scholarly journals Toward Elucidating the Membrane Topology of Helix Two of the Colicin E1 Channel Domain

2006 ◽  
Vol 281 (43) ◽  
pp. 32375-32384 ◽  
Author(s):  
Dawn White ◽  
Abdiwahab A. Musse ◽  
Jie Wang ◽  
Erwin London ◽  
A. Rod Merrill
Keyword(s):  
Fluorescence Emission ◽  
Fluorescence Labeling ◽  
Interfacial Region ◽  
Closed Channel ◽  
Membrane Bilayer ◽  
Colicin E1 ◽  
Aqueous Solvent ◽  
Least Squares Fit ◽  
Membrane Bound ◽  
Α Helix

The membrane-bound closed state of the colicin E1 channel domain was investigated by site-directed fluorescence labeling using a bimane fluorophore attached to each single cysteine residue within helix 2 of each mutant protein. The fluorescence properties of the bimane fluorophore were measured for the membrane-associated form of the closed channel and included fluorescence emission maximum, fluorescence anisotropy, apparent polarity, surface accessibility, and membrane bilayer penetration depth. The fluorescence data show that helix 2 is an amphipathic α-helix that is situated parallel to the membrane surface, but it is less deeply embedded within the bilayer interfacial region than is helix 1 in the closed channel. A least squares fit of the various data sets to a harmonic wave function indicated that the periodicity and angular frequency for helix 2 in the membrane-bound state are typical for an amphipathic α-helix (3.8 ± 0.1 residues per turn and 94 ± 4°, respectively) that is located at an interfacial region of a membrane bilayer. Dual quencher analysis also revealed that helix 2 is peripherally membrane associated, with one face of the helix dipping into the interfacial region of the lipid bilayer and the other face projecting outwardly into the aqueous solvent. Finally, our data show that helices 1 and 2 remain independent helices upon membrane association with a short connector link (Tyr363–Gly364) and that short amphipathic α-helices participate in the formation of a lipid-dependent, toroidal pore for this colicin.

scholarly journals Secondary structure of NADPH: protochlorophyllide oxidoreductase examined by circular dichroism and prediction methods*

1996 ◽  
Vol 317 (2) ◽  
pp. 549-555 ◽  
Author(s):  
Simon J. BIRVE ◽  
Eva SELSTAM ◽  
Lennart B.-Å. JOHANSSON
Keyword(s):  
Secondary Structure ◽  
Fluorescence Emission ◽  
Random Coil ◽  
Prolamellar Body ◽  
Interfacial Region ◽  
Protochlorophyllide Oxidoreductase ◽  
Loop Region ◽  
Rossmann Fold ◽  
Α Helix ◽  
Β Sheet

To study the secondary structure of the enzyme NADPH: protochlorophyllide oxidoreductase (PCOR), a novel method of enzyme isolation was developed. The detergent isotridecyl poly(ethylene glycol) ether (Genapol X-080) selectively solubilizes the enzyme from a prolamellar-body fraction isolated from wheat (Triticum aestivumL.). The solubilized fraction was further purified by ion-exchange chromatography. The isolated enzyme was studied by fluorescence spectroscopy at 77 K, and by CD spectroscopy. The fluorescence-emission spectra revealed that the binding properties of the substrate and co-substrate were preserved and that photo-reduction occurred. The CD spectra of PCOR were analysed for the relative amounts of the secondary structures, α-helix, β-sheet, turn and random coil. The secondary-structure composition was estimated to be 33% α-helix, 19% β-sheet, 20% turn and 28% random coil. These values are in agreement with those predicted by the Predict Heidelberg Deutschland and self-optimized prediction method from alignments methods. The enzyme has some amino acid identity with other NADPH-binding enzymes containing the Rossmann fold. The Rossmann-fold fingerprint motif is localized in the N-terminal region and at the expected positions in the predicted secondary structure. It is suggested that PCOR is anchored to the interfacial region of the membrane by either a β-sheet or an α-helical region containing tryptophan residues. A hydrophobic loop-region could also be involved in membrane anchoring.

scholarly journals Scanning the Membrane-bound Conformation of Helix 1 in the Colicin E1 Channel Domain by Site-directed Fluorescence Labeling

