scholarly journals Isolation and biophysical characterization of GSU0105, a triheme c-type cytochrome from Geobacter sulfurreducens

2020 ◽  
Author(s):  
Tyler J. Brittain ◽  
Matthew C. O’Malley ◽  
Coleman M. Swaim ◽  
Reilly A. Fink ◽  
Oleksandr Kokhan
Keyword(s):  
Charge Transfer ◽  
Charge Transfer Band ◽  
Geobacter Sulfurreducens ◽  
Size Exclusion ◽  
Multiple Sequence ◽  
Biophysical Characterization ◽  
Methionine Residue ◽  
Vis Spectroscopy ◽  
Cyt C

AbstractC-type cytochromes play an important role in respiration of dissimilatory metal-reducing bacteria. They form extended conduits for charge transfer between the cellular metabolism and external electron acceptors such as particles of iron oxide, metal ions, and humic substances. Out of more than a hundred c-type cytochromes in Geobacter sulfurreducens, only a small fraction has been previously characterized. Here we present our results on expression and biophysical characterization of GSU0105, a novel 3-heme cytochrome, important for Fe(III) respiration in G. sulfurreducens. We successfully cloned the gene and achieved ~3 mg/L of culture GSU0105 expression in E.coli. Despite a similar size (71 amino acids) and the same number of c-type hemes to the members of the cytochrome (cyt) c7 family, multiple sequence alignment suggests that GSU0105 does not belong to the cyt c7 family. UV-Vis spectroscopy revealed typical c-type cytochrome spectral features, including a weak iron-sulfur charge transfer band suggesting that at least one heme is ligated with a methionine residue. Far UV circular dichroism studies demonstrate approximately 35% content of α-helices and β-sheets, each, as well as thermal aggregation occurring above 60 °C. A combination of SAXS and analytical size exclusion chromatography data shows that GSU0105 is monomeric in solution. Finally, affinity pull-down assays demonstrate high binding affinity to PpcD and weaker binding to the other members of the cyt c7 family.

scholarly journals Dissociation Mechanics and Stability of Type a Botulinum Neurotoxin Complex by Means of Biophysical Evaluation

2022 ◽  
Author(s):  
Shavron Hada ◽  
Jae Chul Lee ◽  
Eun Chae Lee ◽  
Sunkyong Ji ◽  
Jeong Sun Nam ◽  
...  
Keyword(s):  
Light Scattering ◽  
Botulinum Neurotoxin ◽  
Type A ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Biophysical Characterization ◽  
Exclusion Chromatography ◽  
Associated Proteins ◽  
Dissociated State

Abstract Biophysical characterization of type A botulinum neurotoxin (BoNT/A) complex along with its thermodynamic stability was assessed through a combination of various methods. BoNT/A exists as large complexes in association with neurotoxin associated proteins (NAPs). To evaluate its biophysical behavior, size-exclusion chromatography (SEC), multi-angled light scattering (MALS), enzyme linked immunosorbent assay (ELISA), and dynamic light scattering (DLS) were utilized. Initially, a single peak (peak 1) of SEC was observed at pH 6.0, and an additional peak (peak 2) appeared at pH 7.4 with a decrement of peak 1. Through MALS and ELISA, the peak 2 was determined to be BoNT/A dissociated from its complex. The dissociation was accelerated by time and temperature. At 37°C, dissociated BoNT/A self-associated at pH 7.4 in the presence of polysorbate 20. On the other hand, the dissociation was partly reversible when titrated back to pH 6.0. Overall, BoNT/A was more stable when associated with NAPs at pH 6.0 compared to its dissociated state at pH 7.4. The conventional analytical methods could be utilized to relatively quantify its amount in different formulations.

Chemische Charakterisierung des [py2X]+-Ions (X = Br, J) / Chemical Characterization of the [py2X]+ Ion (X = Br, J)

1973 ◽  
Vol 28 (11-12) ◽  
pp. 731-735 ◽  
Author(s):  
Karl-Heinz Tytko ◽  
Martin Schmeisser
Keyword(s):  
Charge Transfer ◽  
Charge Transfer Band ◽  
Chemical Characterization ◽  
Chloride Ions ◽  
Chemical Behaviour ◽  
Species Being

The chemical behaviour of the [py2X]+ ion in acetonitrile as solvent can be well characterized by its intense charge transfer band. The two pyridine molecules of the ion can be substituted successively by chloride ions, the resulting species being py · XCl and XCl2-. In the order of stability of the species [py2X]+ < py · XCl < XCl2- the adduct py · XCl thus is placed near the XCl2- ion. Consequently even this ion can act as Cl donor against the [py2X]+ ion. The XCl and pyridine arising from this reaction on their part form the adduct py · XCl as well. The ionization of the adduct py · JCl giving [py2J]+ and JCl2-, as proposed in the literature, cannot be correct.

