Assessing changes in the expression levels of cell surface proteins with a turn-on fluorescent molecular probe

Article abstract of DOI:10.1039/d0cc07095e

Tri-nitrilotriacetic acid (NTA)-based fluorescent probes were developed and used to image His-tagged-labelled outer membrane protein C (His-OmpC) in liveEscherichia coli. One of these probes was designed to light up upon binding, which provided the means

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DOI: 10.1039/D0CC07095E
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Assessing Changes in the Expression Levels of Cell Surface Proteins with a
Turn-on Fluorescent Molecular Probe
Joydev Hatai,‡a Pragati Kishore Prasad,‡a Naama Lahav-Mankovski,a Noa Oppenheimer-Low,a
Tamar Unger,b Yael Fridmann Sirkis,b Tali Dadosh,c Leila Motiei,*a and David Margulies*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Tri-nitrilotriacetic acid (NTA)-based fluorescent probes were change the intensity and/or the wavelength of emission upon
developed and used to image His-tagged-labelled outer binding. Because these changes largely depend on the
membrane protein C (His-OmpC) in live Escherichia coli. One molecular environment of the fluorescent dyes, such probes
of these probes was designed to light up upon binding, which could be used to distinguish between protein conformational
provided the means to assess changes in the His-OmpC states and binding interactions,5e as well as between
expression levels by taking a simple fluorescence spectrum.
structurally similar isoforms that have distinct surface
characteristics.5a-d,5f The versatility of this approach was
demonstrated with protein surface sensors based on tri-NTA-
Ni2+ complexes that can analyze a wide range of His-tagged
labeled proteins.5e,f By attaching the tri-NTA unit to fluorescent
DNA oligonucleotides, we have recently obtained structurally
programmable probes.5f,6 These DNA-based probes were used
to differentiate between His-tagged glycoforms5f or label His-
tagged cell surface proteins (CSPs) of living bacteria and to
provide the bacteria with new properties.6
There is considerable interest in developing small-molecule-
basedprobes that, byselectivelybindingshortpeptide tags, can
fluorescently label proteins in their native environment.1 The
main advantages of using these probes over labelling with
fluorescent protein (FPs) is their small size, which does not
disrupt the normal function of the proteins of interest (POIs), as
well as their structural versatility, which enables them to be
tailored for different applications.2 One of the most common
tags used in recombinant protein production is the hexa-
histidine tag (His-tag), which is widely used to purify His-tagged
proteins following their expression.3 Owing to its prevalence,
small size, and ability to coordinate to transition metal ions,
One of the advantages of using turn-on probes (or sensors)
over non-responsive probes (or labels) is the ability of the first
totrackdynamicchangesand affordrapid, wash-freedetection.
Although a few turn-on NTA-based sensors for His-tagged
fluorescent probes based on nitrilotriacetic acid (NTA), which proteins were developed,7 these systems generally rely on low-
can coordinate to oligo-histidine tags via metal coordination,
have been developed.2,4 Of particular interest are multivalent
affinity mono-NTA binders,7a-d or require pre-incubation with a
competing quencher that reduces their binding affinity.7e,f In
probes that can bind the His-tagged POIs with high affinity.4g-o our previous work we used responsive tri-NTA-based probes5e,f
The versatility and wide applicability of such probes was to study structural dynamics5e and glycosylation states5f of His-
demonstrated by Tampé, Piehler, and co-workers,4m-p who tagged proteins. However, these probes only operated in vitro.
developed a range of tri-NTA-dye conjugates for studying In contrast, our non-responsive probes, which can selectively
trafficking and structural dynamics of His-tagged proteins, and attach to a His-tagged CSP in living bacteria,6 can only serve as
for imaging them with super resolution (SR) microscopy.
Our group has been developing fluorescent probes that
respond to changes that occur on protein surfaces.5 Unlike
probes that merely label the POI, protein surface sensors5
labels. Herein we present a new tri-NTA-based probe (Fig. 1a,
probe 1) that exhibits a turn-on response upon binding to a His-
tagged CSP of living bacteria. To demonstrate the advantage of
using this probe over labels, we generated two additional tri-
NTAprobesthatbearnon-responsivefluorescentreporters(Fig.