2005 ◽  
Vol 281 (2) ◽  
pp. 885-895 ◽  
Author(s):  
Abdiwahab A. Musse ◽  
Jie Wang ◽  
Gladys P. deLeon ◽  
Gerry A. Prentice ◽  
Erwin London ◽  
...  
Keyword(s):  
Fluorescence Labeling ◽  
Colicin E1 ◽  
Membrane Bound

scholarly journals Very Cool Clathrin

2005 ◽  
Vol 13 (6) ◽  
pp. 3-7
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Plasma Membrane ◽  
Coated Vesicles ◽  
Membrane Bilayer ◽  
Membrane Bound ◽  
Membranous Component

Clathrin-coated vesicles are the shuttle containers within cells. The vesicles carry lipids and proteins between membrane-bound compartments. Clathrin forms a cage-like structure around the membrane-bound vesicle that is pinched off from the plasma membrane (in endocytosis) or a membranous component of the cytoplasm. Clathrin recruits cargo that is within a vesicle through intermediary proteins known as adaptors that help select membrane-anchored protein and form an interface between the clathrin cage and the membrane bilayer.

Conformational Changes of the Membrane-Bound ATPase of Bacterial Chromatophores Revealed by Fluorescence Changes of Fluorescamine-Labelled Coupling Factors

1978 ◽  
Vol 33 (5-6) ◽  
pp. 421-427
Author(s):  
Peter Gräber ◽  
Sathamm Saphon
Keyword(s):  
Conformational Changes ◽  
Fluorescence Emission ◽  
Continuous Light ◽  
Coupling Factor ◽  
Single Turnover ◽  
Fluorescence Change ◽  
Transfer Inhibitor ◽  
Membrane Bound ◽  
Coupling Factors ◽  
Kinetics Of

Abstract The solubilized coupling factor (F1) of Rps. sphaeroides chromatophores was allowed to react with fiuorescamine which led to a fluorescence labelled F1 . After reconstitution with the depleted membranes the fluorescence-labelled F1 was shown to restore photophosphorylation in continuous light in a similar way to the non-labelled F1 . In parallel, a decrease of the fluorescence emission of the labelled and reconstituted coupling factor was observed. The solubilized and labelled F1 showed also a fluorescence decrease as the polarity of the medium was increased. In single turnover flashes the fluorescence change was found to be inhibited by an uncoupling agent such as FCCP. The kinetics of the change were sensitive to phosphorylating agents and to an “energy transfer inhibitor” such as venturicidin. It is suggested that the observed fluorescence changes reflect conformational changes of the ATPase enzyme complex.

scholarly journals Bombyx mori Midgut Membrane Protein P252, Which Binds to Bacillus thuringiensis Cry1A, Is a Chlorophyllide-Binding Protein, and the Resulting Complex Has Antimicrobial Activity

2008 ◽  
Vol 74 (5) ◽  
pp. 1324-1331 ◽  
Author(s):  
Ganesh N. Pandian ◽  
Toshiki Ishikawa ◽  
Makoto Togashi ◽  
Yasuyuki Shitomi ◽  
Kohsuke Haginoya ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Bacillus Thuringiensis ◽  
Bombyx Mori ◽  
Conformational Changes ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Binding Characteristics ◽  
Α Helix ◽  
Negligible Contribution

ABSTRACT The epithelial cell membrane 252-kDa protein (P252) isolated in our laboratory from Bombyx mori midgut was shown to bind strongly with Cry1Aa, Cry1Ab, and Cry1Ac toxins of Bacillus thuringiensis (15). In the current paper, P252 was shown to bind with chlorophyllide (Chlide) to form red fluorescent protein (RFP) complex, termed Bm252RFP, with absorbance and fluorescence emission peaks at 600 nm and 620 nm, respectively. P252 at a concentration of 1 μM is shown to bind with about 50 μM Chlide in a positively cooperative reaction to form Bm252RFP under aerobic conditions and in the presence of light at 37°C. Various parameters influencing this reaction have been optimized for efficient in vitro chemical synthesis of Bm252RFP. Circular dichroism spectra revealed that P252 is composed of a β-structure (39.8% ± 2.2%, based on 5 samples) with negligible contribution of α-helix structure. When bound to Chlide, the β-structure content in the complex is reduced to 21.6% ± 3.1% (n = 5). Since Chlide had no secondary structure, the observed reduction suggests significant conformational changes of P252 during the formation of Bm252RFP complex. Bm252RFP had antimicrobial activity against Escherichia coli, Serratia marcescens, B. thuringiensis, and Saccharomyces cerevisiae with 50% effective concentrations of 2.82, 2.94, 5.88 μM, and 21.6 μM, respectively. This is the first report ever to show clear, concrete binding characteristics of the midgut protein to form an RFP having significant antimicrobial activity.

