An ESIPT fluorescent probe sensitive to protein α-helix structures

Article abstract of DOI:10.1039/c4ob00405a

A large majority of membrane proteins have one or more transmembrane regions consisting of α-helices. Membrane protein levels differ from one type of cell to another, and the expression of membrane proteins also changes from normal to diseased cells. For example, prostate cancer cells have been reported to have downregulated expression of membrane proteins, including zinc transporters, compared with normal prostate cells. These reports inspired us to design a fluorescence probe sensitive to protein α-helical structures to discriminate individual prostate cancer cells from normal ones. A benzazole derivative (1 in this study) was observed to emit strong fluorescence resulting from an excited-state intramolecular proton transfer (ESIPT) in protein α-helical environments. The intensity of ESIPT fluorescence of 1 was observed to be positively correlated with the α-helix content of proteins. The molecular docking simulation suggested that it had low energy for the binding of 1 to proteins when the binding sites were localized within the α-helical regions of protein via H-bonds. Furthermore, 1 was found to be localized in cell membranes through binding to transmembrane α-helical regions of membrane proteins, and was capable of probing differences in the α-helix contents of membrane proteins between normal and cancerous prostate cells through changes in the ESIPT emission intensity. These results indicated that 1 could distinguish individual prostate cancer cells from normal ones, as the changes in the ESIPT fluorescence intensity of 1 could reflect the regulation in expression of the membrane proteins including zinc transporters. This recognition strategy of individual prostate cancer cells might contribute to early diagnosis techniques for prostate cancer.

Full text of DOI:10.1039/c4ob00405a

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An ESIPT fluorescent probe sensitive to protein
α-helix structures†
Cite this: Org. Biomol. Chem., 2014,
12, 5250
Nan Jiang, Chanli Yang, Xiongwei Dong, Xianglang Sun, Dan Zhang* and
Changlin Liu*
A large majority of membrane proteins have one or more transmembrane regions consisting of α-helices.
Membrane protein levels differ from one type of cell to another, and the expression of membrane pro-
teins also changes from normal to diseased cells. For example, prostate cancer cells have been reported
to have downregulated expression of membrane proteins, including zinc transporters, compared with
normal prostate cells. These reports inspired us to design a fluorescence probe sensitive to protein
α-helical structures to discriminate individual prostate cancer cells from normal ones. A benzazole deriva-
tive (1 in this study) was observed to emit strong fluorescence resulting from an excited-state intramole-
cular proton transfer (ESIPT) in protein α-helical environments. The intensity of ESIPT fluorescence of 1
was observed to be positively correlated with the α-helix content of proteins. The molecular docking
simulation suggested that it had low energy for the binding of 1 to proteins when the binding sites were
localized within the α-helical regions of protein via H-bonds. Furthermore, 1 was found to be localized in
cell membranes through binding to transmembrane α-helical regions of membrane proteins, and was
capable of probing differences in the α-helix contents of membrane proteins between normal and can-
cerous prostate cells through changes in the ESIPT emission intensity. These results indicated that 1 could
distinguish individual prostate cancer cells from normal ones, as the changes in the ESIPT fluorescence
intensity of 1 could reflect the regulation in expression of the membrane proteins including zinc transpor-
ters. This recognition strategy of individual prostate cancer cells might contribute to early diagnosis tech-
niques for prostate cancer.
Received 22nd February 2014,
Accepted 1st May 2014
DOI: 10.1039/c4ob00405a
www.rsc.org/obc
N* emission (Scheme 1).2 ESIPT molecules that exhibit charac-
teristic two-band fluorescence spectra in suitable microenvir-
Introduction
Excited-state intramolecular proton transfer (ESIPT) is a reac- onments can act as fluorescence probes to obtain insight into
tion that occurs in certain fluorescent molecules in the excited some biological processes.3–5 Demchenko’s group used a
state.1 In the ground state, an ESIPT molecule exists in the derivative of the most typical ESIPT fluorescent molecule,
enol tautomer state (E); upon irradiation with light, the mole- 3-hydroxyflavone, as a site-sensitive fluorescence label for
cule is driven to the excited state (E*) and emits fluorescence α-crystallin, and proposed that this labelling method might
(called N* emission) in a short wavelength range. When the offer a possibility to study structures, dynamics and intermole-
excited molecule is localized in a suitable microenvironment, cular interactions of the labelled proteins.6–9 Stefani’s group
a proton can be transferred from its H-bonding donor group to reported the use of benzazole derivatives to stain Candida albi-
its H-bonding acceptor group to generate the corresponding cans ATCC 10231 cells and characterize nanocapsule polymeric
keto tautomer (K*). The fluorescent molecule in the excited walls, as well as to probe rice protein and bovine serum
keto state emits fluorescence (called T* emission) with a large albumin (BSA) in solutions, but the detailed mechanisms are
Stokes shift in a longer wavelength range relative to the still to be explored.10–14
A large majority of membrane proteins have one or more
transmembrane regions consisting of α-helices. Membrane
Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, School of
protein levels differ from one type of cell to another, and the
Chemistry, Central China Normal University, Chemistry Building, Central China
expression of membrane proteins also changes from normal to
Normal University, Wuhan, China. E-mail: liuchl@mail.ccnu.edu.cn,
diseased cells.15 For example, in the development of prostate
danzhang@mail.ccnu.edu.cn; Tel: (+86)-27-67867232
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
cancer, the intracellular mobile zinc ion levels are notably
decreased,16–19 along with the downregulation in the
c4ob00405a
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to the suitable distance between their H-bonding acceptor
N atoms and OH groups. In order to examine changes in the
ESIPT properties of 1 and 3 in different microenvironments,
three organic solvents, the protic solvent ethanol and the
aprotic solvents chloroform and dichloromethane, were
chosen in light of their polarity and H-bonding ability.25 Both
polarity and H-bond acceptor ability decreased in the order
ethanol > dichloromethane > chloroform.
