Substrate selectivity and its mechanistic insight of the photo-responsive non-nucleoside triphosphate for myosin and kinesin

Article abstract of DOI:10.1039/c8ob02714e

Linear motor proteins including kinesin and myosin are promising biomaterials for developing nano-devices. Photoswitchable substrates of these biomotors can be used to optically regulate the motility of their associated cytoskeletal filaments in in vitro

Full text of DOI:10.1039/c8ob02714e

Organic &
Biomolecular Chemistry
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PAPER
Substrate selectivity and its mechanistic insight
of the photo-responsive non-nucleoside
triphosphate for myosin and kinesin†
Cite this: Org. Biomol. Chem., 2019,
17, 53
Md. Jahirul Islam,
‡
a,b Kazuya Matsuo,‡a,b Halley M. Menezes,a,b
Masayuki Takahashi,c Hidehiko Nakagawa, d Akira Kakugo, c Kazuki Sadac and
a,b
Nobuyuki Tamaoki
*
Linear motor proteins including kinesin and myosin are promising biomaterials for developing nano-
devices. Photoswitchable substrates of these biomotors can be used to optically regulate the motility of
their associated cytoskeletal filaments in in vitro systems. Here, we describe the discovery of the myosin
selective azobenzene-tethered triphosphate. It enables the specific photocontrol over myosin in a revers-
ible mode with the composite motility assay composed of both kinesin and myosin. The mechanistic
insight into this myosin selectivity is also explained with the docking simulation study.
Received 1st November 2018,
Accepted 3rd December 2018
DOI: 10.1039/c8ob02714e
rsc.li/obc
Gliding motility assay is a well-established model system to
Introduction
study and engineer bio-molecular motors in vitro.4,10 In this
Molecular motor proteins including the linear and rotary versatile approach, the gliding motility of fluorescently labeled
motors are mechanical enzymes that can move by catalytically microtubules or actin filaments driven by bio-motors (kinesin
converting chemical energy of adenosine triphosphate (ATP) or myosin) which are bound to the glass surface can be visual-
into mechanical work.1–3 In particular, cytoskeletal motor pro- ized by fluorescence microscopy. To develop controllable nano-
teins including conventional kinesin-14,5 and myosin II6,7 that devices using bio-molecular motors, various external stimuli
can move unidirectionally along their associated cytoskeletal including electricity,11–13 heat,14–21 light22–27 and so on have
filaments are pivotal for maintaining the cellular functions. been applied. In particular, photo-controllable approaches
Kinesin-1 mainly conducts the intracellular active transport of should be drawing the center of attention due to their high
organelles and macromolecular assemblies along micro- spatiotemporal operability and high switchability. For
tubules from the cell center towards the cell periphery (i.e. instance, Higuchi et al.22 and Hess et al.23 demonstrated caged
anterograde transport). Myosin II moving along actin filaments ATP (photolabile protected ATP) as the photocontrolled one-
toward the barbed end is a leading contributor to muscle con- way switch that was from motility OFF to ON using ultraviolet
traction and cell migration. In spite of the different associated light. In addition to these excellent methods, our group
filaments, these two biomolecular motors possess similar recently reported the reversibly photocontrollable ATP ana-
engines using ATP to generate the mechanical force. Due to logues, i.e. non-nucleoside triphosphate molecules based on
this immense potential of producing force with nano-sized azobenzene (AzoTPs), for kinesin-128 and myosin II.29 AzoTPs
motors, numerous attempts have been made to develop func- in the trans state of their azobenzene moiety can be recognized
tional nano-devices.8,9
as the enzyme substrate for both kinesin and myosin as well as
natural energy molecule ATP for powering the motor proteins,
whereas they in the cis state cannot be recognized (Fig. 1).
These AzoTPs are rapidly photoconverted without any side pro-
ducts due to the photoisomerization reaction of azobenzene
with the picosecond time scale.30,31 In contrast, caged ATP
including the 2-nitrobenzyl type of the photoreactive group is
photoactivated with a millisecond time scale32 to produce ATP
with a highly reactive nitroso compound with proteins as the
byproduct. Using these highlighted photochemical properties
of AzoTPs, we demonstrated the repetitive switching of kinesin
or myosin-based cytoskeletal filament translocation with exter-
aResearch Institute for Electronic Science, Hokkaido University, Kita 20, Nishi 10,
Kita-Ku, Sapporo, Japan. E-mail: tamaoki@es.hokudai.ac.jp
bGraduate School of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku,
Sapporo, Japan
cFaculty of Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Japan
dGraduate School of Pharmaceutical Sciences, Nagoya City University, 3-1, Tanabe-
dori, Mizuho-ku, Nagoya, Japan
†Electronic supplementary information (ESI) available: Fig. S1–S17, Tables S1
and S2, Supplementary movies, and NMR and HR-MS spectra. See DOI: 10.1039/
c8ob02714e
‡These authors contributed equally to this work.
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Results and discussion
Design and synthesis of AzoTP derivatives and their
photoresponsibility
To explore the powering properties towards kinesin and
myosin, four AzoTP derivatives (1a–1e) except for 1c with
different types of linkages and azobenzene substituents were
prepared according to our previous reports.28,29 AzoTP deriva-
tive 1c was newly synthesized as depicted in Scheme 1
(Experimental section) with the details of synthetic pro-
cedures. The characterization (1H NMR, 13C NMR, 31P NMR
and HR-MS) and the purity of 1c are also shown in Fig. S1 and
S9–S17 (ESI†). These AzoTPs were designed to compare the
length and flexibility of linkers in ethyl (1b), propyl (1c) and
ethyl ether linkages (1d) with the original acetamide linker in
1a and the steric and hydrophobic effects of 3′- and 4′-methyl
groups on an azobenzene ring in 1e.