1a, probes 2 and 3). We show that with 1, changes in the
expression levels of His-OmpC can be straightforwardly
assessed by taking a simple fluorescence measurement.
Analytical methods for determining protein expression
levels are important for studying their biological function,8 as
well as for determining the optimal condition for recombinant
a. Department of Organic Chemistry, Weizmann Institute of Science, Rehovot
7610001, Israel. email: leila.motiei@weizmann.ac.il,
david.margulies@weizmann.ac.il
b.Life Sciences Core Facilities, Weizmann Institute of Science, Rehovot 7610001,
Israel.
c. Chemical Research Support, Weizmann Institute of Science, 7610001, Rehovot,
Israel.
‡ Authors with equal contribution.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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and 3, which are appended with non-responsivVeiewfluAroticrleesOcnelinine
DOI: 10.1039/D0CC07095E
(Ex/Em: 465/520 nm) or sCy5 (Ex/Em: 620/665 nm) dyes, were
a)
designed to serve as control probes, namely, fluorescent labels
whose emission should not change upon binding to the His-POI
(Fig. 1b, right). Our expectation that the binding of 1 to His-tag
proteins would induce a turn-on emission signal (Fig. 1b, left) is
basedon our previous studies showingthat molecules linked to
specific protein binders tend to ‘stick’ to the surface of POI.5,13
In addition, it is based on the ability of solvatochromicdyes to
lightupuponbindingtohydrophobicdomainsonproteins.5d-f,14
TheoutermembraneproteinC(OmpC)ofE.coliwasselected
as the protein target for this study because its expression can
be controlled by culturing the bacteria under different
temperatures.15 This should enable us to test the main
hypothesis in our design, namely, that changes in the His-OmpC
expressionlevelscanbestraightforwardlyanalyzedbyfollowing
changes that occur in the fluorescence of 1 (Fig. 1c). Specifically,
we expected that bacteria expressing low levels of His-OmpC
(Fig. 1c, top) should activate fewer probes than bacteria with
high His-OmpC levels (Fig. 1c, bottom). As a result, bacteria with
high expression levels should induce a stronger fluorescence
response.
b)
c)
a)
Fig. 1 (a). Chemical structure of turn-on probe 1 and non-responsive probes 2 and 3. (b)
Schematic illustration of the labelling of His-OmpC in living bacteria using probe 1 (left)
or probes 2 and 3 (right). (c) Illustration of the way that changes in the OmpC expression
levels can be straightforwardly assessed using probe 1. Bacteria expressing low levels of
OmpC (top) will induce a smaller fluorescence response than bacteria with high levels of
OmpC expression (bottom).
b)
protein production.9 Although protein expression levels can be
determined using common analytical techniques, such as
western blotting (WB) or fluorescence cell imaging, these
methodsdonotprovidehigh-throughputanalysis.WithWB,the
cells must be lysed and the detection processinvolves gel
electrophoresis and cumbersome procedures.10 Labeling the
POI with fluorescent probes, such as NTA-based probes,2,4,11 or
fluorescentlylabeledantibodies,12 canbeusedtoassessprotein
expression in intact cells. These methods, however, require the
cells to be washed and plated over coated surfaces, as well as
expertise in operating fluorescence microscopesand image
processingtools,whicharenotaccessibletomanylaboratories.
To demonstrate the advantage of using turn-on probes for
assessing changes in protein expression levels in intact cells,
three probesthatsharethe same tri-NTA unit, but differin their
fluorescent reporters were synthesized (Fig. 1a). According to
ourdesign,probe1,whichisappendedwithanenvironmentally
sensitive solvatochromic dye (Nile Red, Ex/E m: 545/655 nm),
should act as a turn-on fluorescent sensor that responds to
binding to a His-tagged POI (Fig. 1b, left). In contrast, probes 2
c)
20 μm
Fig. 2 (a) Merged bright-field and fluorescence images of bacteria expressing His-OmpC
(top) and OmpC (that lacks the His-tag) (bottom) following incubation with probes 1-3
(500ꢀnM) and washing. (b) STORM images of His-tagged bacteria labelled with probe 3.
