Photoclickable dendritic molecular glue: Noncovalent-to-covalent photochemical transformation of protein hybrids

Article abstract of DOI:10.1021/ja401059w

A water-soluble dendron with a fluorescein isothiocyanate (FITC) fluorescent label and bearing nine pendant guanidinium ion (Gu ⁺)/benzophenone (BP) pairs at its periphery (Glue^BP-FITC) serves as a photoclickable molecular glue . By multivalent salt-bridge formation between Gu⁺ ions and oxyanions, Glue ^BP-FITC temporarily adheres to a kinesin/microtubule hybrid. Upon subsequent exposure to UV light, this noncovalent binding is made permanent via a cross-linking reaction mediated by carbon radicals derived from the photoexcited BP units. This temporal-to-permanent transformation by light occurs quickly and efficiently in this preorganized state, allowing the movements of microtubules on a kinesin-coated glass plate to be photochemically controlled. A fundamental difference between such temporal and permanent bindings was visualized by the use of optical tweezers .

Full text of DOI:10.1021/ja401059w

Communication
pubs.acs.org/JACS
Photoclickable Dendritic Molecular Glue: Noncovalent-to-Covalent
Photochemical Transformation of Protein Hybrids
Noriyuki Uchida,† Kou Okuro,*,† Yamato Niitani,‡ Xiao Ling,‡ Takayuki Ariga,‡ Michio Tomishige,*,‡
and Takuzo Aida*,†
†Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo
113-8656, Japan
‡Department of Applied Physics, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
S
* Supporting Information
ABSTRACT: A water-soluble dendron with a fluorescein
isothiocyanate (FITC) fluorescent label and bearing nine
pendant guanidinium ion (Gu+)/benzophenone (BP)
pairs at its periphery (GlueBP-FITC) serves as a “photo-
clickable molecular glue”. By multivalent salt-bridge
formation between Gu+ ions and oxyanions, GlueBP-
FITC temporarily adheres to a kinesin/microtubule
hybrid. Upon subsequent exposure to UV light, this
noncovalent binding is made permanent via a cross-linking
reaction mediated by carbon radicals derived from the
photoexcited BP units. This temporal-to-permanent trans-
formation by light occurs quickly and efficiently in this
preorganized state, allowing the movements of micro-
tubules on a kinesin-coated glass plate to be photochemi-
cally controlled. A fundamental difference between such
temporal and permanent bindings was visualized by the
use of “optical tweezers”.
Figure 1. Schematic structure of the “photoclickable molecular glue”
GlueBP-FITC. Orange-and-green circles represent pendant adhesive
n living systems, fusion of certain biological motifs is known
to occur in a stepwise fashion through intermediates upon
guanidinium ion (Gu+)/photoreactive benzophenone (BP) pairs.
I
temporal adhesion.1−7 Such a dynamic nature is essential to
prevent unfavorable kinetic traps from interfering with the
cascade sequence of assembly events. An example of stepwise
fixation in biology is immobilization of leukocytes at vascular
endothelial cells,1−3 where leukocytes are immobilized
temporarily via selectin at the initial stage and then
permanently via integrin. A similar mechanism operates for
blood coagulation,4,5 where fibrinogen utilizes its cell-adhesive
peptide sequence to adhere onto cell surfaces before being
polymerized for permanent fixation.
Here we report GlueBP-FITC (Figure 1) as a “photoclickable
molecular glue” that allows for efficient transformation of
temporal (noncovalent) adhesion into permanent (covalent)
fixation by UV light (Figure 2). This molecular glue is
composed of a water-soluble dendron that accommodates
guanidinium ion (Gu+) pendants at its periphery. For optical
visualization, the core part of this dendron is fluorescently
labeled with fluorescein isothiocyanate (FITC). We recently
reported that a prototype of this dendritic molecular glue
adheres to proteins,8,9 phospholipid membranes,10 and clay
nanosheets11 via formation of a multivalent salt bridge between
the Gu+ pendants and oxyanions.12 In proximity to each Gu+
pendant, the newly designed GlueBP-FITC bears a benzophe-
Figure 2. Schematic illustration of the “photoclickable molecular glue”
concept: GlueBP-FITC covalently binds to a protein via photo-
excitation of benzophenone (BP) units after noncovalent adhesion
mediated by formation of salt bridges between guanidinium (Gu+)
ions and protein oxyanions.
