scholarly journals The Native Monomer of Bacillus Pumilus Ribonuclease Does Not Exist Extracellularly

2018 ◽  
Vol 2018 ◽  
pp. 1-7 ◽  
Author(s):  
Olga Ilinskaya ◽  
Vera Ulyanova ◽  
Irina Lisevich ◽  
Elena Dudkina ◽  
Nataliya Zakharchenko ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Concentration ◽  
Bacillus Pumilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Culture Fluid ◽  
Size Exclusion ◽  
High Protein Concentration ◽  
Exclusion Chromatography ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Hypochromic Effect

Supported by crystallography studies, secreted ribonuclease of Bacillus pumilus (binase) has long been considered to be monomeric in form. Recent evidence obtained using native polyacrylamide gel electrophoresis and size-exclusion chromatography suggests that binase is in fact dimeric. To eliminate ambiguity and contradictions in the data we have measured conformational changes, hypochromic effect, and hydrodynamic radius of binase. The immutability of binase secondary structure upon transition from low to high protein concentration was registered, suggesting the binase dimerization immediately after translocation through the cell membrane and leading to detection of binase dimers only in the culture fluid regardless of ribonuclease concentration. Our results made it necessary to take a fresh look at the binase stability and cytotoxicity towards virus-infected or tumor cells.

scholarly journals Molecular Characterization of MexL, the Transcriptional Repressor of the mexJK Multidrug Efflux Operon in Pseudomonas aeruginosa

2005 ◽  
Vol 49 (5) ◽  
pp. 1844-1851 ◽  
Author(s):  
Rungtip Chuanchuen ◽  
Jared B. Gaynor ◽  
RoxAnn Karkhoff-Schweizer ◽  
Herbert P. Schweizer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Exclusion ◽  
Mutant Protein ◽  
Mobility Shift ◽  
Promoter Sequences ◽  
Exclusion Chromatography ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Gel Mobility Shift ◽  
Carboxy Terminal

ABSTRACT The Pseudomonas aeruginosa mexJK efflux operon is constitutively expressed in mutants with defects in the upstream mexL gene, which encodes a repressor of the TetR family. MexL and a MexLA47D mutant protein were purified from Escherichia coli as fusion proteins with carboxy-terminal hexahistidine tags. Native polyacrylamide gel electrophoresis and size exclusion chromatography revealed that MexL is a tetramer in solution. MexL and MexLA47D oligomerization was confirmed using a genetic approach, and the MexLA47D mutant protein was not impaired in multimerization. Gel mobility shift and footprinting assays demonstrated that MexL, but not MexLA47D, binds specifically to the 94-bp mexL-mexJ intergenic region to sequences located between positions −84 and −20 from the mexJ initiation codon. MexL protected about 60 nucleotides on each strand, and the protected regions overlapped almost perfectly, a finding consistent with MexL regulating the expression of both mexL and mexJK, which was ascertained by gene fusion analyses. The protected region contains predicted −10 and −35 promoter sequences for both mexL and mexJ, with partially overlapping −10 regions. The mexL promoter assignment was verified by mapping the mexL transcription start site, and the mexJ promoter was localized to the predicted regions using lacZ fusions. The MexL-protected region contains two inverted GTATTT repeats, and their location in the protected region and overlap with the mexL and mexJ promoter sequences strongly support a role in MexL binding.

Gamma radiation effects on α-lactalbumin: structural modifications

2001 ◽  
Vol 79 (2) ◽  
pp. 154-157 ◽  
Author(s):  
A Chapelier ◽  
M Desmadril ◽  
C Houée-Levin
Keyword(s):  
Thermodynamic Properties ◽  
Conformational Changes ◽  
Radiation Effects ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Aromatic Amino Acids ◽  
Size Exclusion ◽  
Structural Modifications ◽  
Exclusion Chromatography ◽  
Radiation Induced

α-Lactalbumin was irradiated in the lyophilized state in air at ambient temperature. The irradiated protein was examined by size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, circular dichroism, and microcalorimetry. Irradiation induced the loss of aromatic amino acids and of helicity so that fragmentation and aggregation products were obtained. The thermodynamic properties of the protein were also modified. The irradiated protein had lower stability, however, the temperature at which denaturation occurred process remained constant.Key words: ionizing radiation, radiation-induced conformational changes, protein structural modifications, protein thermodynamic properties.

