of the molecules appeared globular on hydrophobic surfaces. The distribution of secondary structures in adsorbed fibrinogen was quantified by in situ Fourier-transform infrared (FTIR) analysis. Substrates of identical chemical bulk composition but different surface hydrophobicity permit direct comparison among them. Adsorption properties of fibrinogen are different for each degree of hydrophobicity. Although there is some increase of turn structure and decrease of beta-sheet structure, the secondary structure of adsorbed fibrinogen on hydrophilic surface turned out to be rather similar to that of the protein in solution phase with a major alpha-helix content. Hydrophilic surfaces exhibit superior blood compatibility as required for medical applications.

Adsorption↗
PubMed2006

Conformational analysis of protein secondary structure during spray-drying of antibody/mannitol formulations.

Inhalation of spray-dried particles is a promising delivery route for proteins as an alternative to injection. Changes in the protein structure and aggregation have to be avoided. The effect of mannitol, a stabilizing agent typically used in both liquid and lyophilized protein formulations, on an antibody (IgG1) in a spray-dried powder was studied using different biophysical and chromatographic techniques. At first, different solutions composed of antibody (IgG1) and mannitol at a ratio between 20/80 and 100/0 IgG1/mannitol (100 mg/ml total solid) were investigated for their stability. Protein solutions containing the IgG1 showed mannitol-dependent aggregation. High amounts of mannitol (50-80%) exerted a destabilizing effect on the antibody and the aggregate 9 level increased to 2.6-4.2%. In contrast, solutions with only 20-40% mannitol showed the same amount of aggregates as the pure antibody solution. The antibody mannitol solutions were investigated by circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) to evaluate whether changes in the protein secondary structure can be correlated with aggregation. Considering the sensitivity of the used methods and data evaluation, FTIR spectra did not reveal structural changes in the IgG1/mannitol solutions compared to the pure antibody, despite varying aggregate levels. Thermal stress was reflected in perturbations of the secondary structure, but mannitol-dependent aggregation could not be correlated to detectable alterations in the FTIR spectra. Analyzing the CD spectra revealed no distinctive change in the shape of the CD curve, indicating that the protein secondary structure is mainly retained. This is in agreement with the infrared data. Subsequently, the IgG1/mannitol solutions were spray-dried at Tin/Tout of 90/50 degrees C. Using ATR-FTIR for the investigation of the protein amide I band in the spray-dried powder revealed changes in the sub-components of the amide I band. This indicates that the peptide groups (CO and NH) of the protein are found in a different environment in the solid state, compared to the liquid protein formulation. After redissolution of the powders, the native structure of the pure antibody solution was found identical to the protein secondary structure before spray-drying, indicating that the protein secondary structure is not strongly altered in the dry state, and not affected by the spray-drying process. Thus, from the presented study it can be concluded that the formation of antibody aggregates in mannitol formulations cannot be correlated with significant perturbations of the protein secondary structure elements.

Chemistry, Pharmaceutical↗
PubMed2004

Comparative studies on trifluoroethanol (TFE) state of a thermophilic alpha-amylase and its mesophilic counterpart: limited proteolysis, conformational analysis, aggregation and reactivation of the enzymes.

Detailed circular dichroism (CD), scattering and quenching studies, 1-anilinonaphthalene-8-sulfonate (ANS) binding, irreversible thermoinactivation, activity measurements and proteolytic digestion of bacterial alpha-amylases have been carried out to elucidate the effect of trifluoroethanol (TFE) on the structure of these enzymes. Under high concentrations of TFE both of the alpha-amylases, a thermostable alpha-amylase from Bacillus licheniformis (BLA) and its mesophilic counterpart from Bacillus amyloliquefaciens (BAA), acquire partially folded state characterized by an enhanced content of the secondary structure (helix) and reduced tertiary structures. According to ANS binding studies, we suggest that the TFE states induced by TFE/water mixture are not the molten globule state in the alpha-amylase folding pathway. In addition, data shows significant reversible aggregation of both enzymes in TFE/water mixtures with concentration between 10 and 60% (v/v). However, reversibility is more in case of BAA. As expected, in the absence of TFE, the thermophilic enzyme compared to mesophilic enzyme, shows a greater resistance to digestion by thermolysin. With respect to fluorescence quenching by acrylamide and potassium iodide, the thermophilic enzyme, BLA, is characterized by higher structural flexibility as compared to the BAA. On the other hand, in the presence of TFE, the enzymes are digested by protease to produce large protein fragments. It is proposed that highly helical secondary structures, acquired by BAA and BLA when dissolved in aqueous TFE, prevent binding and adaptation of the protein substrate at the active site of the protease.

Anilino Naphthalenesulfonates↗
PubMed2004

Ion mobility-mass spectrometry applied to cyclic peptide analysis: conformational preferences of gramicidin S and linear analogs in the gas phase.

In this paper, we present an investigation of the gas-phase structural differences between cyclic and linear peptide ions by matrix-assisted laser desorption ionization-ion mobility-mass spectrometry. Specifically, data is shown for gramicidin S (cyclo-VOLFPVOLFP where phenylalanines are D rather than L-type amino acids and the O designates the non-standard amino acid ornithine) and five linear gramicidin S analogues. Results are interpreted as evidence for a beta-sheet (or beta-hairpin) conformational preference in both linear-protonated and sodiated-cyclic gramicidin S gas-phase peptides, and a preference for the protonated-cyclic peptide to adopt a collapsed, random coil-type conformation. A comparison with solution-phase circular dichroism measurements is performed, and structures similar to those observed in the gas phase appear to be favored in low-dielectric solvents such as 2,2,2-triflouroethanol. The utility of ion mobility-mass spectrometry (IM-MS) as a means of rapidly distinguishing between linear and cyclic peptide forms in also discussed.