none (BP) unit, whose photoexcited triplet state can generate a
reactive carbon radical.13 When GlueBP-FITC adheres to a
Received: January 30, 2013
Published: March 12, 2013
© 2013 American Chemical Society
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Journal of the American Chemical Society
Communication
protein, this carbon radical efficiently gives rise to the formation
of a covalent bond between GlueBP-FITC and the target protein
(permanent fixation; Figure 2).14 In a proof-of-concept study,
we utilized GlueBP-FITC for photocontrol of the translational
movements of microtubules on a kinesin-coated glass surface15
driven by ATP-fueled hand-over-hand motions of kinesin.16 We
also highlight results of an investigation using “optical tweezers”
that shows how the affinity of microtubules with kinesin
changes upon photoclicking of adhering GlueBP-FITC.
is also noteworthy that the secondary structure of BSA, as
observed by its circular dichroism (CD) spectral profile at
200−250 nm (Figure S4), was virtually unchanged by this
covalent immobilization process.17
GlueBP-FITC can covalently unite different proteins. For a
demonstration, we chose a microtubule/kinesin biomachinery
system. Kinesin is a motor protein that walks hand-over-hand
along a microtubule strand,16,19 fueled by repeated cycles of
binding of ATP followed by its hydrolysis to ADP.20 This
machinery system plays a vital role in biological events such as
cell division21,22 and substance transportation,23−25 for which
kinesin and the microtubule are programmed to undergo
noncovalent heterotropic hybridization. For the purpose of
exploring possible effects of GlueBP-FITC on the machinery
motion, we prepared a flow cell with an interior volume of ∼20
μL in which Cy5-labeled fluorescent microtubules (8.8 ng/mL)
were placed on a kinesin-coated glass plate.17 The flow cell was
then treated with GlueBP-FITC (10 μM) and UV-irradiated for
2 min centered at 300 nm (handy UV lamp).17 As shown in
Figure 4a(iii) and b(iii), even after the admission of ATP buffer
Photoclickable GlueBP-FITC (Figure 1)17 was synthesized by
incorporation of FITC into the focal core of a dendron carrying
azide groups, a “click” reaction between the resultant FITC-
labeled, azide-bearing dendron and dendrons carrying both an
alkyne-substituted focal core and pendant Boc-protected Gu+/
BP pairs, and removal of the Boc groups. To investigate the
photoinduced reactivity of GlueBP-FITC, a mixture of GlueBP-
FITC (3 μM) and bovine serum albumin (BSA) (10 μM) was
irradiated with a Xe arc lamp (305−315 nm bandpass filter) in
Tris-HCl buffer ([Tris] = 20 mM, pH 7.0) over a period of 10
min. In electronic absorption spectroscopy, we observed a
decrease in the absorption intensity at 270−320 nm (Figure S3
in the Supporting Information),17 analogous to the case
reported for photochemical covalent binding of BP with
actin.18 The sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) profiles developed by staining
with Coomassie brilliant blue (CBB) (Figure 3a) did not
Figure 3. SDS-PAGE profiles of BSA (5.0 μM) in (i) the absence and
(ii−vi) the presence of GlueBP-FITC (3.0 μM) (ii) before and (iii−vi)
after UV irradiation at 310 nm (Xe arc lamp, 305−315 nm bandpass
filter) for (iii) 1, (iv) 2, (v) 5, and (vi) 10 min, developed by (a)
staining with CBB or (b) fluorescence emission of FITC excited at 488
nm.