Whether 1–3 underwent an ESITP reaction was obvious
from their fluorescence spectra in the selected solvents
(Fig. 1a–c). Two well-separated, intense fluorescence peaks
were observed for 1 in both chloroform and dichloromethane,
and for 3 in all three solvents (Fig. 1a,c), corresponding
respectively to enol (N*) and keto tautomer (T*) emissions, as
previously reported,10,26 but 2 showed only normal emission
under the conditions tested (Fig. 1b). These results indicate
that 1 and 3 underwent an ESIPT reaction, but 2 did not in the
solvents tested. In order to confirm the occurrence of the
ESIPT reactions in 1 and 3, the intensity ratios of T* to N*
emission (IT*/IN*) were determined (Fig. 1d). Obviously, the
IT*/IN* ratios were significantly reduced with an increase in the
H-bonding ability of solvents, and were greater in chloroform
than in either dichloromethane or ethanol. This fact indicated
that the ESIPT process occurred much more easily in aprotic
solvents with no H-bonding ability, because H-bonds between
a fluorescent molecule and a solvent molecule could interfere
in the formation of keto tautomers. This could lead to dis-
appearance of ESIPT emission, which occurred for 1 in
ethanol (Fig. 1a). Moreover, the hydroxyl group in the ortho-
position of benzoxazolyl in 1 and 3 was essential for ESIPT. In
addition, the electron-donating group NH2 in 1 could enhance
the electron density on the OH group and reduce the intra-
molecular proton transfer, which resulted in the decreased
intensity of ESIPT emission and the IT*/IN* ratio of 1 compared
to 3 under the conditions tested.
Scheme 1 (a) Structures of 1–3. (b) ESIPT reaction in 1. E* and K* rep-
resent the excited enol and keto tautomers of 1, respectively.
expression of Zip-family proteins (hZip1, hZip2, hZip3) which
have been reported to be responsible for the accumulation of
zinc in prostate cells.20–22 The zinc transporters including
hZip1–3 (UniProt, Q9NY26, Q9NP94 and Q9BRY0) have eight
potential transmembrane α-helices.23 Obviously, downregula-
tion in the expression of zinc transporters can lead to a
decrease in protein α-helix content in prostate cancer cell
membranes. Therefore, the changes in α-helix contents in cell
membranes might be a potential biomarker for the recog-
nition of individual prostate cancer cells, given that the
expression of membrane proteins other than zinc transporters
is not significantly changed in the transformation of normal
prostate cells to cancer cells.
In this study, we report an ESIPT molecule 1 (5-amino-2-
benzooxazol-2-yl-phenol, a derivative of 2-(2′-hydroxyphenyl)-
benzazole) that emits strong ESIPT fluorescence in both
organic solvents and proteins with a high α-helix content
(Scheme 1). The strong ESIPT fluorescence was brought about
by the biding of 1 to proteins with a high α-helix content. The
sensitivity of 1’s ESIPT fluorescence to the protein α-helix
structures could be used to probe changes in the protein
α-helix content in prostate cell membranes. Because the
changes in the α-helix content could reflect the expression of
membrane proteins, individual prostate cancer cells could be
distinguished from normal cells by utilizing the changes in
the ESIPT fluorescence intensity of 1.
