ArticleJournal of amino acids2013
Binding Stoichiometry of a Recombinant Selenophosphate Synthetase with One Synonymic Substitution E197D to a Fluorescent Nucleotide Analog of ATP, TNP-ATP.
Article in Journal of amino acids, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 1 paper.
What it found
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.
Who cites it
1 citing paper in PubMed, 2 citations in OpenAlex.
- A review of TNP-ATP in protein binding studies: benefits and pitfalls.Biophysical reports · 2021Review
Corrections and comments
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Authors and funding
4 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The transformation of the strain DH5α (TM)-T1(R) 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 × 10(8) CFU/ μ g 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.
Identifiers
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Registered trials
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