Left: Transverse cut. Right: Whole bacteria. (c) Images of bacteria expressing His-OmpC
following incubation with 500 nM of probe 2 , 4, or 5.
2 | J. Name., 2012, 00, 1-3
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Next, we determined whether, as expected from our design,
DOI: 10.1039/D0CC07095E
the emission of probe 1 would enhance upon binding to the
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a)
b)
engineered bacteria (Fig. 1b, left) and whether the change in
fluorescence could be used to straightforwardly determine
changes in the His-OmpC expression (Fig. 1c). For this purpose,
we measured the emission spectrum of 100 nM of 1 with Ni (II)
before and after adding the engineered bacteria (His-bacteria)
(Fig. 4a, left) and compared the optical responses to the
responses of probes 2 (Fig. 4a, middle) and 3 (Fig. 4a, right)
under the same conditions. As a control, we also measured the
responses of the three probes to the bacteria that lack a His-tag.
The results show that the emission intensity of probes 2 and 3
slightly changed (1.2- and 1.5-fold, respectively) upon
incubation withthe His-tagged E. coli. However, because similar
changes were observed when 2 and 3 were subjected to
bacteria that lack His-tag, these experiments indicate that the
small changes in the emission resulted from non-specific
interactions, which should not be affected by alterations in the
His-OmpC expression levels. In contrast, subjecting probe 1 to
the His-bacteria led to a hypsochromic shift and a 4.25-fold
increase in the emission intensity (at 655 nm). The fact that
these profound changes were only induced by the bacteria that
express a His-tag indicates that probe 1 acts as a sensor that
selectively responds to His-OmpC.
The benefit of using a turn-on sensor (probe 1) over labels
(probes 2 and 3) for assessing changes in the local
concentrations of His-OmpC was demonstrated by recording
the emission spectra of the three probes in the presence of the
His-tagged bacteria cultured at 8, 15, and 37 °C (Fig. 4b). Unlike
withthefluorescencecellimagingexperiment(Fig.3b), inwhich
differences in His-OmpC expression could be determined by
both the sensor (1) and the label (3), there was no change in the
emission spectrum of labels 2 (Fig. 4b, middle) or 3 (Fig. 4b,
right) upon incubation with the different samples of bacteria. In
contrast, probe 1 exhibited an increase in emission (Fig. 4b, left)
that is proportional to the enhancement in the His-OmpC
expression level (Fig. 3). Although the temperature-dependent
increase in His-OmpC expression was also determined by gel
Fig. 3 Assessing changes in His-OmpC expression level in bacteria cultured at 8, 15, or 37
°C. (a) Gel electrophoresis, or (b) Fluorescence imaging. The bars correspond to the
fluorescence intensity values measured from living bacteria labelled with probes 1 or 3.
Initially, we tested the ability of the three probes to
fluorescently label His-OmpC by incubating them (500 nM) with
the engineered bacteria (His-bacteria) and Ni(II), followed by
washing and imaging (Fig. 2a, top). As a control, the probes
were also incubated with E. coli-expressing OmpC (that lacks
His-tag) (Fig. 2a, bottom). The results show that only the His-
tagged bacteria were labelled, each with a distinct emission
color, indicating specific interactions between the probes and
His-OmpC. Super resolution STORM (stochastic optical
reconstruction microscopy) imaging with probe 3 showed that,
as expected, the labelling occurs only on the outer membrane
(Fig.2b, left)andthat His-OmpCspansacrosstheentire bacteria
(Fig. 2b, right). The contribution of the multivalent interaction
between the three NTA groups and the His-tag to the labelling
efficiency of the probes was determined by comparing the
fluorescence images taken following incubation of the His-
bacteria with 500 nM of 2 (Fig. 2c, left) to the ones taken
following incubation with 500 nM of probes 4 (middle) or 5
(right), which possess only two or one NTA group, respectively.