Results and discussion
ESIPT fluorescence in organic solvents
Three benzoxazole molecules 1–3 (Scheme 1) were prepared
and characterized according to the previously reported syn-
thetic approach (see ESI, Fig. S1† for detailed data).10,24 Com-
Fig. 1 Fluorescence spectra of 8 mM 1 (a, λex = 335 nm), 2 (b, λex
310 nm) and 3 (c, λex = 320 nm) in EtOH (—), CH2Cl2 ), CHCl3 ) at
25 °C, respectively. (d) Relative emission intensity ratios of T* to N*
=
(
(
pared to 2, 1 and 3 underwent ESIPT reactions more easily due bands of 1 (I460 nm/I390 nm) and 3 (I483 nm/I367 nm) in the three solvents.
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Table 1 The simulation data for the molecular docking of 1 into the selected proteins
Distance of intermolecular
hydrogen bonds (Å)
Binding energy
Protein (PDB)
HSA (1BM0)
Helix content (%)
Angle (°)
(kcal mol−1
)
7131
41
N1–H⋯OE2 (Glu425)
O2⋯H–ND1 (His146)
N1–H⋯OE1 (Glu35)
N1–H⋯OD2 (ASp52)
N1–H⋯O (Gly214)
N1–H⋯O (Gly212)
O2⋯H–N (Gly193)
N1–H⋯O (Thr74(A))
—
2.678
2.989
3.018
2.921
3.103
3.077
3.167
2.664
139.4751
48.451
158.2631
36.161
140.6591
46.58315
2.173
−5.33
Lysozyme (3RT5)
Trypsin (3ATK)
−5.1
1032
−4.03
Potassium ion channel (1BL8)
Glucose transporter (4GBZ)
6733
7634
142.308
—
−5.83
−5.35
ESIPT fluorescence in protein α-helix environments
to the normal band at ∼390 nm, but not in the other two
protein solutions (Fig. 2a). However, 2 and 3 emitted only
either strong enol or keto tautomer fluorescence in all four
protein solutions under the conditions tested, as observed in
the buffer (Fig. 2b and c). These results revealed that 1 under-
went ESIPT reaction in the proteins which had high α-helix
contents (66% for BSA and 71% for HSA), suggesting that the
ESIPT fluorescence of 1 might be sensitive to the protein
α-helical structures.
Analogues of 1–3 were reported to emit ESIPT fluorescence at
∼460 nm in BSA solutions.10 The hydrophobic and low polar
sites within a protein are similar to the environments found in
organic solvents with low polarity, and should facilitate the
ESIPT emission. Therefore, fluorescence spectra were utilized
to examine whether 1–3 undergo ESIPT reactions in the solu-
tions of the proteins containing various α-helix contents. We
selected four proteins, BSA, human serum albumin (HSA),
lysozyme and trypsin, and their α-helix contents are listed in
Table 1. The fluorescence spectra of 1–3 were first measured in
the buffer used as control. The results showed that the normal
fluorescence band at ∼400 nm arose from the enol tautomers
of excited 1 and 2, whereas the fluorescence at ∼485 nm was
the ESIPT emission arising from the keto tautomers of excited
3 (Fig. 2a–c). These results indicated that the fluorescence
from either enol or keto tautomer could be observed in the
buffer. 1 was observed to produce a well-separated fluore-
scence band at ∼460 nm in BSA and HSA solutions in addition
In order to prove the sensitivity of 1’s ESIPT emission to
protein α-helical structures, the effects of BSA concentration,
temperature, and protein denaturing agents on fluorescence
spectra of 1 were examined. Firstly, the fluorescence spectra
were recorded at a fixed concentration of 1 and varied BSA con-
centrations. The results showed that the IT*/IN* ratios increased
significantly with increasing molar ratio of BSA : 1 (Fig. 3a and
b). The intensity of both T* and N* emissions from solutions
containing BSA and 1 at a given concentration was observed to
decrease with increasing temperature (Fig. 3c and d), as did
the IT*/IN* ratios (Fig. 3d). These results could be attributed to
loss of the natural α-helix structures in BSA due to heating. To
rule out the possibility that the temperature sensitivity of the
ESIPT emission was an artifact of the heating, the fluorescence
spectra of 1 in the absence of BSA were measured over the
given temperature range. As expected, only normal emission
was observed for 1 under the conditions tested (Fig. S3†).
Moreover, the intensity of this emission was reduced with
heating, and the extent of the reduction was similar to that
observed in the presence of BSA. These facts demonstrate that
the ESITP emission of 1 is positively correlated with the
content of ordered α-helix structures in a protein. As a result,
the combination of the BSA concentration and temperature
experiments suggested that the ESITP emission intensity of 1
is sensitive to the α-helix content in a protein.
