ORIGINAL RESEARCH PAPER
Detection of glucose-induced conformational changein hexokinase II using fluorescence complementation assay
Eun-Ju Jeong Æ Kyoungsook Park ÆHyou-Arm Joung Æ Chang-Soo Lee ÆDai-Wu Seol Æ Bong Hyun Chung Æ Moonil Kim
Received: 24 October 2006 / Revised: 22 December 2006 / Accepted: 23 December 2006 /Published online: 24 February 2007� Springer Science+Business Media B.V. 2007
Abstract 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 fluores-
cence 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 NEG-
FP:HXK:CEGFP recombinant protein were trea-
ted 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 fluores-
cence reconstitution assays, coupled with a quick
and easy fluorescent read-out protocol.
Keywords Conformational change �Fluorescence complementation assay �Glucose-induced fit � Hexokinase II �Split EGFP
Introduction
Hexokinases not only catalyze the first step in the
glycolytic pathway, but also senses glucose levels
in most organisms (Stulke and Hillen 1999;
Rolland and Sheen 2005). Mammalian genomes
encode for one low-affinity glucokinase (type IV)
and three high-affinity hexokinases (type I, II, and
III) (Wilson 2003). Given the amino acid similar-
ity of the type I–III isozymes between the N- and
C-terminal regions, the evolutionary hypothesis
of mammalian hexokinases that the type I–II
isozymes resulted from duplication and fusion of
a gene encoding an 50-kDa type IV hexokinase
have been suggested. For instance, the type I
hexokinase (hexokinase I) is a 100 kDa protein
with its C-terminal region possessing the catalytic
function, whereas the N-terminal was associated
with a regulatory role (Arora et al 1993).
Many lines of evidence suggest that conforma-
tional changes in hexokinase are induced by the
E.-J. Jeong � K. Park � H.-A. Joung � C.-S. Lee �B. H. Chung (&) � M. KimBioNanotechnology Research Center, KoreaResearch Institute of Bioscience and Biotechnology,P.O. Box 115, Yuseong, Daejeon 305-600,Republic of Koreae-mail: [email protected]
M. Kim (&)e-mail: [email protected]
D.-W. SeolDepartment of Surgery, University of PittsburghSchool of Medicine, Pittsburgh, PA 15261, USA
123
Biotechnol Lett (2007) 29:797–802
DOI 10.1007/s10529-007-9313-x
binding of glucose (Aleshin et al 1998; Kamata
et al 2004), mechanistically caused by the closing of
the cleft between the lobes into which the glucose
binds. This is an excellent example of the ‘induced
fit’ model. Recently, human hexokinase I has been
determined to adopt a dimeric form in the crystal
structure (Mulichak et al 1998; Rosano et al 1999).
Upon the binding of glucose to hexokinase, the
attendant movement and rotation of the small and
large subdomains cause the enzyme to adopt its
closed conformation. From the reported 3-D
structures of hexokinase I, a homology modeling
of the structure of human hexokinase II has been
also suggested, thereby showing that the N-terminal
half from one subunit parallel the C-terminal
domain of the second subunit in the dimeric
conformation (Pastorino and Hoek 2003).
In this study, in order to investigate in detail
the glucose-induced structural transition of hexo-
kinase II on the basis of its antiparallel dimeric
structure model, we employed a split-enhanced
green fluorescent protein (EGFP) complementa-
tion assay. Our results demonstrate that glucose-
induced conformational change in the dimeric
hexokinase II can be successfully monitored
in vivo using fluorescence reconstitution assays,
with a rapid and simple detection method.