Fig. 1 Schematic representation of cis–trans photo isomerization of
1a–1e with UV and VIS light irradiation.
nal light stimuli without any significant fatigue of cytoskeletal
filament motility.
Photochromic azobenzene triphosphates can undergo
reversible cis–trans isomerization by absorbing ultraviolet
(UV) and visible (VIS) light, respectively (Fig. 1). UV-VIS
absorption spectra of 1a–1e in BRB-80 buffer before
irradiation (BI) and after UV (365 nm) or VIS (436 nm)
irradiation are shown in Fig. 2 and Fig. S2 (ESI†). By the illu-
mination of UV light to form cis rich states of AzoTPs (UV
photostationary state, UV PSS), the π–π* band at around
327 nm in absorbance spectra decreased, while the n–π*
band at around 430 nm increased. An opposite trend (namely,
the increment of the π–π* band and the decrement of the
n–π* band) was observed by applying VIS light to reach the
trans rich states (VIS PSS). The cis–trans isomer ratio of 1a–1e
at UV PSS and VIS PSS was calculated by 1H NMR measure-
ments as shown in Table S1 and Fig. S3 (ESI†). The reversible
photoisomerization of 1a–1e could be recycled several times
by alternating UV and VIS light irradiation without any
fatigue. cis isomers of all AzoTPs shown here were stable in
BRB-80 buffer at 25 °C (t1/2 > 3 days) enough to show an insig-
Due to the well-conserved engine mechanism in kinesin
and myosin,33 ATP can efficiently power each kinesin and
myosin. In sharp contrast, other nucleotides including syn-
thetic ATP analogues exhibited unique specificity towards
kinesin and/or myosin.34–43 The specific substrate should be a
useful tool for analyzing which proteins are responsible for the
motor-based motility in a crude system. Herein, we describe
the successful discovery of the myosin-selective photoswitch-
able energy molecule. Through the structure–activity relation-
ship study in both microtubule–kinesin and actin–myosin
systems, we found the myosin-selective AzoTP derivative (1e)
with hydrophobic bulky substitution on the azobenzene
moiety retaining high photoswitchability, which was also
demonstrated in the kinesin–myosin composite system.44
Compared with ATP as a standard substrate, 1e exhibited 9.4-
fold higher selectivity toward the actin–myosin system than
the microtubule–kinesin system. This selectivity mechanism
was rationally explained through the docking simulation
study.
Scheme 1 Synthetic scheme of 1c. Reagent and condition: (a) CuI, bis(triphenylphosphine)palladium(II) dichloride, 2-propyn-1-ol, triethylamine, RT,
18 h. (b) 10% Pd/C, H2, MeOH, RT, 48 h. (c) Nitrosobenzene, AcOH, toluene, N2 atmosphere, 60 °C, 21 h. (d) Di-tert-butyl N,N-diisopropyl-
phosphoramidite, 1H-tetrazol, dry THF, Ar atmosphere, RT, 6 h, then mCPBA, 0 °C, 1 h. (e) TFA, dry DCM, Ar atmosphere, RT, 6 h. (f) Tributylamine,
carbonyldiimidazole, tributylammonium pyrophosphate, dry DMF, Ar atmosphere, RT, overnight.
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Fig. 2 UV-VIS absorption spectra of 1a and 1e before irradiation and at UV photo-stationary state (PSS) and VIS PSS. UV-VIS absorption spectra of
(a) 1a (4.4 × 10−4 M) and (b) 1e (5.2 × 10−4 M) in BRB-80 buffer at 25 °C. Before irradiation (black line), UV PSS (red line), and VIS PSS (dark green line).
Insets: Absorbance changes after the alternate irradiation with UV (20 s) and VIS (150 s) light for 5 cycles.
nificant thermal back reaction during the motility experi- gliding velocity (Vmax) of actin filaments with 1c was 1.4 µm
ments (Fig. S4, ESI†).
s−1, which was 27% of Vmax (5.1 µm s−1) compared with ATP
(Fig. S5†). The gliding velocity of actin filaments was reversibly
controlled for several cycles with repeated UV and VIS light
illumination (Fig. 3b). Similarly to other AzoTPs in the actin–
myosin system,29 the cis isomer of 1c had no contribution to
powering the myosin. The motility at UV PSS originated from
the remaining trans isomer of 1c (12%) as plotted in the
theoretical curves (dashed black line in Fig. 3a). Therefore, we
AzoTP derivatives in actin–myosin in vitro motility assay
Very recently, we reported that AzoTPs 1a, 1b, 1d and 1e
worked as photo-responsive myosin substrates in a photorever-
sible mode in actin–myosin gliding motility assay
(Table S2†).29 The newly synthesized 1c was studied in the
actin–myosin gliding motility assay (Fig. 3). The maximum
●
Fig. 3 Photoresponsive actin gliding motility with 1c (a) Gliding velocity depending on the concentration of 1c. (Filled circle ( ): BI condition and
○
open circle ( ): after the UV irradiation condition). Solid black line: curve fitting using the Michaelis–Menten equation (Km = 0.28 0.09 mM, Vmax
=
1.4 0.2 µm s−1 for 1c) and dashed black line: theoretical curve derived from the solid black line for the remaining trans isomer (12%) in the cis rich
state of 1c. (b) Reversible photoregulation of the actin gliding velocity using 1c (0.30 mM) with repeated UV and VIS light irradiation. Error bars rep-
resent the standard deviation of 10 actin filaments in a single flow cell.