Amino Acid Sequence↗
PubMed2005

Conformational analysis and comparison between theoretical and experimental vibration spectra for isocyanate species on Ag/Al2O3 catalyst.

Density functional theory (DFT) calculations were performed to study the structure and vibrational frequencies of the intermediate for the isocyanate (NCO) surface species on Ag/Al(2)O(3) catalyst using the GAUSSIAN 98 suite of programs. Thermal decomposition of adsorbed CH(3)NO(2) species leads to the formation of two types of NCO species (NCO on Ag and NCO on Al(2)O(3)) above 298K. In order to investigate the reaction mechanism, we have designed the calculated models (a-g) for the NCO intermediate. Through the analysis of the vibration mode and the vibration frequency of the calculated models (a-g), we conclude that there is an excellent agreement between the calculated vibration spectrum of the model (e) containing the -AgNCO group or model (g) containing the -AlNCO group and the experimental vibration spectrum.

Aluminum Oxide↗
PubMed1997

Purification, characterization and conformational analysis of a haemorrhagin from the venom of Agkistrodon acutus.

AaHIV, a medium-sized toxin with a mol. wt of 44,000, a pI of 5.0 and a low cysteine content, was isolated from the venom of Agkistrodon acutus by ion-exchange and gel filtration chromatography. It had haemorrhagic, lethal and caseinolytic activities, the last of which was inhibited by EDTA, Zn(CH3COO)2 or CuSO4. The circular dichroism spectrum at pH 7.0 showed two negative bands at 210 nm and 219 nm, corresponding to secondary structure contents of 18.2% alpha-helix, 31.0% beta-sheet, 17.2% beta-turn and 33.6% random-coil. The effect of various pH values or the addition of EDTA or Zn(CH3COO)2 on the circular dichroism spectrum was also investigated.

Agkistrodon↗
PubMed1998

Design, synthesis, and conformational analysis of a novel series of HIV protease inhibitors.

A combination of structure-based design and both solution, and solid-phase synthesis were utilized to derive a potent (nM) series of HIV-1 protease inhibitors bearing a structurally novel backbone. Detailed structural analysis of several inhibitors prepared in this series has suggested that rigidification of the P1/P2 region of this class of molecules may result in compounds with improved potency.

Anti-HIV Agents↗
PubMed1993

Purification, characterization, and conformational analysis of rabbit plasma lipid transfer protein.

A procedure for rapid isolation of lipid transfer protein (LTP) from commercially available rabbit plasma is described. Use of protease inhibitors was important for obtaining intact, stable LTP. After lipoproteins were precipitated from the plasma by dextran sulfate, column chromatographies through Butyl-Toyopearl 650M, CM-Toyopearl 650M, and Butyl-Toyopearl 650M were employed. Overall purification from plasma was (3830 +/- 710)-fold with a yield of 3-5%. The isolated LTP migrated as a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with M(r) = 74K and had an NH2-terminal amino acid sequence and amino acid composition closely matching those predicted by its cDNA. This band was recognized by immunoblotting with an anti-human LTP monoclonal antibody, TP2. Gel permeation chromatography revealed that LTP behaved as a globular protein of M(r) = 83K. Isoelectric focusing of the isolated LTP demonstrated a ladder of bands with pI's of 5.7-5.9. The specific activity of rabbit LTP was similar to that of human LTP. Monoclonal antibody TP2, that blocked human plasma LTP activity almost completely, only partially inhibited purified rabbit LTP, and rabbit plasma LTP activity to a similar extent. By a centrifugation binding assay, rabbit LTP was shown to predominantly associate with lipid microemulsion in its presence. Circular dichroism spectroscopy indicated a high content of beta structure, and Provencher and Glöckner analysis gave estimated fractional values of 0.30, 0.39, 0.12, and 0.19 for alpha-helix, beta-sheet, beta-turn, and remainder content, respectively. Upon lipid binding, the helical content did not change drastically, although there was some disordering of beta structure.

Amino Acid Sequence↗
PubMed1988

High-resolution NMR studies of fibrinogen-like peptides in solution: resonance assignments and conformational analysis of residues 1-23 of the A alpha chain of human fibrinogen.

The proton resonances of the following synthetic linear human fibrinogen-like peptides were completely assigned with two-dimensional NMR techniques in solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp(7)-Phe-Leu-Ala-Glu-Gly(12)-Gly(13)-Gly(14)- Val(15)-Arg(16)-Gly-Pro-Arg-Val-Val-Glu-Arg (F10), Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly(13)-Gly(14)-Val-Arg (F11), and Gly-Pro-Arg-Val-Val-Glu-Arg (F12). No predominant structure was found in the chain segment from Ala(1) to Gly(6) for F10 in both H2O and dimethyl sulfoxide. The previous suggestion that there is a hairpin loop involving residues Gly(12) to Val(15) in the A alpha chain of human fibrinogen is supported by the slow backbone NH exchange rates of Gly(14) and Val(15), by an unusually small NH chemical shift of Val(15), and by strong sequential NOE's involving this region in F10. This local chain fold within residues Asp(7) to Val(20) may place the distant Phe residue near the Arg(16)-Gly(17) peptide bond which is cleaved by thrombin.

Amino Acid Sequence↗