The effect of Zn(2+) on the secondary structure of a histidine-rich fusogenic peptide and its interaction with lipid membranes.
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Membrane fusion between uncharged lipid vesicles can be triggered by the peptide sequence 'B18' from the fertilization protein 'bindin', but it only proceeds efficiently in the presence of Zn(2+) ions. We studied (i) the interaction of Zn(2+) with the fusogenic peptide B18, (ii) the binding of B18 to 1-palmitoyl-2-oleoylglycero-3-phosphocholine (POPC), and (iii) the ternary system POPC/B18/Zn(2+). The complex formation of Zn(2+) with the central histidine-rich motif of B18 appears to shift the secondary structure away from a beta-sheet towards an alpha-helical conformation. Here we observe for the first time an essentially alpha-helical structure of the peptide when immersed in POPC bilayers which appears to represent its functional fusogenic state. Infrared linear dichroism suggests a peripheral, oblique insertion mode of B18, mediated by the hydrophobic patches along one side of the amphipathic peptide. Furthermore, the hydration level of the peptide is reduced, suggesting that the hydrophobic region of the bilayer is involved in the lipid/peptide interactions. The hydration capacity of the POPC/B18/Zn(2+) system is distinctly smaller than that of POPC/Zn(2+) without peptide. The accompanying decrease in the number of tightly bound water molecules per lipid can be interpreted as a reduction in the repulsive 'hydration' forces, which usually prevent the spontaneous fusion of lipid vesicles. Binding of the B18 peptide in the presence of Zn(2+) effectively renders the membrane surface more hydrophobic, thus allowing fusion to proceed. (+info)
Excluded volume effects on the refolding and assembly of an oligomeric protein. GroEL, a case study.
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We have studied the effect of macromolecular crowding reagents, such as polysaccharides and bovine serum albumin, on the refolding of tetradecameric GroEL from urea-denatured protein monomers. The results show that productive refolding and assembly strongly depends on the presence of nucleotides (ATP or ADP) and background macromolecules. Nucleotides are required to generate an assembly-competent monomeric conformation, suggesting that proper folding of the equatorial domain of the protein subunits into a native-like structure is essential for productive assembly. Crowding modulates GroEL oligomerization in two different ways. First, it increases the tendency of refolded, monomeric GroEL to undergo self-association at equilibrium. Second, crowding can modify the relative rates of the two competing self-association reactions, namely, productive assembly into a native tetradecameric structure and unproductive aggregation. This kinetic effect is most likely exerted by modifications of the diffusion coefficient of the refolded monomers, which in turn determine the conformational properties of the interacting subunits. If they are allowed to become assembly-competent before self-association, productive oligomerization occurs; otherwise, unproductive aggregation takes place. Our data demonstrate that the spontaneous refolding and assembly of homo-oligomeric proteins, such as GroEL, can occur efficiently (70%) under crowding conditions similar to those expected in vivo. (+info)
Three types of mycolic acid from Mycobacterium tuberculosis Brevanne: implications for structure-function relationships in pathogenesis.
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Saponification of the chloroform-soluble wax from Mycobacterium tuberculosis Brevanne led to the isolation of three classes of mycolic acid containing characteristic functional groups along the methylene backbone: type alpha (two cyclopropane rings); type beta (methoxyl, methyl, and cyclopropane); and type gamma (ketone, methyl, and cyclopropane). The structures of these acids were elucidated principally by mass spectrometry. The high mass region of the keto mycolate is presented showing the meromycolal and molecular ion regions. This is first time a molecular peak for this mycolic acid has been reported. The structure of the keto mycolate was further substantiated by study of the mass spectral fragmentation of its dithioketal derivative. Within each type of acid, a series of homologs was encountered, varying according to the number of methylene units in the backbone chain. Chromatographic and infrared spectrophotometric evidence is presented for the alkali-induced isomerization of the three types of mycolates. (+info)
Spectroscopic techniques in the study of membrane solubilization, reconstitution and permeabilization by detergents.
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This review focuses on the use of spectroscopic techniques for the study of membrane solubilization, reconstitution, and permeabilization by detergents. Turbidity and light scattering, visible and infrared spectroscopic methods, fluorescence, nuclear magnetic resonance, electron spin resonance and X-ray diffraction are examined from the point of view of their applicability to the above detergent-mediated phenomena. A short introduction is provided about each of the techniques, and references are given for further study. (+info)
Influence of aerobic oxidation of mouse erythrocytes on their recognition by macrophages.
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Membrane protein modification can change cell surface properties which can be correlated with altered macrophage-erythrocyte interactions. Mouse erythrocytes were incubated in phosphate buffer for different times to induce protein modification. Mouse erythrocyte membrane changes were analyzed by infrared analyses and gel electrophoresis. Proteolytic digestion of membrane proteins was observed. After 22 hours preliminary incubation, the number of erythrocytes adhering to a monolayer of macrophages reached a maximum, the majority of which had not been phagocytosed. Most of the erythrocytes incubated for 40 hours underwent phagocytosis after adhesion to the macrophages. (+info)
In vivo chitin-cadmium complexation in cell wall of Neurospora crassa.
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Fungal cell wall, mainly composed of chitin, an N-acetylglucosamine polymer, is known to participate in heavy metal detoxification. In the present study, an effort was made to elucidate the sites involved in complexation of cadmium by the chitin material of cell wall of Neurospora crassa. Based on the results of physical techniques, such as solid-state 13C-NMR, X-ray diffraction, IR and molecular modeling, a structure was proposed for the chitin-cadmium complex. The ring and C-3 hydroxyl oxygens of N-acetylglucosamine were implicated in the complexation of cadmium by the chitin of the fungal cell wall. The studies further revealed that the conformation of chitin did not alter after cadmium complexation. (+info)
Use of a single glycine residue to determine the tilt and orientation of a transmembrane helix. A new structural label for infrared spectroscopy.
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Site-directed dichroism is an emerging technique for the determination of membrane protein structure. However, due to a number of factors, among which is the high natural abundance of (13)C, the use of this technique has been restricted to the study of small peptides. We have overcome these problems through the use of a double C-deuterated glycine as a label. The modification of a single residue (Gly) in the transmembrane segment of M2, a protein from the Influenza A virus that forms H(+)-selective ion channels, has allowed us to determine its helix tilt and rotational orientation. Double C-deuteration shifts the antisymmetric and symmetric stretching vibrations of the CD(2) group in glycine to a transparent region of the infrared spectrum where the dichroic ratio of these bands can be measured. The two dichroisms, along with the helix amide I dichroic ratio, have been used to determine the helix tilt and rotational orientation of M2. The results are entirely consistent with previous site-directed dichroism and solid-state NMR experiments, validating C-deuterated glycine (GlyCD(2)) as a structural probe that can now be used in the study of polytopic membrane proteins. (+info)
gamma-phosphate protonation and pH-dependent unfolding of the Ras.GTP.Mg2+ complex: a vibrational spectroscopy study.
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The interdependence of GTP hydrolysis and the second messenger functions of virtually all GTPases has stimulated intensive study of the chemical mechanism of the hydrolysis. Despite numerous mutagenesis studies, the presumed general base, whose role is to activate hydrolysis by abstracting a proton from the nucleophilic water, has not been identified. Recent theoretical and experimental work suggest that the gamma-phosphate of GTP could be the general base. The current study investigates this possibility by studying the pH dependence of the vibrational spectrum of the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes. Isotope-edited IR studies of the Ras.GTP.Mg(2+) complex show that GTP remains bound to Ras at pH as low as 2.0 and that the gamma-phosphate is not protonated at pH > or = 3.3, indicating that the active site decreases the gamma-phosphate pK(a) by at least 1.1 pK(a) units compared with solution. Amide I studies show that the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes partially unfold in what appear to be two transitions. The first occurs in the pH range 5.4-2.6 and is readily reversible. Differences in the pH-unfolding midpoints for the Ras.GTP.Mg(2+) and Ras.GDP.Mg(2+) complexes (3.7 and 4.8, respectively) reveal that the enzyme-gamma-phosphoryl interactions stabilize the structure. The second transition, pH 2.6-1.7, is not readily reversed. The pH-dependent unfolding of the Ras.GTP.Mg(2+) complex provides an alternative interpretation of the data that had been used to support the gamma-phosphate mechanism, thereby raising the issue of whether this mechanism is operative in GTPase-catalyzed GTP hydrolysis reactions. (+info)