کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
866293 1470959 2015 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Ratiometric analyses at critical temperatures can magnify the signal intensity of FRET-based sugar sensors with periplasmic binding proteins
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Ratiometric analyses at critical temperatures can magnify the signal intensity of FRET-based sugar sensors with periplasmic binding proteins
چکیده انگلیسی


• We examined the critical temperature effect on FRET-based sensors.
• The FRET ratio change of maltose sensor was dramatically increased at critical temperature.
• The temperature effect was also observed in other FRET-base sugar sensors.
• The proposed mechanism is thermal relaxation affecting ligand-binding states.

Fluorescence resonance energy transfer (FRET)-based sensors transduce ligand recognition into a change in the fluorophore spectrum, as ligand binding alters the distance between and orientation of two fluorescent proteins. Here, we report a dramatic increase in the signal intensity of FRET-based sugar sensors with bacterial periplasmic binding proteins (PBPs) in the binding moiety, by increasing the analysis temperature, usually higher than 50 °C. The increased signal intensity results from a sudden decrease in background signal at critical temperatures, while recovering the maximum FRET ratios in the presence of ligands. When tested with a maltose sensor using a maltose-binding protein as the binding moiety, the FRET ratio at the critical temperature, 55 °C, was 17-fold higher than at ambient temperatures. Similar effects were observed using analogous sensors for allose, arabinose, and glucose, providing highly dynamic and quantitative ratio changes at the critical temperatures. The proposed mechanism underlying the signal improvement is thermal relaxation of the binding proteins at the critical temperature; this hypothesis was supported by the results of intrinsic tryptophan fluorescence and circular dichroism experiments. In summary, this study shows that the conformational relaxation of proteins under specific conditions can be leveraged for highly sensitive and rapid measurements of ligands using FRET-based sensors.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biosensors and Bioelectronics - Volume 72, 15 October 2015, Pages 37–43
نویسندگان
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