change with the exposure time (iii−iv) but were substantially
identical to those of (i) BSA and (ii) its non-irradiated mixture
with GlueBP-FITC. However, by reference to the SDS-PAGE
profiles developed by FITC emission (λex = 488 nm) without
CBB (Figure 3b), we found that the protein fractions
corresponding to the bands in Figure 3a(iii−vi) were
fluorescent. Since the bands were not fluorescent unless the
mixture of BSA and GlueBP-FITC was UV-irradiated [Figure 3a
and b(ii)], we conclude that GlueBP-FITC was covalently
attached to BSA upon UV irradiation. This photochemical
reaction is rather quick, considering that the band was highly
emissive even after UV irradiation for 1 min [Figure 3b(iii)]. It
Figure 4. Effects of photoclickable GlueBP-FITC (10 μM) on the
translational movements of Cy5-labeled microtubules (8.8 ng/mL) on
a kinesin-coated glass plate at 26 °C in ATP buffer.26 Conditions: (i)
+microtubules; (ii) +microtubules → +GlueBP-FITC; (iii) +micro-
tubules → +GlueBP-FITC → +UV exposure for 2 min; (iv) +GlueBP-
FITC → +UV exposure for 2 min → +microtubules. For the UV
exposure, samples were irradiated with UV light centered at 300 nm
using a handy UV lamp. Before fluorescence microscopy, all of the
samples were rinsed with ATP buffer. (a) Fluorescence microscopy
traces (λex = 635 nm) of Cy5-labeled microtubules in 0−80 s. Scale
bars = 3 μm. (b) Average velocities of the microtubules.
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Journal of the American Chemical Society
Communication
([ATP] = 2 mM) to induce the hand-over-hand motions of
kinesin,26 the microtubules became completely immobile on
the kinesin-coated glass plate, as observed by fluorescence
microscopy (λex = 635 nm). In contrast, without irradiation
[Figure 4a(ii)], the microtubules moved at an average velocity
of 52.8 nm/s [Figure 4b(ii)], which is comparable to the
average velocity of 58.7 nm/s observed in a control experiment
without GlueBP-FITC [Figure 4a and b(i)]. On the other hand,
when the kinesin-immobilized glass plate was first treated with
GlueBP-FITC (10 μM) and then irradiated for 2 min at 300 nm
prior to hybridization with microtubules,17 admission of ATP
buffer as a fuel resulted in movement of the microtubules
[Figure 4a and b(iv)]. Therefore, the GlueBP-FITC-mediated
photoreaction did not deteriorate the motor function of
kinesin. BP in GlueBP-FITC was essential for the covalent
immobilization, as the use of a dendritic glue without BP10
under identical conditions resulted in no photoinduced arrest
of the microtubules (Figures S6 and S7).17
The above observations prompted us to investigate how the
binding strength between kinesin and the microtubules
changed upon adhesion with GlueBP-FITC and subsequent
photoclicking. For this purpose, we made use of “optical
tweezers”, which utilize a focused laser beam to generate a
trapping force in the piconewton range27,28 for holding a
polystyrene (PS) bead. Figure 5a shows a schematic illustration
of the principle of optical tweezers for our experiment. Through
a strong His6 tag (kinesin)/anti-His6 antibody (PS bead)
interaction, PS beads (4.7 pM) were attached to adenosine 5′-
(β,γ-imido)triphosphate (AMP-PNP)−kinesin (no ATPase
activity; 5.7 nM) that had been hybridized beforehand with
microtubules (0.4 mg/mL) immobilized on a glass plate.17 The
above setup was constructed within a flow cell having an
interior volume of ∼10 μL (Figure 5a). With a 1064 nm laser
beam, 20 individual PS beads were held one-by-one and pulled
to check whether they could be peeled off at a given trapping
force, as determined by bright-field optical microscopy. The
trapping force for holding a PS bead was varied over the range
30−200 pN,17 where junction points other than that formed by
the kinesin/microtubule hybridization are supposed not to
break up.29−31 Thus, when the trapping force is insufficient for
the kinesin/microtubule hybrid to break up, the PS bead, which
may have initially moved along a pulled direction, is eventually
released from the optical tweezers and returned to the origin
spontaneously (Figure 5c). On the other hand, when the
kinesin/microtubule hybrid break up, the bead loses positional
control after the laser trapping is turned off (Figure 5d). It
should be noted that 1:1 kinesin/microtubule hybrids have
been reported to break up when the kinesin part is pulled by an
applied force of ∼17 pN.32,33 Nevertheless, the stoichiometry of
the kinesin/microtubule hybridization was unspecified and
differed from bead to bead. Thus, we conducted three sets of
the above experiments, each using a separately prepared flow-
cell setup, and the data at each individual trapping force were
averaged (Figure 5b).