scholarly journals Colicin E1 forms a dimer after urea-induced unfolding

1999 ◽  
Vol 340 (3) ◽  
pp. 631-638 ◽  
Author(s):  
Brian A. STEER ◽  
Ariel A. DINARDO ◽  
A. Rod MERRILL
Keyword(s):  
Intermediate State ◽  
Hydrophobic Interactions ◽  
Fluorescence Emission ◽  
Cd Spectroscopy ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Terminal Portion ◽  
Colicin E1 ◽  
Tryptophan Residues

Unfolding of the soluble colicin E1 channel peptide was examined with the use of urea as a denaturant; it was shown that it unfolds to an intermediate state in 8.5 M urea, equivalent to a dimeric species previously observed in 4 M guanidinium chloride. Single tryptophan residues, substituted into the peptide at various positions by site-directed mutagenesis, were employed as fluorescent probes of local unfolding. Unfolding profiles for specific sites within the peptide were obtained by quantifying the shifts in the fluorescence emission maxima of single tryptophan residues on unfolding and plotting them against urea concentration. Unfolding reported by tryptophan residues in the C-terminal region was not characteristic of complete peptide denaturation, as evidenced by the relatively blue-shifted values of the fluorescence emission maxima. Unfolding was also monitored by using CD spectroscopy and the fluorescent probe 2-(p-toluidinyl)-naphthalene 6-sulphonic acid; the results indicated that unfolding of helices is concomitant with the exposure of protein non-polar surface. Unfolding profiles were evaluated by non-linear least-squares curve fitting and calculation of the unfolding transition midpoint. The unfolding profiles of residues located in the N-terminal region of the peptide had lower transition midpoints than residues in the C-terminal portion. The results of unfolding analysis demonstrated that urea unfolds the peptide only partly to an intermediate state, because the C-terminal portion of the channel peptide retained significant structure in 8.5 M urea. Characterization of the peptide's global unfolding by size-exclusion HPLC revealed that the partly denatured structure that persists in 8.5 M urea is a dimer of two channel peptides, tightly associated by hydrophobic interactions. The presence of the dimerized species was confirmed by SDS/PAGE and intermolecular fluorescence resonance energy transfer.

scholarly journals Initial Characterization of the Photosynthetic Apparatus of “Candidatus Chlorothrix halophila,” a Filamentous, Anoxygenic Photoautotroph

2007 ◽  
Vol 189 (11) ◽  
pp. 4196-4203 ◽  
Author(s):  
Allison M. L. van de Meene ◽  
Tien Le Olson ◽  
Aaron M. Collins ◽  
Robert E. Blankenship
Keyword(s):  
Photosynthetic Bacteria ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Photosynthetic Apparatus ◽  
Fluorescence Emission ◽  
Photosynthetic Characteristics ◽  
Green Bacteria ◽  
Green Photosynthetic Bacteria ◽  
Antenna Complex ◽  
Membrane Bound