UV-vis and Binding Studies of Cobalt Tetrasulfophthalocyanine–Thiolate Complexes as Intermediates of the Merox Process

1999 ◽  
Vol 03 (07) ◽  
pp. 654-666 ◽  
Author(s):  
ALI NAVID ◽  
EDUARD M. TYAPOCHKIN ◽  
CHARLES J. ARCHER ◽  
EVGUENII I. KOZLIAK
Keyword(s):  
Charge Transfer ◽  
Charge Transfer Band ◽  
Broad Band ◽  
Spectral Feature ◽  
Binding Constants ◽  
Kinetic Studies ◽  
Binding Studies ◽  
Π Stacking ◽  
Vis Spectroscopy ◽  
Major Factors

Intermediates of the cobalt tetrasulfophthalocyanine ( CoTSPc )-catalyzed thiol autoxidation were studied by UV-vis spectroscopy. All thiolates react with CoTSPc resulting in the formation of 1:1 complexes. Three major factors control both the stability and aggregation of the complexes: thiolate basicity, metal-to-ligand charge transfer (MLCT) and π stacking. Basic thiolates partially reduce C oII TSPc , whereas CoTSPc complexes with low-basicity aliphatic thiolates ( p K a < 4) do not exhibit Co (II) reduction, based on the absence of the characteristic Co (I) charge transfer band at 450 nm. CoTSPc complexes with aliphatic and bulky aromatic thiolates appear to be aggregated in aqueous solutions and are characterized by a broad band at 650 nm. Non-bulky aromatic thiolates of low basicity ( p K a < 6) form unique stable monomeric Co II TSPc complexes. This unique spectral feature can be attributed to π stacking between the phthalocyanine ring and thiolate. Comparison of binding constants shows that the partial reduction of Co (II) significantly contributes to the thiolate binding. A combination of aromatic π stacking and MLCT appears to be responsible for the observed 1000-fold stronger binding of non-basic aromatic thiolates as compared with aliphatic ligands of similar basicity. Kinetic studies confirm the importance of the thiolate binding type for catalysis.

scholarly journals Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28

Viruses ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 15
Author(s):  
Emilio Reyes-Aldrete ◽  
Erik A. Dill ◽  
Cecile Bussetta ◽  
Michal R. Szymanski ◽  
Geoffrey Diemer ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Analytical Ultracentrifugation ◽  
Atp Hydrolysis ◽  
Size Exclusion ◽  
Radius Of Gyration ◽  
Biophysical Characterization ◽  
X Ray ◽  
Double Stranded Dna ◽  
Symmetric Complex

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.

Preparation and Characterization of Porphyrin-Acridine Conjugates as Bifunctional Antitumor Agents

1995 ◽  
Vol 48 (10) ◽  
pp. 1693 ◽  
Author(s):  
G Karagianis ◽  
JA Reiss
Keyword(s):  
Mass Spectrometry ◽  
Size Exclusion Chromatography ◽  
Pure State ◽  
Antitumor Agents ◽  
Size Exclusion ◽  
Diode Array ◽  
Physical Measurements ◽  
Vis Spectroscopy ◽  
Exclusion Chromatography

The porphyrin-acridine conjugates (22) and (23) were prepared by condensation of porphyrin (17) with the 9-aminoacridine derivative (6) or (15), respectively. The conjugate drugs were isolated by size-exclusion chromatography in yields of up to 35% and further purified by semipreparative h.p.l.c . to 98% purity. Characterization of the conjugates was effected by 1H n.m.r. and u.v./vis . spectroscopy, f.a.b. mass spectrometry and u.v./vis . diode array h.p.l.c. Attempts to condense acridine (6) or (15) to porphyrin (16), (18) or (19) resulted in the isolation of the porphyrin-acridine conjugates (20), (21), (24) and (25) in low yields (≤ 25%), but the products could not be obtained in a pure state. However, their presence was identified by several physical measurements.

