scholarly journals Spermidine Binding to the Acetinobacter baumannii Efflux Protein AceI Observed by Near-UV Synchrotron Radiation Circular Dichroism Spectroscopy

2022 ◽  
Author(s):  
Simon Patching
Keyword(s):  
Circular Dichroism ◽  
Synchrotron Radiation ◽  
Antimicrobial Agents ◽  
Foodborne Pathogen ◽  
Multidrug Efflux ◽  
Experimental Conditions ◽  
E Coli ◽  
Plasmid Construct ◽  
Hospital Acquired ◽  
Circular Dichroïsm

The aim of this work was to test polyamines as potential natural substrates of the Acinetobacter baumannii chlorhexidine efflux protein AceI using near-UV synchrotron radiation circular dichroism (SRCD) spectroscopy. The Gram-negative bacterium A. Baumannii is a leading cause of hospital-acquired infections and an important foodborne pathogen. A. Baumannii strains are becoming increasingly resistant to antimicrobial agents, including the synthetic antiseptic chlorhexidine. AceI was the founding member of the recently recognised PACE family of bacterial multidrug efflux proteins. Using the plasmid construct pTTQ18-aceI(His6) containing the A. Baumannii aceI gene directly upstream from a His6-tag coding sequence, expression of AceI(His6) was amplified in E. coli BL21(DE3) cells. Near-UV (250-340 nm) SRCD measurements were performed on detergent-solubilised and purified AceI(His6) at 20 °C. Sample and SRCD experimental conditions were identified that detected binding of the triamine spermidine to AceI(His6). In a titration with spermidine (0-10 mM) this binding was saturable and fitting of the curve for the change in signal intensity produced an apparent binding affinity (KD) of 3.97 +/- 0.45 mM. These SRCD results were the first experimental evidence obtained for polyamines as natural substrates of PACE proteins.

scholarly journals Investigation by Synchrotron Radiation Circular Dichroism of the Secondary Structure Evolution of Pepsin under Oxidative Environment

Foods ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 998
Author(s):  
Laetitia Théron ◽  
Aline Bonifacie ◽  
Jérémy Delabre ◽  
Thierry Sayd ◽  
Laurent Aubry ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Synchrotron Radiation ◽  
Secondary Structure ◽  
Proteolytic Activity ◽  
Digestive Enzyme ◽  
Structure Evolution ◽  
Food Protein ◽  
Maldi Tof ◽  
Kinetics Of ◽  
Circular Dichroïsm

Food processing affects the structure and chemical state of proteins. In particular, protein oxidation occurs and may impair protein properties. These chemical reactions initiated during processing can develop during digestion. Indeed, the physicochemical conditions of the stomach (oxygen pressure, low pH) favor oxidation. In that respect, digestive proteases may be affected as well. Yet, very little is known about the link between endogenous oxidation of digestive enzymes, their potential denaturation, and, therefore, food protein digestibility. Thus, the objective of this study is to understand how oxidative chemical processes will impact the pepsin secondary structure and its hydrolytic activity. The folding and unfolding kinetics of pepsin under oxidative conditions was determined using Synchrotron Radiation Circular Dichroism. SRCD gave us the possibility to monitor the rapid kinetics of protein folding and unfolding in real-time, giving highly resolved spectral data. The proteolytic activity of control and oxidized pepsin was investigated by MALDI-TOF mass spectrometry on a meat protein model, the creatine kinase. MALDI-TOF MS allowed a rapid evaluation of the proteolytic activity through peptide fingerprint. This study opens up new perspectives by shifting the digestion paradigm taking into account the gastric digestive enzyme and its substrate.

scholarly journals Effects of Trehalose on Thermodynamic Properties of Alpha-synuclein Revealed through Synchrotron Radiation Circular Dichroism

Biomolecules ◽  
2015 ◽  
Vol 5 (2) ◽  
pp. 724-734 ◽  
Author(s):  
Paolo Ruzza ◽  
Rohanah Hussain ◽  
Barbara Biondi ◽  
Andrea Calderan ◽  
Isabella Tessari ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Synchrotron Radiation ◽  
Thermodynamic Properties ◽  
Alpha Synuclein ◽  
Circular Dichroïsm

Circular Dichroism and Synchrotron Radiation Circular Dichroism Applications to Biomaterials

2019 ◽  
pp. 147-172
Author(s):  
Rohanah Hussain ◽  
Tamás Jávorfi ◽  
Charlotte S. Hughes ◽  
Giuliano Siligardi
Keyword(s):  
Circular Dichroism ◽  
Synchrotron Radiation ◽  
Circular Dichroïsm

scholarly journals EfrAB, an ABC Multidrug Efflux Pump in Enterococcus faecalis

2003 ◽  
Vol 47 (12) ◽  
pp. 3733-3738 ◽  
Author(s):  
Eun-Woo Lee ◽  
M. Nazmul Huda ◽  
Teruo Kuroda ◽  
Tohru Mizushima ◽  
Tomofusa Tsuchiya
Keyword(s):  
Enterococcus Faecalis ◽  
Antimicrobial Agents ◽  
Efflux Pump ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Multidrug Efflux ◽  
Chromosomal Dna ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Dna Fragment

ABSTRACT A DNA fragment responsible for resistance to antimicrobial agents was cloned from the chromosomal DNA of Enterococcus faecalis ATCC 29212 by using drug-hypersensitive mutant Escherichia coli KAM32 as a host cell. Cells of E. coli KAM32 harboring a recombinant plasmid (pAEF82) carrying the DNA fragment became resistant to many structurally unrelated antimicrobial agents, such as norfloxacin, ciprofloxacin, doxycycline, acriflavine, 4′,6-diamidino-2-phenylindole, tetraphenylphosphonium chloride, daunorubicin, and doxorubicin. Since the sequence of the whole genome of E. faecalis is known, we sequenced several portions of the DNA insert in plasmid pAEF82 and identified two open reading frames within the insert. We designated the genes efrA and efrB. A search of the deduced amino acid sequences of EfrA and EfrB revealed that they are similar to each other and that they belong to the ATP-binding cassette (ABC) family of multidrug efflux transporters. Transformed E. coli KAM32 cells harboring efrAB showed energy-dependent efflux of acriflavine. The efflux activity was inhibited by reserpine, verapamil, and sodium-o-vanadate, known inhibitors of ABC efflux pumps.

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