scholarly journals Isolation of HDL by sequential flotation ultracentrifugation followed by size exclusion chromatography reveals size-based enrichment of HDL-associated proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jack Jingyuan Zheng ◽  
Joanne K. Agus ◽  
Brian V. Hong ◽  
Xinyu Tang ◽  
Christopher H. Rhodes ◽  
...  
Keyword(s):  
Structural Integrity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cholesterol Acyltransferase ◽  
Density Lipoprotein ◽  
Size Exclusion ◽  
High Yield ◽  
Phospholipid Transfer ◽  
Exclusion Chromatography ◽  
Associated Proteins ◽  
Alpha 1 Antitrypsin

AbstractHigh-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase

2000 ◽  
Vol 350 (1) ◽  
pp. 139-147 ◽  
Author(s):  
Diego F. GÓMEZ CASATI ◽  
Miguel A. AON ◽  
Alberto A. IGLESIAS
Keyword(s):  
Conformational Changes ◽  
Tertiary Structure ◽  
Fluorescence Emission ◽  
Maximal Activity ◽  
Size Exclusion ◽  
Exclusion Chromatography ◽  
Adp Glucose Pyrophosphorylase ◽  
Kinetics Of ◽  
Allosteric Regulators

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor Pi. Molecular ‘crowding’renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

scholarly journals Enhancement of the Structural Stability of Full-Length Clostridial Collagenase by Calcium Ions

2012 ◽  
Vol 78 (16) ◽  
pp. 5839-5844 ◽  
Author(s):  
Naomi Ohbayashi ◽  
Noriko Yamagata ◽  
Masafumi Goto ◽  
Kimiko Watanabe ◽  
Youhei Yamagata ◽  
...  
Keyword(s):  
Melting Point ◽  
Structural Stability ◽  
Conformational Changes ◽  
Full Length ◽  
Size Exclusion ◽  
Content Type ◽  
Protein Size ◽  
Exclusion Chromatography ◽  
Domain Arrangement ◽  
Thermal Melting

ABSTRACTThe clostridial collagenases G and H are multidomain proteins. For collagen digestion, the domain arrangement is likely to play an important role in collagen binding and hydrolysis. In this study, the full-length collagenase H protein fromClostridium histolyticumwas expressed inEscherichia coliand purified. The N-terminal amino acid of the purified protein was Ala31. The expressed protein showed enzymatic activity against azocoll as a substrate. To investigate the role of Ca2+in providing structural stability to the full-length collagenase H, biophysical measurements were conducted using the recombinant protein. Size exclusion chromatography revealed that the Ca2+chelation by EGTA induced interdomain conformational changes. Dynamic light scattering measurements showed an increase in the percent polydispersity as the Ca2+was chelated, suggesting an increase in protein flexibility. In addition to these conformational changes, differential scanning fluorimetry measurements revealed that the thermostability was decreased by Ca2+chelation, in comparison with the thermal melting point (Tm). The melting point changed from 54 to 49°C by the Ca2+chelation, and it was restored to 54°C by the addition of excess Ca2+. These results indicated that the interdomain flexibility and the domain arrangement of full-length collagenase H are reversibly regulated by Ca2+.

scholarly journals Impact of Botrytis cinerea Contamination on the Characteristics and Foamability of Yeast Macromolecules Released during the Alcoholic Fermentation of a Model Grape Juice

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 472
Author(s):  
Richard Marchal ◽  
Thomas Salmon ◽  
Ramon Gonzalez ◽  
Belinda Kemp ◽  
Céline Vrigneau ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Alcoholic Fermentation ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Grape Juice ◽  
Size Exclusion ◽  
Periodic Acid Schiff ◽  
Exclusion Chromatography ◽  
The Impact