To further confirm the correlation of the ESITP emission
intensity of 1 with the α-helix content in a protein, the
α-helical protein BSA was treated with varying concentrations
of urea. Because urea can disrupt the secondary and tertiary
structures of proteins, the α-helix content in BSA would be
reduced by the increasing urea concentration.27,28 Treatment
with 0–8 M urea led to the reduction of α-helix content from
67.3% to 48.1% in 32 mM BSA, as demonstrated by circular
dichroism (CD) spectra (Fig. 3e). With the reduction of the
Fig. 2 (a) Fluorescence spectra of 8 mM 1 (λex = 335 nm) in the pres-
ence of 32 mM BSA, HSA, lysozyme and trypsin, respectively. (b) Fluore-
scence spectra of 0.5 mM 2 (λex = 310 nm) in the presence of 2 mM BSA,
HSA, lysozyme and trypsin, respectively. (c) Fluorescence spectra of
8 mM 3 (λex = 320 nm) in the presence of 32 mM BSA, HSA, lysozyme
and trypsin, respectively. All fluorescence experiments were performed
in 100 mM Tris-HCl buffer (pH 7.4, 150 mM NaCl) at 25 °C.
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2 or 3 in the BSA solution (Fig. 2b and c) because of the lack of
intra- or intermolecular H-bonds.
Obviously, the formation of intramolecular H-bonds in 2
was difficult owing to the large separation between its benzoxa-
zolyl N atom and OH group, despite the favorable formation of
intermolecular H-bonds between 2 and the proteins. However,
it was possible for 3 to form the intramolecular H-bond in
both the presence and absence of BSA, because of the lack of
an NH2 group, leading to the presence of 3 in the keto form
even in the absence of proteins. Therefore, 2 and 3 could not
undergo the ESIPT reaction like that in 1 when exposed to BSA
and HSA. In addition, the differences in the fluorescence
spectra of 1 and 3 revealed the essential role of the NH2 group
in the ESIPT fluorescence of 1 in the protein-bound state. On
the other hand, the ordered hydrophobic sites in a protein
consisting of α-helices can offer a low polarity microenviron-
ment for fluorescence emission. Thus, N* and T* bands, as
well as the IT*/IN* ratio of 1, were observed to be enhanced in
the BSA and BSA solutions compared with the other two pro-
teins with low α-helix contents (41% for lysozyme and 10% for
trypsin) (Fig. 2a). As a result, it is reasonable to conclude that
the ESIPT fluorescence of 1 could be ascribed to the suitable
localization of 1 in the α-helical environments within a
protein, likely via the intermolecular H-bonds between the
NH2 group on 1 and an amide group in the protein.
Fig. 3 (a) Fluorescence spectra of 8 mM 1 upon increased addition of
BSA (0–160 mM). (b) The effect of BSA : 1 molar ratio on T* to N* emis-
sion intensity ratios (I460 nm/I390 nm) of 1, calculated using the fluore-
scence spectra in (a). (c) Fluorescence spectra of 8 mM 1 in the
presence of 32 mM BSA in the temperature range 20–80 °C. (d) The
effect of temperature on the I460 nm/I390 nm ratios of 1, calculated using
the fluorescence spectra in (c). (e) CD spectra of 32 mM BSA exposed to
0–8 M urea. (f) I460 nm/I390 nm ratios of 8 mM 1 were reduced with the
decreasing α-helix content of BSA exposed to 0–8 M urea. The α-helix
contents of BSA exposed to 0–8 M urea were calculated based on the
CD spectra in (e). All fluorescence experiments were performed in
Molecular docking into α-helical sites in a protein
In order to support the hypothesis that 1 could associate with
the α-helical proteins BSA and HSA via intermolecular
H-bonds between the NH2 or OH group on 1 and amide
groups in the proteins, molecular docking simulations were
100 mM Tris-HCl buffer (pH 7.4, 150 mM NaCl) at 25 °C. The error bars carried out using the AutoDock program (version 4.2.0)29,30
were the results of at least three parallel measurements.
based on the protein structural data. The results delineated
above indicated that the ESIPT fluorescence of 1 was sensitive
to the α-helix structures in BSA and HSA. HSA was selected for
the molecular docking with 1, as the structural data of HSA are
α-helix content in BSA, the IT*/IN* ratios of 1 were observed to
significantly decrease from 0.76 to 0.15 (Fig. 3f). The results of
the protein denaturing experiment, together with the results
described above, proved that the ESIPT emission intensity of 1
is dominated by the α-helix content in a protein.
available in the Protein Data Bank (PDB), and HSA is homolo-
gous to BSA. The interactions between 1 and lysozyme or
trypsin were also simulated for comparison. The 1-bound
hydrophobic sites in the proteins were visualized by molecular
surface models (Fig. 4a, c and e), indicating that the shapes of
the sites for 1 binding differ from one type of protein structure
to another. This result highlights the fact that α-helical sites or
sites rich in α-helices are appropriate for 1 binding in
proteins.
