Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein.
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The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment. (+info)
Quantitation of secondary structure in ATR infrared spectroscopy.
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Polarized attenuated total reflection infrared spectroscopy of aligned membranes provides essential information on the secondary structure content and orientation of the associated membrane proteins. Quantitation of the relative content of different secondary structures, however, requires allowance for geometric relations of the electric field components (E(x), E(y), E(z)) of the evanescent wave, and of the components of the infrared transition moments, in combining absorbances (A and A(perpendicular)) measured with radiation polarized parallel with and perpendicular to, respectively, the plane of incidence. This has hitherto not been done. The appropriate combination for exact evaluation of relative integrated absorbances is A + (2E(z)(2)/E(y)(2) - E(x)(2)/E(y)(2))A(perpendicular), where z is the axis of ordering that is normal to the membrane plane, and the x-axis lies in the membrane plane within the plane of incidence. This combination can take values in the range approximately from A - 0.4A(perpendicular) to A + 2.7A(perpendicular), depending on experimental conditions and the attenuated total reflection crystal used. With unpolarized radiation, this correction is not possible. Similar considerations apply to the dichroic ratios of multicomponent bands, which are also treated. (+info)
Two-dimensional IR correlation spectroscopy: sequential events in the unfolding process of the lambda cro-V55C repressor protein.
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A question often posed in protein folding/unfolding studies is whether the process is fully cooperative or whether it contains sequential elements. To address this question, one needs tools capable of resolving different events. It seems that, at least in certain cases, two-dimensional (2D) IR correlation spectroscopy can provide answers to this question. To illustrate this point, we have turned to the Cro-V55C dimer of the lambda Cro repressor, a protein known to undergo thermal unfolding in two discrete steps through a stable equilibrium intermediate. The secondary structure of this intermediate is compatible with that of a partially unfolded protein and involves a reorganization of the N terminus, whereas the antiparallel beta-ribbon formed by the C-terminal part of each subunit remains largely intact. To establish whether the unfolding process involves sequential events, we have performed a 2D correlation analysis of IR spectra recorded over the temperature range of 20-95 degrees C. The 2D IR correlation analysis indeed provides evidence for a sequential formation of the stable intermediate, which is created in three (closely related) steps. A first step entails the unfolding of the short N-terminal beta-strand, followed by the unfolding of the alpha-helices in a second step, and the third step comprises the reorganization of the remaining beta-sheet and of some unordered segments in the protein. The complete unfolding of the stable intermediate at higher temperatures also undergoes sequential events that ultimately end with the breaking of the H bonds between the two beta-strands at the dimer interface. (+info)
Horseradish peroxidase monitored by infrared spectroscopy: effect of temperature, substrate and calcium.
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Horseradish peroxidase was examined as a function of Ca and substrate binding using infrared spectroscopy in the temperature range of 10-300 K. The Ca complex could be identified by the carboxylate stretches. The amide peak positions indicate that the protein remains stable from room temperature to 10 K. Shifts in these peaks are consistent with increased hydrogen bonding as temperature decreases, but the protein conformation is maintained at cryogenic temperatures. The substrate, benzohydroxamic acid, produced no detectable change in the infrared spectrum, consistent with X-ray crystallography results. With removal of Ca, the protein maintained its overall helicity. (+info)
Attraction of the oriental fruit fly, Dacus dorsalis, to methyl eugenol and related olfactory stimulants.
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The attraction of male oriental fruit flies to methyl eugenol and 34 analogues was investigated quantitatively using the characteristic feeding response. Methyl eugenol was the most active compound studied, with a feeding response to 0.01 mug, but saturation of the allyl side chain or replacement of allyl by allyloxy produced compounds almost as effective. Replacement of the methoxy groups by methylenedioxy, methyl, or chloro groups abolished all response. The ring geometry of the methoxy groups was critical, with orthodimethoxy most active and meta-dimethoxy inactive. Replacement of methoxy with hydroxy, methylthio, or amino groups did not abolish the response. The failure of the oriental fruit fly to respond to the methyl and chloro isosteres of methyl eugenol was contrasted with the response of a human odor panel which perceived these compounds as having weak floral odors. (+info)
Preparation and microbial decomposition of synthetic [14C]ligins.
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A definitive assay for microbiological and biochemical research on the biodegradation of lignin was developed using radioactive synthetic lignins specifically labeled in the side chains, aromatic rings or in the methoxyl groups. The [14C]lignins were prepared by oxidative polymerization with peroxidase and H2O2 Of specifically labeled coniferyl alcohol (4-hydroxy-3-methyoxycinnamyl alcohol). The synthetic polymers were shown by spectroscopic and chemical methods to contain the same intermonomer linkages found in natural lignins. Incubation of the [14C]lignins with known lignin-degrading fungi and with a forest soil resulted in 14CO2 evolution. (+info)
Application of isotope-selective non-dispersive infrared spectrometry for the evaluation of the 13C-urea breath test: comparison with three concordant methods.
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The aim of this work was to compare the performance of isotope-selective non-dispersive infrared spectrometry (IRIS) for the 13C-urea breath test with the combination of the 14C-urea breath test (14C-UBT), urease test and histologic examination for the diagnosis of H. pylori (HP) infection. Fifty-three duodenal ulcer patients were studied. All patients were submitted to gastroscopy to detect HP by the urease test, histologic examination and 14C-UBT. To be included in the study the results of the 3 tests had to be concordant. Within one month after admission to the study the patients were submitted to IRIS with breath samples collected before and 30 min after the ingestion of 75 mg 13C-urea dissolved in 200 ml of orange juice. The samples were mailed and analyzed 11.5 (4-21) days after collection. Data were analyzed statistically by the chi-square and Mann-Whitney test and by the Spearman correlation coefficient. Twenty-six patients were HP positive and 27 negative. There was 100% agreement between the IRIS results and the HP status determined by the other three methods. Using a cutoff value of delta-over-baseline (DOB) above 4.0 the IRIS showed a mean value of 19.38 (minimum = 4.2, maximum = 41.3, SD = 10.9) for HP-positive patients and a mean value of 0.88 (minimum = 0.10, maximum = 2.5, SD = 0.71) for negative patients. Using a cutoff value corresponding to 0.800% CO2/weight (kg), the 14C-UBT showed a mean value of 2.78 (minimum = 0.89, maximum = 5.22, SD = 1.18) in HP-positive patients. HP-negative patients showed a mean value of 0.37 (minimum = 0.13, maximum = 0.77, SD = 0.17). IRIS is a low-cost, easy to manage, highly sensitive and specific test for H. pylori detection. Storing and mailing the samples did not interfere with the performance of the test. (+info)
Amyloid-beta-sheet formation at the air-water interface.
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An amyloid(1-40) solution rich in coil, turn, and alpha-helix, but poor in beta-sheet, develops monolayers with a high beta-sheet content when spread at the air-water interface. These monolayers are resistant to repeated compression-dilatation cycles and interaction with trifluoroethanol. The secondary structure motifs were detected by circular dichroism (CD) in solution and with infrared reflection-absorption spectroscopy (IRRAS) at the interface. Hydrophobic influences are discussed for the structure conversion in an effort to understand the completely unknown reason for the natural change of the normal prion protein cellular (PrP(C)) into the abnormal prion protein scrapie (PrP(Sc)). (+info)