Materials and methods
Construction of the pNEGFP:HXK:CEGFP
plasmid
In order to construct the pNEGFP:HXK:CEGFP
plasmid, the partial gene encoding for the EGFP
N-terminal fragment (1–157 amino acids) was
initially amplified with the 5¢ primer (CGG GAT
CCA TGG TGA GCA AGG GCG GAG) and the
3¢ primer (GGA ATT CTC CTC CTC CTC CGC
GCT TCT CGT TGG GGT C) via polymerase
chain reaction (PCR). The 5¢ and 3¢ termini were
designed to harbor the BamHI and EcoRI restric-
tion enzyme cleavage sites, respectively. In order to
generate the CEGFP fragment, the partial gene
encoding for the GFP C-terminal fragment (158–
238 amino acids) was amplified via PCR with the 5¢primer (CCC AAG CTT GGA GGA GGA GGA
GAT CAC ATG GTC CTG CTG) and the 3¢
primer (ATA AGA ATG CGG CCG CCA CAT
TGA TCC TAG CAG AG) using the HindIII and
NotI restriction enzyme cleavage sites, respec-
tively. The human type II hexokinase gene was
then PCR-amplified using the 5¢ primer (CGG
AAT TCA TGA TTG CCT CGC ATC TGC) and
the 3¢ primer (ACT AAG CTT TCG CTG TCC
AGC CTC ACG) with the EcoRI and HindIII
restriction enzyme cleavage sites, respectively. The
PCR products were then purified using a DNA
purification kit (Qiagen), and digested with the
indicated restriction enzymes. The resultant DNA
fragments were then ligated with the pET 28a
vector, using a ligation kit (Takara, Japan)
(pNEGFP:HXK:CEGFP), and cloned into the
pET 28 a vector using the BamHI and NotI sites,
thereby generating the NEGFP:HXK:CEGFP in-
frame fusion. The NEGFP:HXK:CEGFP fusion
gene was verified via DNA sequencing.
Expression of the recombinant
NEGFP:HXK:CEGFP protein
The pNEGFP:HXK:CEGFP construct was trans-
formed into E. coli DH5a. The ampicillin-resistant
colonies were screened via the DNA isolation of
3 ml overnight cultures, followed by restriction
mapping. Plasmid DNA was prepared and purified
using a QIAprep spin miniprep kit (Qiagen,
Germany). The plasmids were then transferred to
the expression host, E. coli BL21 (DE3) (Strata-
gene, CA), then plated onto LB plates. A single
colony from a fresh plate was picked and grown at
37�C in 3 ml Luria–Bertani (LB) broth containing
30 mg kanamycin/ml, to an OD600 of 0.6. This was
then inoculated into 100 ml LB broth containing
kanamycin. The cells were grown at 37�C with
shaking to an OD600 of 0.6. The cells were induced
with 1 mM IPTG, and grown at 18�C for 12 h with
shaking. The cells were then harvested by centri-
fugation (6,000 g for 10 min at 4�C).
SDS-PAGE and Western blotting analysis
The E. coli cells were harvested and suspended in
50 mM Tris/HCl buffer (pH 8.0). After cell
disruption by sonication, the soluble and insoluble
fractions were separated by centrifugation
(10 min at 10,000 g). The total cell lysates and
798 Biotechnol Lett (2007) 29:797–802
123
soluble and insoluble fractions were resolved on
10% (v/v) SDS gel and transferred onto nitrocel-
lulose membranes. Western blotting analysis was
conducted using antibodies against human Type
II hexokinase (Santa Cruz, CA).
In vivo visualization of glucose-induced
conformational changes in hexokinase II
For the detection of fluorescence, 3 ll of E. coli
cells expressing NEGFP:HXK:CEGFP protein,
either with or without IPTG treatment, were
spread onto a glass slide stuck to sticky tape, with
wells approx 3 mm diam and 100 lm deep to
compartmentalize to E. coli culture. The samples
were then treated with 10 mM glucose for 1 h at
room temperature. After 1 h, the fluorescence
images of the E. coli cells generating the chimeric
NEGFP:HXK:CEGFP protein were acquired
using a GenePix 4200A 488 nm laser, a micro-
array scanner (Axon Inc., CA).
