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Title 

Ratiometric analyses at critical temperatures can magnify the signal intensity of FRET-based sugar sensors with periplasmic binding proteins

 

분석온도 조절을 통한 당 감지 센서의 감지능 향상

Authors 

Jongsik GamJae-Seok HaHaseong KimDae-Hee LeeJ LeeSeung Goo Lee

Publisher 

Elsevier

Issue Date 

2015

Citation 

Biosensors & Bioelectronics, vol. 72, no. 1, pp. 37-43

Keywords 

Conformation relaxationCritical temperatureFluorescence resonance energy transferMolecular biosensor

Abstract 

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.

ISSN 

0956-5663

Link 

http://dx.doi.org/10.1016/j.bios.2015.04.083

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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