The notable increase in fluorescence with an increase in the
number of NTA units correlates well with the enhancement in
the binding affinities of the three probes (Kd-5 = 5578  7 nM, Kd-
4 =297  1 nM, Kd-2 =14.5  0.6 nM) toward the His-tag, which
were determined by microscale thermophoresis (MST) (Fig. S1,
ESI).
a)
To obtain bacterial cells expressing different levels of His-
OmpC on the outer membrane, the bacteria were cultured at 8,
15, or 37 °C. The expected increase in His-OmpC expression15b
was verified by gel electrophoresis (Fig. 3a and Fig, S2, ESI),
showing a notable enhancement in the His-OmpC levels when
the temperature increased from 8 to 37°C. Precisely, at 8 °C,
there was poor expression, whereas at 37 °C, maximal
expression levels were observed. Another way to analyze these
changes, which does not require cell lysis and gel
electrophoresis, is by labelling His-OmpC with our probes and
comparingthefluorescenceintensitygeneratedbythedifferent
samples. To this end, the bacteria that were cultured under
differenttemperatures werelabelledwith probes 1or 3 and the
fluorescence generated by the labelled cells was quantified. As
shown in Figure 3b, the changes in His-OmpC expression
determined by fluorescence imaging are in agreement with the
ones observed using the gel electrophoresisanalysis.
b)
Fig. 4 (a). Fluorescence spectra of probes 1 (left), 2 (middle), and 3 (right) in the absence
(black) and presence of bacteria expressing His-OmpC (His-bacteria) or bacteria that
express unmodified OmpC (bacteria). (b) Fluorescence spectra of probes 1 (left), 2
(middle), and 3 (right) to His-bacteria cultured at different temperatures.
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be readily analyzed by performing a single incubation step and
by taking a single fluorescence measurement.
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To summarize, a turn-on fluorescent probe (1) integrating a
tri-NTA group and a solvatochromic dye was developed and
used to analyze changes that occur in the expression levels of
His-OmpC in living bacteria. Additional probes that combine
NTA groups with non-responsive fluorescence dyes (2-5) were
also developed and used to confirm the principles underlying
the sensor design. We showed, for example, that although all
probes (1-5) can label His-OmpC, only sensor 1 displayed
fluorescence enhancement upon binding to the engineered
bacteria, indicating the important role the environmentally
sensitive dye plays in the sensor’s response. The contribution of
the tri-NTA unit to the sensor’s labeling efficiency was also
demonstrated by showing that the mono- and bis-NTA probes
(4 and 5) poorly label the bacteria compared with the tri-NTA
probes. Our results also show different applications that can be
achieved with the new tri-NTA probe family developed in this
study (probes 1-3). We have shown that probes 1-3 can be used
to label His-tagged CSPs with different colors; probe 3 can be
used to image them with super resolution, and probe 1 can
straightforwardly analyze changes that occur in their
properties. Probe 1 thus adds a new capabilityto our previous
tri-NTA-basedprobesthatcouldeithersensestructuralchanges
of His-tagged proteins in vitro,5e,for could only label them in
living cells.6 The ability of 1 to determine changes in the
expression level of His-tagged proteins in living bacteria
indicates the potential to expand the analytical toolscurrently
used to monitor changes in protein expression, such as WB or
fluorescence cell imaging. In our future work we aim to test
whether such sensors can be used to quantify protein
expressionlevels inliving cells andwhethertheycanbe applied
to investigate the dynamic properties of cell surface receptors.
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This work was supported by the Israel Science Foundation.
Conflicts of interest
There are no conflicts to declare.
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Table of Content Image
A turn on fluorescent molecular probe was used to assess changes in the expression level of His-
tagged cell surface proteins in living bacteria.