Results and discussion
The rationale for the split fluorescent protein-
based monitoring of glucose-induced
conformational change in hexokinase II
Several lines of evidence appear to suggest that
the generation of fluorescence from partial GFP
fragments, which is attributed to reconstituted
GFP, might also be used to evaluate protein–
protein interactions (Ozawa et al 2001). Based on
this split GFP technology, we previously
described a split GFP reconstitution assay which
could be utilized for the monitoring of conform-
ationally-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. The split GFP method is
principally predicated on the restoration of fluo-
rescence effected when split fluorescent proteins
are brought back into contact with each other.
Upon stimulation with maltose, the two lobes of
MBP are brought closer together, which results in
the complementation of the two split GFP
fragments attached to the N- and C-termini of
MBP. This split GFP system is contingent on the
reconstitution of the NGFP and CGFP fragments,
which results in recovery of fluorescence.
In accordance with the same strategy, we have
attempted herein to evaluate the possibility of
monitoring the conformational alterations atten-
dant to the induced fit of human hexokinase II in
response to glucose stimulation, via the split
fluorescence protocol. This glucose-induced con-
formational alteration is generally referred to as
an ‘embracing mechanism’, which may require
hexokinase to embrace or surround its substrate,
such that the only way in which the substrate can
enter and leave the active site is for the enzyme to
open and close (Blow and Steitz 1970). In
particular, we employed a GFP-modified version,
with the alterations Ser 65–Thr and Phe 64–Leu,
referred to as enhanced green fluorescent protein
(EGFP), as a reporter. This selection was due to
the fact that EGFP produces a fluorescence 35
times more intense than that of wild-type GFP
(Kain and Ma 1999). The split EGFP strategy for
the detection of glucose-induced structural tran-
sition from open form to closed form in hexoki-
nase was based on the introduction of EGFP, as a
visible reporter molecule, which splits into two
non-fluorescent fragments, which are linked to
the N- and C-termini of hexokinase, respectively
(Fig. 1). As shown in Fig. 1, we hypothesized that
the exogamous chromophores would move closer
together when glucose bound to the dimeric
hexokinase II, thereby resulting in the comple-
mentation of the two exogamous split EGFP
fragments. In order to evaluate the veracity of this
hypothesis, the first step was to prepare the fusion
construct of two non-fluorescent fragments,
NEGFP (1–157 amino acids) and CEGFP (158–
238 amino acids), each of which are linked
genetically to the N- and C-terminal halves of
hexokinase II as a single structural molecule, as
described in Materials and Methods.
Expression of the chimeric
NEGFP:HXK:CEGFP protein
In order to monitor the glucose-induced confor-
mational alterations in hexokinase via the split
EGFP method, we initially constructed the
pNEGFP:HXK:CEGFP plasmid to express the
chimeric NEGFP:HXK:CEGFP recombinant pro-
Biotechnol Lett (2007) 29:797–802 799
123
tein (Fig. 2A). As a consequence of the construc-
tion of the chimeric NEGFP:HXK:CEGFP gene,
NEGFP was then fused to the N-terminal end of
hexokinase II, and CEGFP was attached to its
C-terminal end (Fig. 2A). Glucose binding would
then bring the exogamous N- and C-termini of
hexokinase II in a dimeric structure into closer
physical proximity, thereby inducing a subsequent
change in the intensity of fluorescence. For the
expression of the recombinant NEGFP:HXK:CE-
GFP protein, the pNEGFP:HXK:CEGFP plas-
mids were then transformed into the expression
host, E. coli BL21 (DE3). As shown in Fig. 2B,
after the induction of IPTG, we noted an obvious
extra band, which corresponded to a molecular
mass of approximately 126 kDa, consistent with
the expected molecular weight of NEG-
FP:HXK:CEGFP, as compared to the bands
observed with the E. coli strain harboring the
pET 28a control plasmid. The expressed protein
was then validated via Western blotting using the
polyclonal antibody of hexokinase II (Fig. 2C).
