Monitoring of conformational change in maltose binding protein using split green fluorescent protein.
In this study, we describe a novel method for the detection of conformational changes in proteins, which is predicated on the reconstitution of split green fluorescent protein (GFP). We employed fluorescence complementation assays for the monitoring of the conformationally altered proteins. In particular, we used maltose binding protein (MBP) as a model protein, as MBP undergoes a characteristic hinge-twist movement upon substrate binding. The common feature of this approach is that GFP, as a reporter protein, splits into two non-fluorescent fragments, which are genetically fused to the N- and C-termini of MBP. Upon binding to maltose, the chromophores move closer together, resulting in the generation of fluorescence. This split GFP method also involves the reconstitution of GFP, which is determined via observations of the degree to which fluorescence intensity is restored. As a result, reconstituted GFP has been observed to generate fluorescence upon maltose binding in vitro, thereby allowing for the direct detection of changes in fluorescence intensity in response to maltose, in a concentration- and time-dependent fashion. Our findings showed that the fluorescence complementation assay can be used to monitor the conformational alterations of a target protein, and this ability may prove useful in a number of scientific and medical applications.
Jeong
Jinyoung
J
BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yuseong, Daejeon 305-600, Republic of Korea.
Kim
Sang Kyu
SK
Ahn
Junhyoung
J
Park
Kyoungsook
K
Jeong
Eun-Ju
EJ
Kim
Moonil
M
Chung
Bong Hyun
BH
D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D009068:Movement; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins