![](/images/graphics-bg.png)
Binding Stoichiometry of a Recombinant Selenophosphate Synthetase with One Synonymic Substitution E197D to a Fluorescent Nucleotide Analog of ATP, TNP-ATP
Joint Authors
Preobrazhenskaya, Y. V.
Stenko, A. I.
Shvarts, M. V.
Lugovtsev, V. Y.
Source
Issue
Vol. 2013, Issue 2013 (31 Dec. 2013), pp.1-8, 8 p.
Publisher
Hindawi Publishing Corporation
Publication Date
2013-01-30
Country of Publication
Egypt
No. of Pages
8
Main Subjects
Abstract EN
The transformation of the strain DH5αTM-T1R with plasmid vector pET11a containing the cloned gene of bacterial selenophosphate synthetase (SPS), selD, from the E.
coli BL21-Gold (DE3) strain gives an overproducing strain of SPS with one synonymic substitution, E197D.
The transformation efficiency was estimated as 8 × 108 CFU/μg plasmid DNA.
28 mg of highly purified preparation of recombinant SPS capable of binding TNP-ATP was eluted from DEAE-Sephadex column in amount of 15 % from the total soluble protein in crude extract.
The fluorescent derivative of ATP, 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate (TNP-ATP), was used as a synthetic analog of the substrate for the monitoring and quantitative analysis of the functional activity of SPS.
The non-linear regression analysis of the saturation curve of TNP-ATP binding to D197 SPS with GraphPad Prism software fits to a model with 2 distinct binding sites with KDs different in order.
The SPS existence in a form of tetramer in given reaction conditions, in accordance with the concentration stoichiometry of 4 moles of TNP-ATP to 1 mole of recombinant protein, is being discussed.
The tetramer structure was predicted with molecular modelling software YASARA and modelled in vacuum using steepest descent minimization energy method.
We hypothesize here the recombinant SPS exists as a dimer in solution with two active sites capable of ATP binding in each subunit.
American Psychological Association (APA)
Preobrazhenskaya, Y. V.& Stenko, A. I.& Shvarts, M. V.& Lugovtsev, V. Y.. 2013. Binding Stoichiometry of a Recombinant Selenophosphate Synthetase with One Synonymic Substitution E197D to a Fluorescent Nucleotide Analog of ATP, TNP-ATP. Journal of Amino Acids،Vol. 2013, no. 2013, pp.1-8.
https://search.emarefa.net/detail/BIM-513500
Modern Language Association (MLA)
Preobrazhenskaya, Y. V.…[et al.]. Binding Stoichiometry of a Recombinant Selenophosphate Synthetase with One Synonymic Substitution E197D to a Fluorescent Nucleotide Analog of ATP, TNP-ATP. Journal of Amino Acids No. 2013 (2013), pp.1-8.
https://search.emarefa.net/detail/BIM-513500
American Medical Association (AMA)
Preobrazhenskaya, Y. V.& Stenko, A. I.& Shvarts, M. V.& Lugovtsev, V. Y.. Binding Stoichiometry of a Recombinant Selenophosphate Synthetase with One Synonymic Substitution E197D to a Fluorescent Nucleotide Analog of ATP, TNP-ATP. Journal of Amino Acids. 2013. Vol. 2013, no. 2013, pp.1-8.
https://search.emarefa.net/detail/BIM-513500
Data Type
Journal Articles
Language
English
Notes
Includes bibliographical references
Record ID
BIM-513500