The sensitivity of the ESIPT fluorescence of 1 to proteins
with high α-helix content can be understood in light of the
hydrophobic protein sites which consist of α-helices. On the
one hand, if 1 could be localized in a suitable site within an
α-helical protein, it would be able to form H-bonds between its
NH2 group and the amide groups (either CO or NH) on the
protein α-helices. The intermolecular H-bonds could reduce
the electron-donating ability of the NH2 group and weaken the
O–H bond in 1. Obviously, this favors the formation of the
intramolecular O–H⋯N H-bond in 1, which results in readier
proton transfer from the OH group to the benzoxazolyl N atom
on 1 in the protein-bound state than in the protein-free state.
Therefore, the strong fluorescence emission caused by the
ESIPT reaction in 1 could be observed in the BSA- or HSA-con-
taining media. In contrast, no new emission was observed for
The docking calculations showed that binding energy of 1
to HSA (−5.33 kcal mol−1) was lower than those of 1 to both
trypsin (−5.1 kcal mol−1) and lysozyme (−4.03 kcal mol−1
)
(Table 1). The data also demonstrated that the α-helices played
a key role in the binding. As indicated by the docking, the
binding energy was mainly dominated by the intermolecular
energy which was dependent on H-bonds between 1 and the
proteins (Fig. 4b, d and f). Therefore, the differences in the
H-bonding strengths and modes could underlie the change in
the binding energy. It was clear from Fig. 4b, d and f that the
H-bonds could only form between the NH2 group on 1 and the
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Fig. 4 The interactions between 1 and protein molecules visualized by molecular docking simulations. Here, 1 is represented as a stick model;
C: grey, N: blue, O: red. Top: the hydrophobic cavities in HSA (a), lysozyme (c) and trypsin (e) for 1 binding are displayed as molecular surface
models. The residues involved in the H-bonding between 1 and HSA (b), lysozyme (d) or trypsin (f) are highlighted. Bottom: the proposed H-bonds
are shown as green dashed lines. The residues involved in the interactions between 1 and the protein molecules are indicated, and the fragments of
protein secondary structures are shown in different colours.
proteins, and the benzoxazolyl N atom and the OH group on 1 a large majority of membrane proteins consist mainly of
were not involved in the formation of the intermolecular α-helices.35–37 This prompted us to examine the fluorescence
H-bonds, as anticipated. Therefore, the intramolecular spectra of 1 in membrane protein solutions. We extracted
O–H⋯N H-bond in 1 was not affected (Scheme 1). On the membrane and cytosol proteins (0.2 mg mL−1) from HeLa cells
basis of the H-bond length data (Table 1), the H-bonding by cell lysis to perform this examination. The membrane pro-
strength between the NH2 group on 1 and the corresponding teins in the samples consisted of cell and plasma membrane
residues decreased with a fall in the α-helix content in the proteins. The fluorescence spectra of 1 in the samples, as well
tested proteins. Obviously, the strong intermolecular H-bonds as in BSA solution (0.2 mg mL−1), were determined under
decreased the electron density of the OH group on 1, and identical conditions. Compared with 1 in the buffer tested, any
thereby promoted the intramolecular proton transfer in 1. As a significant fluorescence emission at 425–550 nm was not
result, a significant ESIPT reaction occurred in the HSA-bound observed for 1 exposed to the cytosol protein and BSA (Fig. 5a)
form of 1; that is, stronger ESIPT emission could be observed at the given concentrations. Obviously, 1 cannot emit observa-
for HSA exposure of 1 compared with lysozyme or trypsin ble fluorescence at 425–550 nm in solutions with low BSA con-
exposure. In addition, comparison of the H-bonding modes centration because of its BSA concentration-dependent ESIPT
between HSA and lysozyme (Fig. 4b and d) suggested that the reaction (Fig. 3a and b). Meanwhile, this result also indicated
arrangement of the α-helices in proteins could also play a role that the ESIPT reaction was not pronounced in 1 exposed to
in the binding of 1. The parallel and near parallel distributions the cytosol proteins. However, when treated with the mem-
of α-helices could favor the association of 1 with the proteins. brane protein solution, 1 was observed to emit noticeable fluore-
Taking these results together with those obtained from the scence at 425–550 nm (Fig. 5a), demonstrating that an ESIPT
fluorescence experiments performed above, it is reasonable to reaction occured in 1 in a membrane protein environment.
conclude that the ESIPT fluorescence of 1–3 in protein Furthermore, because the ESIPT emission of 1 was positively
α-helical environments was dependent not only on the intra- correlated with the α-helix content in proteins, as indicated
molecular position of the OH group on 1, but also on the local- above, the noticeable ESIPT reaction in 1 suggested that the
ization within the protein molecules of the NH2 group on 1 α-helix content in the membrane proteins was much higher
bound to α-helical sites via H-bonds.