Botrytis cinerea is a fungal pathogen responsible for the decrease in foamability of sparkling wines. The proteolysis of must proteins originating from botrytized grapes is well known, but far less information is available concerning the effect of grape juice contamination by Botrytis. The impact from Botrytis on the biochemical and physico-chemical characteristics of proteins released from Saccharomyces during alcoholic fermentation remains elusive. To address this lack of knowledge, a model grape juice was inoculated with three enological yeasts with or without the Botrytis culture supernatant. Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALLS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques (AgNO3 and periodic acid Schiff staining) was used in the study. When Botrytis enzymes were present, a significant degradation of the higher and medium MW molecules released by Saccharomyces was observed during alcoholic fermentation whilst the lower MW fraction increased. For the three yeast strains studied, the results clearly showed a strong decrease in the wine foamability when synthetic musts were inoculated with 5% (v/v) of Botrytis culture due to fungus proteases.

scholarly journals The use of blue native PAGE in the evaluation of membrane protein aggregation states for crystallization

2008 ◽  
Vol 41 (6) ◽  
pp. 1150-1160 ◽  
Author(s):  
Jichun Ma ◽  
Di Xia
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Quality ◽  
Structural Information ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Exclusion ◽  
Native Page ◽  
Exclusion Chromatography ◽  
Blue Native

Crystallization has long been one of the bottlenecks in obtaining structural information at atomic resolution for membrane proteins. This is largely due to difficulties in obtaining high-quality protein samples. One frequently used indicator of protein quality for successful crystallization is the monodispersity of proteins in solution, which is conventionally obtained by size exclusion chromatography (SEC) or by dynamic light scattering (DLS). Although useful in evaluating the quality of soluble proteins, these methods are not always applicable to membrane proteins either because of the interference from detergent micelles or because of the requirement for large sample quantities. Here, the use of blue native polyacrylamide gel electrophoresis (BN–PAGE) to assess aggregation states of membrane protein samples is reported. A strong correlation is demonstrated between the monodispersity measured by BN–PAGE and the propensity for crystallization of a number of soluble and membrane protein complexes. Moreover, it is shown that there is a direct correspondence between the oligomeric states of proteins as measured by BN–PAGE and those obtained from their crystalline forms. When applied to a membrane protein with unknown structure, BN–PAGE was found to be useful and efficient for selecting well behaved proteins from various constructs and in screening detergents. Comparisons of BN–PAGE with DLS and SEC are provided.

scholarly journals Characterization of a Novel β-Galactosidase fromBifidobacterium adolescentis DSM 20083 Active towards Transgalactooligosaccharides

2000 ◽  
Vol 66 (4) ◽  
pp. 1379-1384 ◽  
Author(s):  
Katrien M. J. Van Laere ◽  
Tjakko Abee ◽  
Henk A. Schols ◽  
Gerrit Beldman ◽  
Alphons G. J. Voragen
Keyword(s):  
Size Exclusion Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Extract ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Bifidobacterium Adolescentis ◽  
Exclusion Chromatography

ABSTRACT This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel β-galactosidase (β-Gal II). In cells grown on TOS, in addition to the lactose-degrading β-Gal (β-Gal I), another β-Gal (β-Gal II) was detected and it showed activity towards TOS but not towards lactose. β-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. β-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. β-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35°C. The enzyme showed highestV max values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. β-Gal II was active towards β-galactosyl residues that were 1→4, 1→6, 1→3, and 1↔1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.

scholarly journals Purification and partial biochemical characterization of normal human interleukin 1.

1984 ◽  
Vol 160 (3) ◽  
pp. 772-787 ◽  
Author(s):  
J A Schmidt
Keyword(s):  
High Performance ◽  
Biochemical Characterization ◽  
Interleukin 1 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Thymocyte Proliferation ◽  
Exclusion Chromatography ◽  
Normal Human

A protocol for the rapid, efficient purification of the major charged species of human interleukin 1 (IL-1) has been developed using high performance anion exchange and size exclusion chromatography. The isolated material is pure as determined by sodium dodecyl sulfate (SDS) gradient polyacrylamide gel electrophoresis (PAGE) and analytical isoelectric focusing (IEF). The molecular weight of the purified material is 15,000 and the isoelectric point (pI) is 6.8, values that are in good agreement with those previously reported for human IL-1. 10(-10) M concentrations of the purified material give half-maximal stimulation in the thymocyte proliferation assay. Amounts of IL-1 sufficient for receptor studies and detailed biochemical analysis can now be produced on a regular basis.

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