Monitoring of conformational change in maltose binding protein using split green fluorescent protein

Jeong, Jinyoung; Kim, Sang Kyu; Ahn, Jun Hyoung; Park, Kyoungsook; Jeong, Eunju; Kim, Moonil; Chung, Bong Hyun

doi:10.1016/j.bbrc.2005.11.056

Cited by 35 publications

(31 citation statements)

References 19 publications

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“…[ 23,24,29,30 ] Subsequently, a suite of proteins has been found to demonstrate such fragment reconstitution behavior, such as ubiquitin, dihydroforlate reductase, and green fl uorescent protein. [ 19,20,22,[29][30][31] Despite the wide ubiquity of proteins able to undergo fragment reconstitution and their application in bioassays, surprisingly, use of fragment reconstitution has not been…”

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confidence: 99%

“…[19][20][21][22][23][24] The small protein GB1 is a good example of such a protein that is capable of undergoing spontaneous reconstitution when split into two fragments [25][26][27][28] ( Figure 1 A). This assisted protein fragment reconstitution property has been used to develop assays to probe protein-protein interactions in vitro as well as in living cells.…”

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confidence: 99%

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Protein Fragment Reconstitution as a Driving Force for Self‐Assembling Reversible Protein Hydrogels

Kong

2015

Adv Funct Materials

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Due to their potential biomedical applications, protein-based hydrogels have attracted considerable interest. Although various methods have been developed to engineer self-assembling, physically-crosslinked protein hydrogels, exploring novel driving forces to engineer such hydrogels remains challenging. Protein fragment reconstitution, also known as fragment complementation, is a self-assembling mechanism by which protein fragments can reconstitute the folded conformation of the native protein when split into two halves. Although it has been used in biophysical studies and bioassays, fragment reconstitution has not been explored for hydrogel construction. Using a small protein GL5 as a model, which is capable of fragment reconstitution to reconstitute the folded GL5 spontaneously when split into two halves, GN and GC, we demonstrate that protein fragment reconstitution is a novel driving force for engineering self-assembling reversible protein hydrogels. Fragment reconstitution between GN and GC crosslinks GN and GC-containing proteins into self-assembling reversible protein hydrogels. These novel hydrogels show temperature-dependent reversible sol-gel transition, and excellent property against erosion in water. Since many proteins can undergo fragment reconstitution, we anticipate that such fragment reconstitution may offer a general driving force for engineering protein hydrogels from a variety of proteins, and thus signifi cantly expanding the 'toolbox' currently available in the fi eld of biomaterials. DOI IntroductionProtein hydrogels have attracted considerable interest over the last two decades due to their potential applications in a wide range of biomedical applications, including drug delivery, as artifi cial extracellular matricies and regenerative medicines. [1][2][3] Among engineered protein hydrogels, self-assembling hydrogels are of particular interest. [ 2,4,5 ] Inspired by the pioneering work of Tirrell and co-workers, [ 6 ] researchers have been developing novel strategies for engineering self-assembling protein hydrogels [4][5][6][7][8] Adv. Funct explored in engineering self-assembling protein hydrogels or supramolecular protein polymers. For the two split fragments A and B that can undergo fragment reconstitution to reconstitute the native folded structure, it should be feasible to crosslink multifunctional polymers (S-A) n and (S-B) m into a polymer network and form a hydrogel, where S represents spacers, A and B are the two fragments capable of reconstituting, and m and n represent the number of repeating units (Figure 1 B). Using fragments from a loop elongation variant of GB1 as a model system, we demonstrate the proof-of-principle of using protein fragment reconstitution as a driving force to engineer self-assembling protein hydrogels. Using different protein designs, we demonstrate both two-component as well as singlecomponent approaches to engineer protein fragment reconstitution-driven protein hydrogels.

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Protein Fragment Reconstitution as a Driving Force for Self‐Assembling Reversible Protein Hydrogels

Kong

2015

Adv Funct Materials

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“…Based on this split GFP technology, we previously described a split GFP reconstitution assay which could be utilized for the monitoring of conformationally-altered proteins (Jeong et al 2006). In that approach, we elected to utilize the maltose binding protein (MBP) as a model protein for conformational change.…”

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confidence: 99%

Detection of glucose-induced conformational change in hexokinase II using fluorescence complementation assay

et al. 2007

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Conformational changes in hexokinase are induced by its binding to glucose, thus providing an excellent example of an 'induced fit' model. To observe glucose-induced fluorescence restoration in hexokinase II using split-enhanced, green fluorescent protein (EGFP) in a process involving the reconstitution of split EGFP, E. coli cells expressing the chimeric NEGFP:HXK:CEGFP recombinant protein were treated with glucose and visualized via fluorescence read-outs. The reconstituted EGFP generated a strong fluorescence upon glucose stimulation of the bacteria. Moreover, the fluorescence intensity became stronger with increasing glucose up to 10 mM, with a maximum being observed after 60 min in a time- and concentration-dependent manner. Conformational changes associated with glucose-induced fit in human hexokinase II can thus be monitored successfully in vivo via fluorescence reconstitution assays, coupled with a quick and easy fluorescent read-out protocol.

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“…[10] As some of them were already applied to measure conformations of proteins, the continuous recording of the structural state of a protein may become possible as well. [44,49] …”

Section: Altered Protein Conformationsmentioning

confidence: 98%

Split‐Ubiquitin and the Split‐Protein Sensors: Chessman for the Endgame

Müller

Johnsson

2008

ChemBioChem

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Monitoring of conformational change in maltose binding protein using split green fluorescent protein

Cited by 35 publications

References 19 publications

Protein Fragment Reconstitution as a Driving Force for Self‐Assembling Reversible Protein Hydrogels

Protein Fragment Reconstitution as a Driving Force for Self‐Assembling Reversible Protein Hydrogels

Detection of glucose-induced conformational change in hexokinase II using fluorescence complementation assay

Split‐Ubiquitin and the Split‐Protein Sensors: Chessman for the Endgame

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