than those in the cytosol proteins at the given concentration as
expected. In addition, the normal emission of 1 was consider-
ably enhanced by the treatment with the membrane proteins
compared to that with the cytosol proteins (Fig. 5a), indicating
that the α-helical motifs in membrane proteins could create an
ordered environment that facilitated the normal fluorescence
Sensitivity of the ESIPT fluorescence of 1 to intracellular
α-helical proteins
Although intracellular proteins have α-helix contents which
vary with protein structure type, the transmembrane regions in
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Fig. 5 (a) Fluorescence spectra of 1 in the presence of membrane proteins, cytosol proteins and BSA, respectively. The membrane and cytosol pro-
teins were extracted from HeLa cells. The concentrations of the samples were 0.2 mg protein mL−1. (b, c) H-bonds, residues, and secondary struc-
tures involved in interactions between 1 and a potassium channel (b) or glucose transporter (c). The binding modes between 1 and the membrane
proteins were visualized by the molecular docking simulation. (d) Fluorescence spectra of 8 mM 1 (λex = 335 nm) in the presence of 32 mM BSA,
HSA, 320 mM SDS, and 0.1% lecithin (0.01 g lecithin in 10 mL Tris-HCl buffer), respectively. All fluorescence experiments were performed in 100 mM
Tris-HCl buffer (pH 7.4, 150 mM NaCl) at 25 °C.
emission of 1. In contrast, the effects of both lipids and deter- corresponding residues on the α-helices. The strong H-bonds
gents on 1’s ESIPT fluorescence were examined to rule out the of 1 to the membrane proteins, as indicated by bond length
possibility that the hydrophobic environments created by data (Table 1), facilitated the ESIPT reaction in 1 by reducing
these favored the ESIPT fluorescence of 1. Compared with BSA the electron density on its OH group. Thus, the H-bond
and HSA proteins, lecithin (lipid) and SDS (detergent) could between the NH2 and α-helices results in the observed ESIPT
not make 1 undergo the ESIPT reaction (Fig. 5d). As a result, it emission of 1 in membrane proteins. In addition, the hydro-
was concluded that fluorescence of 1 was a sensitive probe for phobic and ordered binding sites comprising α-helices favored
membrane proteins with high α-helical content.
the enhanced normal fluorescence of 1 in membrane proteins.
The enhanced ESIPT and normal fluorescence of 1 implied As a consequence, the combination of the molecular docking
an interaction between 1 and α-helical regions in membrane data and fluorescence determination revealed that the fluore-
proteins. To understand this interaction, the molecular scence intensity of 1 was a sensitive probe to changes in
docking simulation of 1 with typical membrane protein mole- α-helix contents in membrane proteins.
cules was carried out using the same docking approach as
Response of 1’s fluorescence to membrane proteins in living
prostate cells
described above. A potassium ion channel and a glucose trans-
porter were selected for the docking simulation because these
two most abundant membrane proteins have transmembrane
regions that consist entirely of α-helices, as demonstrated by
their X-ray structures.33,34 On the one hand, the docking data
(Table 1) showed that the binding energies of 1 to this pair of
membrane proteins were −5.83 and −5.35 kcal mol−1, respect-
ively, which were lower than that of 1 to HSA. On the other
hand, the results indicated that the binding sites of 1 did loca-
lize within α-helical regions in membrane proteins, and 1 had
a similar binding mode in the α-helical regions (Fig. 5b and c)
to that in HSA (Fig. 4a and b). The binding mode was con-
trolled by the H-bond between the NH2 group on 1 and the
The observations delineated above inspired us to examine
whether the fluorescence of 1 could change with protein
α-helix contents in living prostate cells. In order to con-
veniently perform this examination, prostate normal (RWPE 1)
and cancer cell (DU145) lines were selected, as a notable differ-
ence was reported in the expression of the Zip family mem-
brane proteins between these two types of cells,38 and the
different expression levels of Zip family proteins might be
shown by the interactions with 1. In light of the reported
results,23 the Zip family proteins (hZip1, hZip2, and hZip3) all
have eight potential transmembrane α-helices. To observe the
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Fig. 6 (a) A representative flow cytometry raw histogram for fluore-
scence intensity of 1. (b) Mean fluorescence intensity of 4 mM 1 per
RWPE 1 or DU145 cell after incubation for 1 h. The results are represen-
tative of at least five independent experiments. ***P < 0.001.