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concluded that in the actin–myosin system, a wide range of derivatives, 1b–1e, as the substrates of kinesin-1 to drive micro-
AzoTP derivatives with different linkers and azobenzene moi- tubule gliding in the microtubule–kinesin system (Fig. 4, S6
eties exhibited the myosin-powering activities in a photo- (ESI†) and Table 1). Photoswitchability of gliding velocity of
switchable mode.
microtubules by alternating illumination with UV and VIS
light was also studied. AzoTP derivatives 1b (0.51 µm s−1 at
3.0 mM) and 1c (0.45 µm s−1 at 3.0 mM) exhibited comparable
velocity with 1a under the BI condition. Upon the subsequent
UV irradiation, the gliding velocity decreased to 0.31 µm s−1 at
3.0 mM with 1b and 0.34 µm s−1 at 3.0 mM with 1c (Fig. 4a, b
and Fig. S6, ESI†), but their changes between BI and UV con-
ditions were not drastic compared with the actin–myosin
system (Table S2† and Fig. 3). The reversible photoswitching of
microtubule gliding velocity using 1b and 1c was also achieved
with alternating irradiation of UV and VIS light (Fig. S7, ESI†).
AzoTP derivatives in microtubule–kinesin in vitro motility
assay
So far, only 1a28 was exploited in microtubule–kinesin in vitro
motility assay in our previous study. Using 1a, the microtubule
gliding velocity was drastically photo-switched in concert with
cis–trans photoisomerization of 1a by illumination with two
different wavelengths of light (BI: 0.51 µm s−1, UV: 0.12 µm s−1
at 3.0 mM). Encouraged by successful application of our
AzoTPs to the actin–myosin system, we investigated AzoTP
●
○
Fig. 4 Microtubule gliding velocity (filled circle ( ): BI condition and open circle ( ): UV condition) with AzoTP derivatives (a) 1b, (b) 1c, (c) 1d and
(d) 1e.45 Solid black line: curve fitting using the Michaelis–Menten equation (Km = 0.82 0.07 mM, Vmax = 0.66 0.02 µm s−1 for 1b; Km = 0.93
0.09 mM, Vmax = 0.60 0.02 µm s−1 for 1c, and Km = 1.0 0.4 mM, Vmax = 0.15 0.03 µm s−1 for 1d; Km = 2.1 1 mM, Vmax = 0.058 0.02 µm s−1
for 1e). Dashed black line: theoretical curve calculated from the remaining trans isomer in the cis rich state of the respective AzoTP derivatives. Error
bars represent the standard deviation of 10 microtubules in a single flow cell.
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Table 1 Summary of AzoTP derivatives in the microtubule–kinesin
system
We also confirmed the kinesin selectivity of GTP in our
motility system. GTP could power kinesin to drive micro-
tubules efficiently (0.21 µm s−1 at 1.0 mM), whereas it could
not work efficiently (0.049 µm s−1 at 1.0 mM) in the actin–
myosin motility system (Fig. S5 and S6c, ESI†). This special
trend encouraged us to compare AzoTPs in both myosin
and kinesin systems in a quantitative way. To directly
demonstrate the powering activities and efficiencies of our
AzoTPs for myosin and kinesin, ATP was used as the stan-
dard substrate for both the kinesin and myosin motor
protein systems (Table 2). AzoTPs (1a–1d) exhibited moder-
ate activity towards both myosin (>27%Vmax of ATP) and
kinesin (>16%Vmax of ATP). However, AzoTP 1e showed
different trends toward myosin and kinesin. In the myosin,
1e showed 59% of Vmax and 16% of 1/Km (3.7 mM−1) com-
pared with ATP. In the kinesin, 1e showed 6.3% of Vmax and
3.8% of 1/Km (0.48 mM−1) compared with ATP. This high
activity specificity of 1e (Myo/Kin = 9.4) with high photo-
switchability (Table 2) indicated that 1e was recognized as a
substrate of not kinesin-1 but myosin II, although other
AzoTPs (1a–1d) worked as substrates for both motor
proteins.
AzoTP
derivatives
Vmax
Gliding velocity
switchinga (%)
(µm s−1
)
Km (mM)
1a23
1b
1c
1d
1e
0.83 0.06
0.66 0.02
0.60 0.02
0.15 0.03
0.058 0.02
1.7 0.2
76
39
26
27
0.82 0.07
0.93 0.09
1.0 0.4
2.1
1
N.D.
a Gliding velocity switching between trans and cis-rich states of AzoTP
derivatives at saturated concentration, 3.0 mM for 1a, 1b, 1c and 1d;
2.0 mM for 1e.45 N.D.: not determined.
