Monitoring of conformational change in maltose binding protein using split green fluorescent protein q Jinyoung Jeong a,b , Sang Kyu Kim a,b , Junhyoung Ahn a,b , Kyoungsook Park a , Eun-Ju Jeong a , Moonil Kim a,b, * , Bong Hyun Chung a,b, * a BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yuseong, Daejeon 305-600, Republic of Korea b University of Science and Technology (UST), Yuseong, Daejeon 305-333, Republic of Korea Received 8 November 2005 Available online 18 November 2005 Abstract In this study, we describe a novel method for the detection of conformational changes in proteins, which is predicated on the recon- stitution of split green fluorescent protein (GFP). We employed fluorescence complementation assays for the monitoring of the conform- ationally 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. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Maltose binding protein; Green fluorescent protein; Conformational change Green fluorescent protein (GFP), a small (238 amino acids) 28 kDa protein, with a characteristic barrel-like structure composed of 11 b-sheets slightly twisted around a chromophore [1], has been previously employed in a vari- ety of scientific applications, and has proven invaluable in studies of protein function in cells, as well as a host of cel- lular processes. The most notable applications of GFP are in the areas of molecular and cell biology, in which GFP is utilized as a reporter molecule, a fluorescent tag for fusion proteins, or as a biosensor in the fluorescence resonance energy transfer (FRET) technique [2]. Protein–protein interactions can also be monitored using the protein fragment complementation approach, in which an enzyme is split into two inactive fragments, which are then fused to their respective interaction partners [3]. Enzymes used in this approach include dihydrofolate reductase (DHFR) [4], b-galactosidase [5], and lactamase [6]. When these proteins interact, the split fragments com- plement each other and the function of the relevant enzyme is restored. These split-enzyme systems have been previous- ly applied successfully to the monitoring of protein–protein interactions involving proteins that are anchored to the plasma membrane [7,8]. Recently, a protein fragment complementation method based on the split yellow fluorescence protein (YFP) has 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.11.056 q Abbreviations: GFP, green fluorescent protein; MBP, maltose binding protein; IPTG, isopropyl b-D-thiogalactopyranoside; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. * Corresponding authors. Fax: +82 42 879 8594 (M. Kim). E-mail addresses: kimm@kribb.re.kr (M. Kim), chungbh@kribb.re.kr (B.H. Chung). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 339 (2006) 647–651 BBRC