ABSTRACT “Candidatus Chlorothrix halophila” is a recently described halophilic, filamentous, anoxygenic photoautotroph (J. A. Klappenbach and B. K. Pierson, Arch. Microbiol. 181:17-25, 2004) that was enriched from the hypersaline microbial mats at Guerrero Negro, Mexico. Analysis of the photosynthetic apparatus by negative staining, spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the photosynthetic apparatus in this organism has similarities to the photosynthetic apparatus in both the Chloroflexi and Chlorobi phyla of green photosynthetic bacteria. The chlorosomes were found to be ellipsoidal and of various sizes, characteristics that are comparable to characteristics of chlorosomes in other species of green photosynthetic bacteria. The absorption spectrum of whole cells was dominated by the chlorosome bacteriochlorophyll c (BChl c) peak at 759 nm, with fluorescence emission at 760 nm. A second fluorescence emission band was observed at 870 nm and was tentatively attributed to a membrane-bound antenna complex. Fluorescence emission spectra obtained at 77 K revealed another complex that fluoresced at 820 nm, which probably resulted from the chlorosome baseplate complex. All of these results suggest that BChl c is present in the chlorosomes of “Ca. Chlorothrix halophila,” that BChl a is present in the baseplate, and that there is a membrane-bound antenna complex. Analysis of the proteins in the chlorosomes revealed an ∼6-kDa band, which was found to be related to the BChl c binding protein CsmA found in other green bacteria. Overall, the absorbance and fluorescence spectra of “Ca. Chlorothrix halophila” revealed an interesting mixture of photosynthetic characteristics that seemed to have properties similar to properties of both phyla of green bacteria when they were compared to the photosynthetic characteristics of Chlorobium tepidum and Chloroflexus aurantiacus.

scholarly journals Improved method for the analysis of membrane proteins by mass spectrometry

2007 ◽  
Vol 30 (1) ◽  
pp. 89-94 ◽  
Author(s):  
Shama P. Mirza ◽  
Brian D. Halligan ◽  
Andrew S. Greene ◽  
Michael Olivier
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Peptide Ions ◽  
Membrane Bilayer ◽  
Mass Spectral ◽  
Associated Proteins ◽  
Mass Spectrometric Identification ◽  
Membrane Bound ◽  
Positively Charged

Membrane-bound and membrane-associated proteins are difficult to analyze by mass spectrometry, since the association with lipids impedes the isolation and solubilization of the proteins in buffers suitable for mass spectrometry and the efficient generation of positively charged peptide ions by electrospray ionization. Current methods mostly utilize detergents for the isolation of proteins from membranes. In this study, we present an improved detergent-free method for the isolation and mass spectrometric identification of membrane-bound and membrane-associated proteins. We delipidate proteins from the membrane bilayer by chloroform extraction to overcome dissolution and ionization problems during analysis. Comparison of our results to results obtained by direct tryptic digestion of insoluble membrane pellets identifies an increased number of membrane proteins, and a higher quality of the resulting mass spectral data.

scholarly journals Membrane Binding by MinD Involves Insertion of Hydrophobic Residues within the C-Terminal Amphipathic Helix into the Bilayer

2003 ◽  
Vol 185 (15) ◽  
pp. 4326-4335 ◽  
Author(s):  
Huaijin Zhou ◽  
Joe Lutkenhaus
Keyword(s):  
Emission Spectroscopy ◽  
Membrane Binding ◽  
Fluorescence Emission ◽  
Phospholipid Vesicles ◽  
Amphipathic Helix ◽  
Spectroscopy Analysis ◽  
Membrane Bilayer ◽  
Hydrophobic Residues ◽  
Tryptophan Residues ◽  
The Mind

ABSTRACT MinD binds to phospholipid vesicles in the presence of ATP and is released by MinE, which stimulates the MinD ATPase. Membrane binding requires a short conserved C-terminal region, which has the potential to form an amphipathic helix. This finding has led to a model in which the binding of ATP regulates the formation or accessibility of this helix, which then embeds in the membrane bilayer. To test this model, we replaced each of the four hydrophobic residues within this potential helix with tryptophan or a charged residue. Introduction of a negatively charged amino acid decreased membrane binding of MinD and its ability to activate MinC. In contrast, mutants with tryptophan substitutions retained the ability to bind to the membrane and activate MinC. Fluorescence emission spectroscopy analysis of the tryptophan mutants F263W, L264W, and L267W confirmed that these tryptophan residues did insert into the hydrophobic interior of the bilayer. We conclude that membrane binding by MinD involves penetration of the hydrophobic residues within the C-terminal amphipathic helix into the hydrophobic interior of the bilayer.

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