期刊
BIOSENSORS & BIOELECTRONICS
卷 72, 期 -, 页码 37-43出版社
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2015.04.083
关键词
Molecular biosensor; Fluorescence resonance energy transfer; Critical temperature; Conformation relaxation
类别
资金
- Intelligent Synthetic Biology Center of Global Frontier Project [2011-0031944]
- Next-Generation Biogreen 21 Program [PJ009524]
- Bio-industry Technology Development Program [310006-5]
- KRIBB Research Initiative Program
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 degrees 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 degrees 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. (C) 2015 Elsevier B.V. All rights reserved.
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