differences in fluorescence of intracellular 1 between prostate
normal and cancer cells, the fluorescence intensity (excited at
375 nm) from the cells exposed to 4 mM 1 under the con-
ditions tested was quantified by collecting Hoechst-blue
channel signals with flow cytometry, because the ESIPT reac-
tion in the membrane protein-bound 1 led to blue fluore-
scence (maximum at ∼460 nm, Fig. 2a). The cytometric data
showed that the mean fluorescence intensity per DU145 cell
(at least 5 parallel assays) was reduced by ∼50% relative to that
per RWPE 1 cell after exposure to 1 (Fig. 6). Obviously, this
result, which was consistent with that obtained by using the
extracted membrane proteins provided by living cell lysis, pro-
vided evidence to support the previously reported observation
that the expression of membrane proteins including zinc
transporters was significantly downregulated in DU145 pros-
tate cancer cells. This indicated that the change in ESIPT
fluorescence intensity of 1 was a measure of the alteration in
the expression of membrane proteins in cells.
Fig. 7 Z-axis two-dimensional confocal fluorescence images for scan-
ning analysis of 4 mM 1 and 40 nM ER-Red in RWPE 1 cells. Single
optical sections (at 1.5 μm steps) at various distances from the bottom
surface are shown. Upper: the collected fluorescence signal from 430 to
490 nm emitted only by 1; middle: the collected fluorescence signal
from 581 to 641 nm emitted only by ER-Red; bottom: the merged image
Distinguishing individual prostate cancer cells from normal
ones
▽
of the upper and middle panels. “ ” indicates the cell membrane.
The observations performed above show a significant differ-
ence in the fluorescence intensity of 1 between the stained
RWPE 1 and DU145 cells, indicating that 1 might provide a
novel method to distinguish individual prostate cancer cells ER-Red, it was also distributed in the cell membrane, as
from normal ones by fluorescence imaging of the changes in demonstrated by the clear cell profiles (Fig. 7, upper). More-
membrane protein levels. In order to use the ESIPT fluore- over, 1 also appeared in other cytosol membranes in addition
scence of 1 to recognize prostate cancer cells, localization of to ER, as indicated by the merged images (Fig. 7, bottom). In
1 in prostate cells was examined by two-photon confocal addition, the observations with the artificial phospholipid
microscopy. Firstly, the intracellular distribution of 1 was membrane indicated that 1 can be retained in and penetrate
observed by co-staining the prostate normal cells RWPE 1 with the artificial membrane, but the retention in the membrane
1 and ER-Tracker Red dye (ER-Red). ER-Red is a fluorescent could not lead to observable ESIPT fluorescence (Fig. 5d).40
dye that is capable of visualizing endoplasmic reticulum (ER) These results allowed us to conclude that 1 visualizes cell and
via specific binding to sulphonylurea receptor (SUR) subtypes cytoplasmic membranes via binding to membrane proteins,
in ER.39 The cells were imaged by two-photon excitation at not via retention in membranes.
800 nm for 1 and single-photon excitation at 561 nm for
Then, the cell lines RWPE 1 and DU145, which show a
ER-Red, after exposure to the two fluorescent molecules under notable difference in expression of membrane proteins includ-
the conditions tested. The fluorescence signals from 430 to ing Zip-family proteins, were exposed to 4 mM 1 under identi-
490 nm were acquired for imaging to utilize the ESIPT emis- cal conditions. The obtained confocal images show that the
sion from the membrane protein-bound 1. The Z-axis scanning blue fluorescence in each RWPE 1 cell was much brighter than
images showed that 1 was localized in ER like ER-Red, as illus- that in DU145 cells (Fig. 8). The results, which were in agree-
trated by the blue and red areas in (Fig. 7, middle), but unlike ment with those of the flow cytometry, indicated that the
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Evaluation of protein α-helix contents
Protein α-helix contents were evaluated through CD experi-
ments. CD experiments were carried out with a Chariscan cir-
cular dichroism photomultiplier (Applied Photophysics
Limited, UK) equipped with a Quantum Northwest TC125
temperature controller at 25 °C, and protein α-helix contents
were calculated by the software CDNN2.1-SimpleSpectra
attached to this instrument. The final concentration of pro-
teins in the CD measurements was 5 μM in 100 mM Tris-HCl
buffer (pH 7.4, 150 mM NaCl). For evaluation of α-helix con-
tents in BSA exposed to urea, 0–8 M urea was added into the
BSA-containing buffers and incubated for 40 min at 37 °C
prior to measurements.
Fig. 8 Confocal images of cell lines RWPE 1 and DU145, after treatment
with 4 μM 1, followed by staining with 40 nM ER-Red.
α-helix content decreased in the order RWPE 1 > DU145 in Molecular docking simulation
light of the ESIPT emission intensity of 1. This result revealed
The molecular simulation on the docking of 1 into the three-
that 1 can act as a promising fluorescent probe to distinguish
dimensional structures of different proteins (download from
PDB) was carried out using the program suite AutoDock 4.2.0
(http://www.scripps.edu/mb/olson/doc/autodock). The widely
used Lamarckian Genetic Algorithm (LGA), an efficient and
robust algorithm,41,42 was chosen for the docking calculation.