Then, E. coli cells expressing the chimeric
NEGFP:HXK:CEGFP recombinant protein were
treated with glucose, and visualized further using
a fluorescence read-out for the detection of
structural alterations associated with the glu-
cose-induced fit in hexokinase II.
glucose
Closed conformation
Open conformation
C2
N2
L
N1
L
C1
NEGFP NEGFP
CEGFP
Glycinelinker(GGGG)
C2
N2
L
C1
N1
L
CEGFP
EGFP EGFP
Fig. 1 Schematic diagram of glucose-induced conforma-tional changes in hexokinase II via split EGFP comple-mentation assay. The N- and C-terminal halves of the splitEGFP were fused to the N- and C-termini of the dimerichexokinase II molecule. Upon glucose binding, theexogamous chromophores moved closer together, result-ing in the recovery of EGFP fluorescence. Peptide glycinelinker sequences are shown at the top. N1, N-terminaldomain 1; N2, N-terminal domain 2; C1, C-terminaldomain 1; C2, C-terminal domain 2; L, Linker domain
lacIkan
ori
f1 origin
pET28a-pET28a-NEGFP:HXK:CEGFPNEGFP:HXK:CEGFP
6×His
NEGFP
BamHI NotIGlycinelinker (GGGG)
CEGFPHexokinase
75 -
kDa M 1 2
100 -
150 -1 2
A
B C
Fig. 2 Construction of the pNEGFP:HXK:CEGFP plas-mid and expression of chimeric NEGFP:HXK:CEGFPrecombinant protein. (A) The map of recombinant plasmidpNEGFP:HXK:CEGFP for expression of NEGFP:HXK:CEGFP. kan, kanamycin resistant gene; ori, origin ofreplication; lac I, lactose repressor gene. (B) SDS-PAGEanalysis of recombinant NEGFP:HXK:CEGFP protein. M,protein marker; 1, total cell lysates of non-induced BL21(DE3); 2, total cell lysates of IPTG-induced BL21 (DE3).The arrow indicates the expressed NEGFP:HXK:CEGFPprotein. (C) This identity was confirmed via Westernblotting using the polyclonal antibody of hexokinase II
800 Biotechnol Lett (2007) 29:797–802
123
Fluorescence complementation assay for the
monitoring of conformational transitions in
hexokinase II in vivo
Visualization of glucose-induced conformational
changes in hexokinase II using fluorescence com-
plementation assay (see methods), is shown in
Fig. 3. Cells harboring the pNEGFP:HXK:CE-
GFP plasmid to express the chimeric protein
emitted a strong green fluorescence upon addition
of 10 mM glucose. Cells to which glucose had not
been added manifested no detectable fluores-
cence. In order to confirm whether the fluores-
cence observed in the glucose-treated cells was,
indeed, arising from the addition of glucose, the
transformed E. coli cells were treated with
10 mM maltose as a negative control. As is
shown in Fig. 3, although some fluorescence was
observed in response to maltose, this was a
minimally detectable signal (Fig. 3). This raises
the question as to whether perhaps a small
amount of glucose exists in the maltose solution
as an impurity.
Next, we attempted to assess glucose-depen-
dent alterations in the fluorescence intensity of
NEGFP:HXK:CEGFP-expressing E. coli cells
using a GenePix 4200A laser scanner (Axon
Inc., CA). As is shown in Fig. 4A, glucose-
induced conformational transitions in hexokinase
II caused the production of green fluorescent
protein, in a concentration-dependent fashion.
The observed increases in fluorescence intensity
appear to be the result of glucose-induced con-
formational changes associated with the induced
fit in hexokinase II, thereby indicating that the
complemented EGFP refolded correctly for the
formation of the EGFP fluorophore. As is shown
in Fig. 4A, a minimal increase in fluorescence
intensity was detected after treatment with 1 mM
glucose as the onset of the conformational change
associated with the glucose-induced fit in hexoki-
nase II. This indicated that low concentrations of
glucose quantitatively induce small alterations.
