Detection of conformationally changed MBP using intramolecular FRET.
The principal objective of this study was to explore protein conformational changes using fluorescence resonance energy transfer (FRET) technology. Maltose binding protein (MBP) was adopted as a target model, due to its well-characterized structure and ligand specificity. To the best of our knowledge, this is the first report to provide information regarding the biological distance between the two lobes of MBP upon maltose binding. For the FRET pair, ECFP and EYFP were used as the donor and the acceptor, and were linked genetically to the C-terminal and N-terminal regions of MBP (ECFP:MBP:EYFP), respectively. After the FRET reaction, maltose-treated MBP was shown to exhibit a considerable energy transfer (FRET efficiency (E)= approximately 0.11, Distance (D)= approximately 6.93 nm) at the ensemble level, which was regarded as reflective of the increase in donor quenching and the upshift in acceptor emission intensity, thereby suggesting that the donor and the acceptor had been brought close together as the result of structural alterations in MBP. However, upon glucose treatment, no FRET phenomenon was detected, thereby implying the specificity of interaction between MBP and maltose. The in vitro FRET results were also confirmed via the acceptor photobleaching method. Therefore, our data showed that maltose-stimulated conformational changes of MBP could be measured by FRET, thereby providing biological information, including the FRET efficiency and the intramolecular distance.
Park
Kyoungsook
K
BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea.
Lee
Lan Hee
LH
Shin
Yong-Beom
YB
Yi
So Yeon
SY
Kang
Yong-Won
YW
Sok
Dai-Eun
DE
Chung
Jin Woong
JW
Chung
Bong Hyun
BH
Kim
Moonil
M
D002352:Carrier Proteins / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008320:Maltose / Q000737:chemistry; D058619:Maltose-Binding Proteins; D011487:Protein Conformation
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D008320:Maltose