These results indicated that the flexibility and length of linker
chains between the azobenzene moiety and triphosphate
group (acetamide linker for 1a, ethyl linker for 1b and propyl
linker for 1c) had a trivial effect on powering the micro-
tubule–kinesin system. AzoTP 1d with the ethyl ether linker
showed less activity (BI: 0.07 µm s−1 at 3.0 mM) than the orig-
inal AzoTP 1a. There was no significant change in the micro-
tubule velocity between BI and UV conditions (UV: 0.10 µm
s−1 at 3.0 mM) although the clear photo-isomerization from
the trans isomer to cis isomer was confirmed by the UV-VIS
spectra (Fig. S2c, ESI†). The ethyl ether linker in 1d had
almost the same length as the propyl linker in 1c, but it was
found that the oxygen atom in the ether group of 1d nega-
tively affected the powering activity and photoswitchability in
the microtubule–kinesin system. This might be because the
ether group worked as the acceptor of hydrogen bonding,
which could interrupt the substrate recognition of 1d by
kinesin. To quantitatively assess the powering ability of cis-
isomers of AzoTPs in the microtubule–kinesin system, we
plotted theoretical curves (dashed black line in Fig. 4a–c and
S6a†) corresponding to the remaining trans isomer (Table S1,
ESI†) of AzoTPs under UV irradiation conditions.
Inconsistency in 1b, 1c and 1d between the theoretical curves
and the actual velocity under UV conditions implied that the
cis isomers had significant activity on gliding motility. In con-
trast to 1a–1d (0.47 µm s−1 for 1a, 0.48 µm s−1 for 1b, 0.41 µm
s−1 for 1c and 0.09 µm s−1 for 1d at 2.0 mM concentration), to
our surprise, 1e with two methyl groups in the azobenzene
moiety showed almost no powering activity (0.027 µm s−1 at
2.0 mM) and photoswitchability towards kinesin-1 (Fig. 4d
and 5).45 Representative fluorescence images of the gliding
motility of microtubules with 1e at 8 min interval for both in
the BI state and after UV illumination state are shown in
Fig. 5. Vmax (0.058 µm s−1) with 1e was only 6.3% of Vmax
(0.92 µm s−1) with ATP (Fig. S6, ESI†) in the microtubule–
kinesin motility assay. Therefore, we concluded that the
methyl substitution critically impeded the substrate reco-
gnition of AzoTP in the kinesin motor.
Computational docking simulation of AzoTP derivatives with
the motor domains of kinesin-1 and myosin II
Since 1e displayed high selectivity towards myosin II compared
with kinesin-1, the putative binding modes of AzoTPs 1a (the
good substrate for both kinesin and myosin) and 1e with
kinesin-1 and myosin II were investigated through the docking
simulations (Fig. 6–8). In the simulation results of kinesin-1
and myosin II with the trans isomer of 1a, triphosphate groups
were chelated with magnesium ions at the same binding site
of the triphosphate group in ATP (Fig. 6a and b). In the azo-
benzene moiety, the benzene ring connected with the tripho-
sphate group through an acetamide linker was well-overlapped
with the adenine ring of ADP in the original X-ray structures of
both kinesin and myosin (Fig. S8, ESI†). In myosin, the azo
group (–NvN–) can interact via a hydrogen bond with Arg131
of covering the adenine ring binding site (ARBS), which could
fix the angle of the azobenzene moiety. Another benzene ring
in the azobenzene moiety was located outside of the ATP
binding site to face the solvent direction. Therefore, it was
likely that there was no interaction of that benzene ring with
the surrounding amino acids. In kinesin, His93 was the key
residue for exhibiting ATPase activity.33 From the simulation,
the benzene unit in 1a was estimated to show the π–π inter-
action with His93 as well as ATP. Due to no amino acids cover-
ing ARBS unlike myosin, the azo group (–NvN–) was relatively
flexible. This flexibility possibly contributed to arranging the
outside benzene ring of the azobenzene moiety not to facing
the solvent direction but to being buried in other space near
the ATP binding site (Fig. 6b). Despite slightly different
binding modes of the trans isomer of 1a with kinesin and
myosin, these well-fittings were robustly consistent with the
Comparison of AzoTPs between myosin and kinesin systems
Guanosine triphosphate (GTP) has been reported as a results on the motility assays that 1a exhibited relatively high
kinesin-selective natural nucleoside triphosphate (NTP).35,39 activities for powering kinesin (4.7% of 1/Km and 90% of Vmax
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Fig. 5 Fluorescence images of the gliding microtubules driven by 1e (2.0 mM) on microtubule–kinesin gliding motility assay. (Top) Before the
irradiation state; (bottom) after the UV irradiation state; (left side) starting frame; (right side) frame after 8 min; red arrows: leading points of the
microtubules at the starting frame; blue arrows: leading points of the same microtubules after 8 min.
Table 2 Summary of efficacy of AzoTP derivatives as the photo-responsive energy molecule in microtubule–kinesin and actin–myosin systems
Microtubule–kinesin system
Actin–myosin system
F-actin velocityb (µm s−1
BI
MT velocitya (µm s−1
)
)
Activity specificity
(Myo/Kin) under
BI condition
NTPs/AzoTPs
BI
UVc
UVc
ATP
GTP
1a
1b
1c
1d
1e
0.92 0.01 (100%)
0.55 0.03 (60%)
0.83 0.06 (90%)
0.66 0.02 (72%)
0.60 0.02 (65%)
0.15 0.03 (16%)
0.058 0.02 (6.3%)
0.92 0.01 (100%)
0.55 0.03 (60%)
0.12 0.01 (13%)
0.31 0.03 (34%)
0.34 0.02 (37%)
0.10 0.02 (11%)
N.D.