This increase was sustained with treatments of up
_+_
_
Non-induced
GlucoseInduced Maltose0.0
0.2
0.4
0.6
0.8
1.0
Rel
ativ
e fl
uo
resc
ence
in
ten
sity
(R
IU)
Fig. 3 In vivo visualization of glucose-induced conforma-tional changes in hexokinase II. For the detection offluorescence images, E. coli cells expressing chimericNGFP:HXK:CGFP protein, either with or without IPTGaddition, were initially spread onto glass slides stuck tosticky tape with 3 mm diameter wells in order to partitionthe bacterial cultures. Then, the fluorescence images of theNEGFP:HXK:CEGFP-expressing E. coli cells treatedwith glucose (or maltose as a control) were acquired witha GenePix 4200A laser scanner (Axon Inc., CA)
0 1 2 10Glucose
(mM)
0 30 60 90 120Time
(min)
0
1 10Glucose concentration (mM)
00.0
0.2
0.4
0.6
0.8
1.0
Rel
ativ
e fl
uo
resc
ence
in
ten
sity
(R
IU)
0.0
0.2
0.4
0.6
0.8
1.0
Rel
ativ
e fl
uo
resc
ence
in
ten
sity
(R
IU)
30 60 90 120Time (min)
5
2 5
A
B
Fig. 4 Structural transition associated with the glucose-induced fit in hexokinase II. Glucose-induced conforma-tional changes in hexokinase II were assessed viafluorescence analyses with a GenePix 4200A frombacterial cells generating NEGFP:HXK:CEGFP proteinsat differing (A) concentrations and (B) incubation timesusing glucose as a substrate
Biotechnol Lett (2007) 29:797–802 801
123
to 10 mM, at which point the solution is
completely saturated (Fig. 4A). The maximum
fluorescence was 5.2 times higher than that
observed in the absence of glucose which indi-
cates that glucose did, indeed, elicit a conforma-
tional change in hexokinase II, resulting in a
concomitant increase in the fluorescence inten-
sity. This demonstrates that the optimal glucose
concentration range for the induction of confor-
mational changes in hexokinase II is about
10 mM under our experimental conditions. In
order to detect the time-dependent changes
occurring in the fluorescence intensity, we treated
NEGFP:HXK:CEGFP-expressing bacterial cells
with glucose until the optimal time for glucose-
induced conformational change in hexokinase II
could be determined, at which time the fluores-
cence intensity would be maximally saturated. As
is shown in Fig. 4B, in the presence of 10 mM
glucose, the fluorescence intensity was 3.5 times
higher than that measured in initiation time point
of glucose treatment. After 60 min, as the tem-
poral threshold with regard to total image satu-
ration, the fluorescence intensity reached its
maximum, thereby indicating that the optimal
incubation time for glucose was somewhere
within the 60–90 min range.
In conclusion, we have successfully monitored
the glucose-mediated structural transition, which
is also referred to as the ‘glucose-induced fit’, in
human hexokinase II, via a split EGFP comple-
mentation technique. Upon binding to glucose,
hexokinase II manifests a structural change that
moves the exogamous chromophores closer
together. As a consequence of this glucose-
induced conformational change, the split EGFP
is reconstituted, and recovers its fluorescent
properties. The resultant activity of the recon-
stituted EGFP can be rapidly and directly
measured via fluorescence intensity measure-
ments. Collectively, the results of this study
showed that the conformational change of
dimeric hexokinase II associated with the glu-
cose-induced fit can be rapidly and directly
monitored in vivo via fluorescence complemen-
tation assays, predicated on the reconstitution of
split EGFP.
Acknowledgements This research was supported bygrants from the Nano/Bio Science & Technology Program(MOST, Korea), and the KRIBB Initiative ResearchProgram (KRIBB, Korea).
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