Organometallic Rhodium(III) and -(I) Complexes with 1,3-Diniethyllumazine(DML). The Crystal Structure of [(η2- (O4,N5)DML)(η5-C5Me5)ClRh](PF6)

1994 ◽  
Vol 49 (11) ◽  
pp. 1554-1560 ◽  
Author(s):  
Oliver Heilmann ◽  
Hans-Dieter Hausen ◽  
Wolfgang Kaim
Keyword(s):  
Crystal Structure ◽  
Charge Transfer ◽  
Structure Determination ◽  
Charge Transfer Band ◽  
Crystal Structure Determination ◽  
Bond Lengths ◽  
Electron Reduction

The crystal structure determination of the new rhodium(III)-complex [(DML)(C5Me5)ClRh](PF6). DML = 1,3-dimethyllumazine, reveals O4,N5 coordination of the metal to DML with not very different bond lengths of 213.0(2) pm (Rh-N) and 219.2(2) pm (Rh-O). This result stands in contrast to the previously reported structure of a cationic dihydrobiopterin bound to [Mo(O)Cl3]- which exhibited a very unsymmetrical chelate ar­rangement. Chemical and electrochemical two-electron reduction of the Rh(III) complex led to a very labile Rh(I) species (DML)(C5Me5)Rh which is distinguished by an intense charge transfer band in the visible. The results confirm the characterization of DML as a weak σ donor/π acceptor ligand with a rather low-lying π* orbital.

scholarly journals Insights into the G-rich VEGF-binding aptamer V7t1: when two G-quadruplexes are better than one!

2019 ◽  
Vol 47 (15) ◽  
pp. 8318-8331 ◽  
Author(s):  
Federica Moccia ◽  
Claudia Riccardi ◽  
Domenica Musumeci ◽  
Serena Leone ◽  
Rosario Oliva ◽  
...  
Keyword(s):  
Size Exclusion ◽  
Biophysical Characterization ◽  
Monomeric Species ◽  
Conformational Preferences ◽  
G Quadruplex ◽  
Exclusion Chromatography ◽  
Extracellular Environment ◽  
Annealed Oligonucleotide ◽  
Better Than

Abstract The G-quadruplex-forming VEGF-binding aptamer V7t1 was previously found to be highly polymorphic in a K+-containing solution and, to restrict its conformational preferences to a unique, well-defined form, modified nucleotides (LNA and/or UNA) were inserted in its sequence. We here report an in-depth biophysical characterization of V7t1 in a Na+-rich medium, mimicking the extracellular environment in which VEGF targeting should occur, carried out combining several techniques to analyse the conformational behaviour of the aptamer and its binding to the protein. Our results demonstrate that, in the presence of high Na+ concentrations, V7t1 behaves in a very different way if subjected or not to annealing procedures, as evidenced by native gel electrophoresis, size exclusion chromatography and dynamic light scattering analysis. Indeed, not-annealed V7t1 forms both monomeric and dimeric G-quadruplexes, while the annealed oligonucleotide is a monomeric species. Remarkably, only the dimeric aptamer efficiently binds VEGF, showing higher affinity for the protein compared to the monomeric species. These findings provide new precious information for the development of improved V7t1 analogues, allowing more efficient binding to the cancer-related protein and the design of effective biosensors or theranostic devices based on VEGF targeting.

scholarly journals The Isolation and Characterization of Molecular Complexes Produced from the Reaction of Nitrophenols with Divalent Metal Oxinates

1979 ◽  
Vol 34 (2) ◽  
pp. 230-234 ◽  
Author(s):  
M. M. Aly ◽  
A. M. Shalaby
Keyword(s):  
Hydrogen Bonding ◽  
Charge Transfer ◽  
Charge Transfer Band ◽  
Picric Acid ◽  
Molecular Complexes ◽  
Divalent Metal ◽  
Bonding System ◽  
Isolation And Characterization ◽  
Hydrogen Bonding System

Abstract The reaction of nitrophenols (picric acid, 2,6-dinitrophenol, and 2,4-dinitrophenol) with divalent metal oxinate (metal = Cu, Co, Ni, Mn, and Zn) produced a type of molecular complex, which comprises two molecules of the metal oxinate and one molecule of the nitrophenol. In the case of the Mg complex the ratio is 1:1. A definite charge-transfer band is detected for the Co(II) complex. A hydrogen bonding system O1...H+...O2 is suggested. This includes the proton of the nitrophenol and two oxygen atoms; each is related to a coordinated oxinate of two different metal oxinate molecules. Analytical and spectral evidence are in accordance with the tentatively suggested formulations.

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