2.9 0.124 (100%)
0.049 0.01d (1.0%)
1.5 0.0424 (52%)
1.0 0.124 (35%)
1.4 0.2e (27%)
2.9 0.124 (100%)
0.049 0.01d (1.0%)
0.84 0.124 (29%)
0.13 0.0224 (4.5%)
0.34 0.03e (6.7%)
0.38 0.0424 (13%)
0.39 0.0524 (13%)
1.0
0.02
0.6
0.5
0.4
4.1
9.4
1.9 0.124 (66%)
1.7 0.224 (59%)
a Microtubule (MT) velocity at Vmax b F-actin filament (F-actin) velocity at Vmax c Under UV conditions, the saturated concentration of AzoTPs in
. .
the corresponding motility assays was considered. d Velocity at 1.0 mM of GTP was considered because Vmax is not determinable due to too low
activity. e The different Vmax (5.1 0.2 µm s−1) of ATP was used for the calculation, because we used the different batch of myosin and actin from
our previous report in this new experiment. Activity specificity, Myo/Kin is the ratio of myosin driven actin filament standardized velocity and
kinesin driven microtubule standardized velocity at Vmax. The standardized velocity is shown in the parentheses with the actual velocity where
the Vmax of ATP is considered as the standard velocity (100%) for each of the motor protein systems. N.D.: not determined.
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Fig. 6 Computational docking simulation of trans isomers of 1a with kinesin-1 and myosin II motor domains. Docking simulation of AzoTPs was
conducted with CLC Drug Discovery Workbench (QIAGEN). X-ray crystal structures obtained from RCSB Protein Data Bank (PDB entry code: 4HNA
for kinesin-1 and 1MMD for myosin II) were used. All figures of simulated results were created with pymol (DeLano Scientific). (a) Simulated result
and (b) binding modes of the trans isomer of 1a in kinesin-1. (c) Simulated result and (d) binding modes of the trans isomer of 1a in myosin II. In simu-
lated results, AzoTPs were represented by a stick mode. Magnesium ions were denoted by a yellow ball. In the figures of binding modes, the amino
acid resides composing ARBS were colored in blue and the residues composing the triphosphate binding site were colored in green.
Fig. 7 Computational docking simulation of cis isomers of 1a with (a) kinesin-1 and (b) myosin II motor domains.
compared with ATP) and myosin (43% of 1/Km and 52% of and myosin for explaining the low affinity of the cis isomer of
Vmax compared with ATP) under the BI condition. Conversely, 1a in kinesin and myosin. As shown in Fig. 7a and b, the total
the cis isomer of 1a has almost no contribution to the gliding incompatibility of the cis isomer of 1a in ARBS was observed,
velocity of cytoskeletal filaments in both the motor protein although the triphosphate group was well-bound. This
systems.28,29 We also docked the cis isomer of 1a with kinesin suggested that the bent azobenzene unit prevented the
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Fig. 8 Computational docking simulation of trans isomers of 1e with kinesin-1 and myosin II motor domain. Docking simulation of AzoTPs was con-
ducted with CLC Drug Discovery Workbench (QIAGEN). X-ray crystal structures obtained from RCSB Protein Data Bank (PDB entry code: 4HNA for
kinesin-1 and 1MMD for myosin II) were used. All figures of simulated results were created with pymol (DeLano Scientific). (a) Simulated result and
(b) binding modes of the trans isomer of 1e in kinesin-1. (c) Simulated result and (d) binding modes of the trans isomer of 1e in myosin II. In simulated
results, AzoTPs were represented by a stick mode. Magnesium ions were denoted by a yellow ball. In the figures of binding modes, the amino acid
resides composing ARBS were colored in blue and the residues composing the triphosphate binding site were colored in green.
approach of 1a into ARBS. Almost similar binding modes of over the motor proteins. The gliding motility of fluo-
AzoTPs were applied except for 1e, which exhibited high rescence labeled microtubules and actin filaments driven
selectivity to myosin over kinesin. In the docking results of the by respective motor proteins was observed with a fluo-
trans isomer of 1e with myosin, the benzene ring in 1e was rescence microscope.
well-occupied with ARBS as well as other AzoTPs and the
The gliding motility of microtubules and actin filaments
dimethyl-substituted benzene ring was located facing to sol- in the kinesin–myosin composite assay with 1e (1.0 mM)
vents (Fig. 8). In contrast, the docking of 1e with kinesin was examined under different light irradiation conditions
showed the incompatibility of the benzene ring of the azo- (Fig. 9, Movie S1 in ESI†). AzoTP 1e could power myosin to
benzene moiety with ARBS, because the steric hindrance of drive actin filaments with photoreversibility (BI: 1.1
two methyl groups on the azobenzene moiety interrupted the 0.04 μm s−1, UV: 0.27 0.01 μm s−1 and VIS: 0.88 0.03 μm
access of 1e to ARBS. Thus, these computational docking s−1 at 1.0 mM) in the composite motility assay. In contrast,
studies could explain our experimental results of myosin it could not power kinesin efficiently to drive microtubules
selectivity in 1e over kinesin.
