کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5370580 1503890 2017 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Towards understanding the E. coli PNP binding mechanism and FRET absence between E. coli PNP and formycin A.
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی تئوریک و عملی
پیش نمایش صفحه اول مقاله
Towards understanding the E. coli PNP binding mechanism and FRET absence between E. coli PNP and formycin A.
چکیده انگلیسی


- Incorporation of the dissociation constant is necessary in FRET calculations.
- aromatic amino acid at the position 159 is the key residue in the nucleoside binding.
- Tyr−/Tyr* inhibits or quenches FRET from other Tyr to FA.
- Tyr159 probably enhances the binding efficiency of FA.
- Phosphate buffer at pH 8.3 seems to restore (partially) FRET in PNPF59Y-FA complexes.

The aim of this study is threefold: (1) augmentation of the knowledge of the E. coli PNP binding mechanism; (2) explanation of the previously observed 'lack of FRET' phenomenon and (3) an introduction of the correction (modified method) for FRET efficiency calculation in the PNP-FA complexes.We present fluorescence studies of the two E. coli PNP mutants (F159Y and F159A) with formycin A (FA), that indicate that the aromatic amino acid is indispensable in the nucleotide binding, additional hydroxyl group at position 159 probably enhances the strength of binding and that the amino acids pair 159-160 has a great impact on the spectroscopic properties of the enzyme. The experiments were carried out in hepes and phosphate buffers, at pH 7 and 8.3. Two methods, a conventional and a modified one, that utilizes the dissociation constant, for calculations of the energy transfer efficiency (E) and the acceptor-to-donor distance (r) between FA and the Tyr (energy donor) were employed. Total difference spectra were calculated for emission spectra (λex 280 nm, 295 nm, 305 nm and 313 nm) for all studied systems. Time-resolved techniques allowed to conclude the existence of a specific structure formed by amino acids at positions 159 and 160. The results showed an unexpected pattern change of FRET in the mutants, when compared to the wild type enzyme and a probable presence of a structure created between 159 and 160 residue, that might influence the binding efficiency. Additionally, we confirmed the indispensable role of the modification of the FRET efficiency (E) calculation on the fraction of enzyme saturation in PNP-FA systems.

Graphical Abstract239

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biophysical Chemistry - Volume 230, November 2017, Pages 99-108
نویسندگان
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