The graphical user interface, AutoDockTools (ADT 1.4.6), was
used to set up each ligand–protein interaction, where all
hydrogen atoms were added, Gasteiger charges were calculated
and nonpolar hydrogen atoms were merged to carbon atoms.
The solvent molecules were removed from the three-dimen-
sional protein structures to obtain the docking grid, and the
active site was defined using AutoGrid. The grid size was set to
60 × 60 × 60 points with a grid spacing of 0.375 Å, a van der
Waals well depth of 0.100 kcal mol−1, an iteration of 20043 and
a population size of 100. The grid box was centered on the
center of the ligand from the corresponding protein structures.
Formal structures of the proteins were assigned by the
program SYBYL, and the best ranked pose was selected from
the ChemScore. The conformation with the lowest binding
energy was used to analyze ligand placement.
individual prostate cancer cells from normal ones.
Experimental section
General methods
All reagents were purchased from commercial sources and
used as received unless stated otherwise. The benzoxazole
molecules 1–3 were prepared and characterized according to
the reported synthesis (see Fig. S1 in ESI†).10,24 All solutions
and buffers were prepared using Millipore-purified water. UV-
visible absorption spectra were measured with a SPECORD 210
Ultra-visible spectrophotometer (Analytic Jena AG). H and 13C
NMR spectra were recorded on a Varian VXR 500 spectrometer.
1
Fluorescence experiments
All fluorescence measurements were carried out with a Cary
Eclipse fluorescence spectrophotometer (Varian, USA) at 25 °C.
For studies of 1–3 under different conditions, the fluorescence
spectra of 1–3 were recorded in 100 mM Tris-HCl buffers
(pH 7.4, 150 mM NaCl) containing 32 mM BSA (Roche), HSA
(Sigma), lysozyme or trypsin (Biosharp) in the emission wave-
length range of 350–600 nm (λex = 335 nm, 1 cm cell length).
These proteins have various α-helix contents (from 71% to
10%, Table 1). To examine effects of protein : 1 ratios and
temperature on fluorescence of 1, fluorescence spectra of 1
were acquired for 0–160 mM BSA-containing samples or for
32 mM BSA treated for 10 min by heating in the temperature
range 20–80 °C. To estimate the effects of protein denaturing
reagents on the fluorescence of 1, fluorescence spectra of 1
were recorded for 32 mM BSA exposed to 0–8 M urea (Sigma)
for 40 min at 37 °C. When examining the fluorescence
response to membrane proteins, 1 was added to membrane
and cytosol proteins (0.2 mg protein mL−1) extracted from
HeLa cells, and fluorescence spectra were recorded under the
Cell culture
Human prostate epithelial RWPE 1 cell line, and human HeLa
cell line were purchased from China Center for Type Culture
Collection, and human prostate cancer DU145 cell line was
from Boster Biological Technology, Ltd. RWPE 1 cells were
grown in keratinocyte-serum free medium (K-SFM, Invitrogen)
containing 5 ng mL−1 human recombinant epithelial growth
factor (hEGF, Invitrogen) and 0.05 mg mL−1 bovine pituitary
extract (Invitrogen). DU145 cells were cultured in Iscove’s
modified Dulbecco’s medium (IMDM, Invitrogen) sup-
plemented with 10% fetal bovine serum (FBS, Invitrogen).
HeLa cells were grown in Dulbecco’s modified Eagle medium
(DMEM, Invitrogen) containing 10% FBS.
Extraction and quantification of membrane proteins
same conditions. For all fluorescence studies of 1, fluorescence Extraction of membrane and cytosol proteins from HeLa cells
spectra of all samples were recorded following incubation for was carried out using a Membrane and Cytosol Protein Extrac-
5 min at 37 °C unless stated otherwise.
tion Kit (Beyotime Institute of Biotechnology, China). The
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Organic & Biomolecular Chemistry
protein samples were quantified by a BCA Protein Assay Kit
(Beyotime Institute of Biotechnology, China).
Acknowledgements
This work was supported by NSFC (no. 21302059, 21271079,
21072074) and the PCSIRT (no. IRT0953).
Flow cytometry
Fluorescence responses of 1 to expression levels of membrane
proteins in living cells were detected by flow cytometry experi-
ments performed with a BD FACSAria III flow cytometer (BD,
USA) with an excitation wavelength of 375 nm and a Hoechst-
blue filter (450 50 nm). RWPE 1 and DU145 cells were loaded
at 37 °C for 1 h with 4 μM 1 in K-SFM and IMDM media,
respectively, prior to measurement.
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