(BI: 0.054 0.01 µm s−1 at 1.0 mM). As the control experi-
ment, ATP (0.20 mM) was applied to our kinesin–myosin
composite system. It was shown that both kinesin and
myosin motors were driven efficiently to afford the velocity
Selective photoregulation of myosin motility using 1e in
kinesin–myosin composite motility assay
Since 1e exhibited high selectivity to myosin over kinesin, of microtubules (0.71
0.02 μm s−1) and actin filaments
the selective photoregulation of myosin was explored in (3.3 0.3 μm s−1) (Movie S2, ESI†). These findings suggested
kinesin–myosin composite motility assay,44 where both that AzoTP 1e also worked as the photoswitchable substrate
kinesin and myosin were attached on the same glass with myosin specificity in kinesin–myosin composite moti-
surface and microtubules and actin filaments were glided lity assay.
60 | Org. Biomol. Chem., 2019, 17, 53–65
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Fig. 9 Fluorescence images of the gliding actin filaments and microtubules driven by 1e (1.0 mM) on kinesin–myosin composite motility assay.
(Top) Before irradiation, trans state; (middle) cis-rich state after UV irradiation; (bottom) trans-rich state after VIS light irradiation; (left side) starting
frame; (right side) frame after 30 s. The distance traveled by five actin filaments (green lines) and five microtubules (red lines) were shown using
tracking the path of each actin filament or microtubule tails.
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aminophenyl)propan-1-ol (1c-2) as a dark brown solid. 1H
NMR (400 MHz, CHLOROFORM-D) δ 7.06–7.02 (m, 2H), 6.76
Conclusion
The myosin-specific AzoTP molecule 1e with high photoswitch- (td, J = 7.4, 1.2 Hz, 1H), 6.69 (d, J = 7.4 Hz, 1H), 3.64 (t, J =
ability was discovered through studying the powering activity 6.0 Hz, 2H), 2.64 (t, J = 7.3 Hz, 2H), 1.87 (quin, J = 6.5 Hz, 2H),
of several AzoTPs in the kinesin and myosin motility assays, 1.67 (br, 1H).
respectively. Even in the kinesin–myosin composite system, 1e
(E)-3-(2-(Phenyldiazenyl)phenyl)propan-1-ol (1c-3). A solu-
could drive the myosin selectively. This myosin selectivity in 1e tion of 1c-2 (0.276 g, 1.8 mmol) in toluene (13 mL) was
was explained by its binding modes with kinesin or myosin degassed under a stream of N2 gas for 15 min. To this solu-
motors through the docking simulation. This rational analysis tion, nitrosobenzene (0.193 g, 1.8 mmol) and acetic acid
of binding modes of AzoTPs using both the experimental (0.5 mL) were added under an Ar atmosphere. The reaction
assays and the computational studies might contribute to the mixture was stirred at 60 °C for 21 h. The solvent of the reac-
other motor-specific substrate molecules. We envision that our tion mixture was evaporated and partitioned between water
motor-specific AzoTP can be a chemical tool for unveiling the and DCM. The DCM part was washed several times with water
motor-based biological processes and constructing nanotech- and dried over MgSO4. The solvent was evaporated in vacuo.
nological systems using the microtubule and actin composite The residue was purified by silica gel column chromatography
system.
(hexane/EtOAc, 4/3) to afford 0.374 g (86%) of (E)-3-(2-(phenyl-
diazenyl)phenyl)propan-1-ol (1c-3) as a dark brown solid. 1H
NMR (400 MHz, CHLOROFORM-D) δ 7.89 (dd, J = 8.3, 1.4 Hz,
2H), 7.67 (dd, J = 8.0, 1.2 Hz, 1H), 7.56–7.46 (m, 3H), 7.44–7.36
(m, 2H), 7.31 (t, J = 7.5 Hz, 1H), 3.63 (q, J = 6.1 Hz, 2H), 3.24 (t,
J = 7.2 Hz, 2H), 2.05 (t, J = 6.1 Hz, 1H), 1.97 (quin, J =
6.6 Hz, 2H).
(E)-Di-tert-butyl (3-(2-(phenyldiazenyl)phenyl)propyl) phos-
phate (1c-4). To a solution of 1c-3 (1.22 g, 5.1 mmol) and di-
tert-butyl N,N-diisopropylphosphoramidite (2.1 mL, 6.5 mmol)