Figure 5. (a) Schematic illustration of the experimental setup using
“optical tweezers” for evaluating the peel-off profiles of PS beads at 24
°C in bead buffer.26 In a flow cell (∼10 μL), PS beads (4.7 pM) having
anti-His6 antibodies were allowed to attach noncovalently to the His6
tag sequence of (AMP-PNP)−kinesin (no ATPase activity; 5.7 nM)
that had been hybridized beforehand with microtubules (0.4 mg/mL)
immobilized on the glass substrate. The the cell was then UV-
irradiated at 300 nm (handy UV lamp). (b) Average numbers of
residual PS beads that were not peeled off at various trapping forces
applied. Samples were untreated (green) or treated with GlueBP-FITC
(4 μM) without (blue) or with (red) UV exposure for 3 min. (c, d)
Bright-field microscopy traces of PS beads (c) returning and (d)
peeled off after the laser trapping was turned off. Scale bars = 1 μm.
than 100 pN, the peel-off profile appeared to resemble that
observed without GlueBP-FITC (green circles). If it is supposed
that the binding stoichiometry at each bead remained
unchanged upon treatment with GlueBP-FITC, the observed
difference in the peel-off profiles indicates that even non-
covalent adhesion of GlueBP-FITC reinforced the kinesin/
microtubule hybrid to a certain extent. We then performed UV
irradiation centered at 300 nm (handy UV lamp) for 3 min to
transform the noncovalent adhesion into a covalent one (Figure
2).17 Obviously, the number of residual beads greatly increased,
as only 4.7 PS beads were peeled off at a maximum trapping
force of 200 pN (Figure 5b, red circles). As expected, unless the
sample was pretreated with GlueBP-FITC, the UV irradiation
hardly changed the peel-off profile (Figure S10).17
In conclusion, we have demonstrated that water-soluble
GlueBP-FITC (Figure 1) having nine pendant guanidinium ion
(Gu+)/benzophenone (BP) pairs can serve as a “photoclickable
molecular glue” that covalently attaches to proteins or unites
different proteins through noncovalent preorganization. As
studied by “optical tweezers”, the temporal-to-permanent
photochemical transformation gives rise to a notable change
Before the treatment with GlueBP-FITC, the average number
of residual PS beads that were not peeled off, among 20 beads
tested, decreased monotonically from 15.3 to 5.7 as the applied
trapping force was increased from 33 to 131 pN (Figure 5b,
green circles). However, after the treatment with GlueBP-FITC
(4 μM),17 the number of residual PS beads obviously increased
(Figure 5b, blue circles), and the peel-off profile as a function of
the applied trapping force showed an inflection point at 100
pN. In the range where the applied trapping force was larger
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Communication
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in binding strength. This transformation proceeds quickly and
efficiently, most likely due to the location of the photoreactive
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(26) ATP buffer (pH 6.8): [PIPES] = 80 mM, [ATP] = 2 mM,
[MgCl2] = 1 mM, [EGTA] = 0.5 mM, [glucose] = 4.5 mg/mL,
[glucose oxidase] = 2.16 mg/mL, [catalase] = 36 μg/mL, [creatine
phosphate] = 2 mM, [creatine kinase] = 10 μg/mL, [2-
mercaptoethanol] = 0.5%, [paclitaxel] = 20 μM. Bead buffer (pH
6.8): [PIPES] = 80 mM, [MgCl2] = 1 mM, [EGTA] = 0.5 mM,
[paclitaxel] = 20 μM, [casein] = 4 mg/mL, [bead] = 4.7 pM.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1
Synthesis of GlueBP-FITC; H NMR, 13C NMR, and MALDI-
TOF-MS spectral data; electronic absorption and CD spectra;
preparation of microtubule and kinesin samples for microscopy;
and related experimental procedures. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
okuro@macro.t.u-tokyo.ac.jp; tomishige@ap.t.u-tokyo.ac.jp;
aida@macro.t.u-tokyo.ac.jp
■
(34) Zhang, Y.; Xu, W. Anticancer Agents Med. Chem. 2008, 8, 698.
(35) Fuster, M. M.; Esko, J. D. Nat. Rev. Cancer 2005, 5, 526.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge the Center for NanoBio Integration at the
University of Tokyo. This work was partially supported by
Grants-in-Aid for Research Activity Start-up (23850007 to
K.O.) and Scientific Research on Innovative Areas “Emergence
in Chemistry” (20111010 to T. Aida) from MEXT. N.U. thanks
JSPS for financial support through a Young Scientist Fellow-
ship.
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