in dry THF (20 mL), 1H-tetrazole (1.04 g, 15.0 mmol) was
added. The reaction mixture was stirred for 7 h at room temp-
erature. A solution of m-chloroperoxybenzoic acid (65%, 2.3 g,
8.6 mmol) in dry CH2Cl2 (10 mL) was added. The mixture was
stirred for 1 h in an ice bath and then for 30 min at room
temperature. Saturated aqueous NaHCO3 (60 mL) was added
and the mixture was stirred for further 30 min. The reaction
Experimental
Chemicals
All chemical and biochemical reagents were purchased from
commercial sources (Wako Pure Chemical, Kanto Chemical,
TCI Chemical, Sigma-Aldrich, Biotium and Dojindo Molecular
Technologies) and used without further purification. Thin-
layer chromatography (TLC) was performed on silica gel 60
F
254-precoated aluminum sheets (Merck) and visualized by
fluorescence microscopy. Chromatographic purification was
performed using flash column chromatography on silica gel
60 N (neutral, 63–210 μm, Kanto Chemical).
Synthesis of AzoTP 1c
3-(2-Aminophenyl)prop-2-yn-1-ol (1c-1). A reaction mixture mixture was partitioned between EtOAc and water. The organic
of copper(I) iodide (32 mg, 0.2 mmol), bis(triphenylphosphine) phase was washed with brine (once), dried over MgSO4 and
palladium(II) dichloride (140 mg, 0.2 mmol), and 2-iodoaniline concentrated. The residue was purified through silica gel
(4.4 g, 20 mmol) in 120 mL of triethylamine was stirred under column chromatography (hexane/EtOAc, 3/2) to afford 1.18 g
an Ar atmosphere. After 30 min of stirring, 2-propyn-1-ol (38%) of (E)-di-tert-butyl (3-(2-(phenyldiazenyl)phenyl)propyl)
(1.2 mL, 20 mmol) was added to the reaction mixture and it phosphate (1c-4) as
a
red oil. 1H NMR (400 MHz,
was kept stirring for 18 h at room temperature. The reaction CHLOROFORM-D) δ 7.92 (dd, J = 8.3, 1.4 Hz, 2H), 7.67 (d, J =
mixture was evaporated and partitioned between water and 7.8 Hz, 1H), 7.55–7.34 (m, 5H), 7.33–7.28 (m, 1H), 4.03 (q, J =
DCM. The DCM part was washed several times with water to 6.4 Hz, 2H), 3.25 (t, J = 7.7 Hz, 2H), 2.06 (quin, J = 7.2 Hz, 2H),
remove triethylamine completely, then dried over MgSO4 and 1.47 (s, 18H).
concentrated. The residue was purified by silica gel column
(E)-3-(2-(Phenyldiazenyl)phenyl)propyl phosphate (1c-5).
chromatography (MeOH/DCM, 3/97) to afford 1.95 g (65%) of a Trifluoroacetic acid (1.8 mL) was added to a solution of com-
1
maroon solid 3-(2-aminophenyl)prop-2-yn-1-ol (1c-1). H NMR pound 1c-4 (0.65 g, 1.5 mmol) in dry DCM (16 mL). The reac-
(400 MHz, CHLOROFORM-D) δ 7.28 (d, J = 1.3 Hz, 1H), 7.13 (t, tion mixture was stirred for 6 h at room temperature. MeOH
J = 7.9 Hz, 1H), 6.70–6.66 (m, 2H), 4.55 (d, J = 4.0 Hz, 2H), 4.21 (30 mL) was added and the mixture was evaporated. This pro-
(br, 2H), 1.70 (br, 1H).
cedure was repeated several times to remove CF3COOH com-
3-(2-Aminophenyl)propan-1-ol (1c-2). A solution of 1c-1 pletely. The residue was dried in vacuo. Water (5 mL) was
(1.95 g, 13.2 mmol) in dry MeOH (60 mL) was added to 10% added to the residue and neutralized with 1 M NaOH. This
Pd/C (0.5 g) under a N2 atmosphere at room temperature. crude product was purified by using a DEAE Sephadex A-25
Then N2 gas was changed to H2 gas with a constant pressure column with 0.5 M triethylammonium hydrogencarbonate
from a rubber bladder. The reaction mixture was stirred for (TEAB) buffer (pH 8.0) as an eluent at 4 °C. The fractions con-
48 h and filtered through a Celite pad. The solvent was evapor- taining the product were coevaporated with EtOH to remove
ated and the residue was purified by silica gel column chrom- water and TEAB to afford 0.60 g (96%) of (E)-3-(2-(phenyldiaze-
atography (hexane/EtOAc, 1/1) to afford 1.03 g (51%) of 3-(2- nyl)phenyl)propyl phosphate (1c-5) (triethyl ammonium salt)
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as a red oil. 1H NMR (400 MHz, CD3OD) δ 7.91 (dd, J = 8.4, stoppered 1 mm path length quartz cells. cis–trans photo-
1.3 Hz, 2H), 7.65 (d, J = 7.9 Hz, 1H), 7.57–7.41 (m, 5H), 7.29 (t, isomerization of AzoTP derivatives was performed by using a
J = 8.4 Hz, 1H), 3.94 (q, J = 6.4 Hz, 2H), 3.26 (t, J = 7.8 Hz, 2H), 365 nm UV LED (C11924-101, Hamamatsu) and a mercury
3.17 (q, J = 7.3 Hz, 6H), 2.00 (quin, J = 7.1 Hz, 2H), 1.29 (t, J = lamp (Ushio) with an appropriate bandpass filter for 436 nm.
7.3 Hz, 9H).
Fluorescence imaging of in vitro gliding motility assay was per-
(E)-3-(2-(Phenyldiazenyl)phenyl)propyl
triphosphate, formed in an EMCCD digital camera (DL-604M-0EM-H1,
azopropylTP (1c). The triethylammonium salt of compound Andor Technology) equipped with an epifluorescence micro-
1c-5 (0.58 g, 1.4 mmol) was converted into its tributyl- scope (IX71, Olympus) with a 60×/1.45 NA oil-immersion objec-
ammonium salt through the addition of tributylamine (1 mL, tive lens (PlanApo, Olympus) and an appropriate excitation
4 mmol) in dry MeOH (7 mL). Triethylamine and MeOH were filter set for 640 nm light from a mercury lamp.
evaporated. The tributylammonium salt was dissolved in dry
Preparation of proteins
DMF (12 mL). While stirring, a solution of 1,1′-carbonyldiimi-
dazole (1.2 g, 7.5 mmol) in dry DMF (10 mL) was added under The kinesin used in this study was a recombinant kinesin con-
an Ar atmosphere and then the reaction mixture was left for sisting of 573 amino acid residues from the N-terminus of a
16 h at room temperature. Excess 1,1′-carbonyldiimidazole was conventional human kinesin. This recombinant kinesin, fused
decomposed through the addition of dry MeOH (0.3 mL, with His-tag at the N-terminus (plasmid; pET 30b), was
6 mmol) and stirring for 1 h. This solution was added drop- expressed in E. coli Rossetta (DE3) pLysS and purified through
wise with mixing to a solution of the tributylammonium salt the general method using Ni-NTA-agarose. Purified kinesin
of pyrophosphate, (prepared from tetrasodium pyrophosphate) was distributed in small aliquots and quickly frozen in liquid
(2.0 g, 7.5 mmol) in dry DMF (10 mL). After reacting overnight N2 and stored at −80 °C until use. Tubulin was purified from
at room temperature, the mixture was cooled to 0 °C in an ice porcine brains through two successive cycles of polymerization
bath. Cold water (15 mL, 4 °C) was added with mixing and and depolymerization in the presence of a high-molarity PIPES
then the pH was adjusted to 7 using 1 M NaOH. The reaction buffer.46 To prepare fluorescently labelled microtubules,
mixture was partitioned between diethyl ether and H2O; the tubulin was labelled with CF®633 Succinimidyl Ester (92133,
aqueous phase was evaporated with EtOH at 30 °C and dried. Biotium). CF®633-microtubules were polymerized by copoly-
The crude product was purified by using the DEAE Sephadex merizing labelled and unlabeled tubulin at a ratio 1 : 4 for
A-25 column with the eluent of a linear gradient of 0.2–1.0 M 60 min at 37 °C and stabilized with paclitaxel.
TEAB solution (over 400 min) at 4 °C. The fractions were
Skeletal muscle myosin was purified from chicken pectora-
checked with ESI MS and the desired fractions were mixed lis muscles and heavy meromyosin (HMM) was produced by
with EtOH and evaporated. The resulting residue was dried digestion of myosin using α-chymotrypsin followed by dialy-
in vacuo. The obtained product was dissolved in dry MeOH sis.47 The HMM was distributed in small aliquots and quickly
(1 mL); upon addition of NaI into acetone (1 M, 10 mL) frozen in liquid N2 and stored at −80 °C until use. F-actin was
sodium salt of the product was precipitated. The precipitate extracted from acetone powder of rabbit skeletal muscle and
was washed several times with acetone and lyophilized to prepared by partially modifying the previously reported
afford 0.40 g (50%) sodium salt of compound 1c as a red method48 and labelled with a phalloidin conjugated CF®633
1
powder. H NMR (400 MHz, D2O) δ 8.00 (dd, J = 7.9, 1.7 Hz, dye (00046T, Biotium) at a 1/1.2 (actin/phalloidin) ratio. The
2H), 7.71–7.54 (m, 6H), 7.46 (t, J = 8.4 Hz, 1H), 4.08 (q, J = fluorescently labelled actin was stored on ice for up to 4 weeks.
6.4 Hz, 2H), 3.27 (t, J = 7.8 Hz, 2H), 2.03 (quin, J = 7.1 Hz, 2H). Concentrations of HMM and actin were determined from the
13C NMR (100 MHz, D2O with some drops of CD3OD as a stan- absorbance at 280 nm using the extinction coefficients of 0.63
dard) δC 153.25, 151.32, 141.91, 132.66, 132.62, 132.00, 130.54, and 1.1 for 1 mg ml−1 solutions, respectively.
128.15, 123.57, 116.66, 67.13, 33.18, 28.16. 31P NMR (160 MHz,
In vitro microtubule–kinesin gliding motility assay
D2O (H3PO4 as the external standard)) δP −9.72 (d, J = 19.5 Hz),
−10.71 (d, J = 19.7 Hz), −22.84 (t, J = 19.4 Hz). HR-MS (ESI, Microtubule gliding on surface bound kinesin motors in a
m/z) calculated for C15H19N2O10P3 [M − H]−: 479.01798; found flow cell was observed using fluorescence microcopy at 25 °C,
479.01855.
as described previously.28 A flow chamber was first filled with
3.5 µL solution of kinesin (300 nM) with casein (0.5 mg mL−1
in BRB-80 buffer (pH 6.9; PIPES, 80 mM; MgCl2, 2 mM; EGTA,
)
Physical measurements
NMR spectroscopy (1H, 13C and 31P) was carried out on a 1 mM) and incubated at room temperature for 3 min. Then
400 MHz spectrometer (ECX-400, JEOL). Purity of AzoTP paclitaxel stabilized microtubules (0.25 µM as tubulin dimer)
derivatives was analyzed using a reversed phase HPLC system in BRB-80 buffer containing paclitaxel (10 µM) and casein
(Prominence, Shimadzu). High-resolution mass spectroscopy (0.1 mg mL−1) were perfused and incubated at room tempera-
(HR-MS) experiments were performed by using a Thermo ture for 3 min. After washing with 3.5 µL of BRB80 buffer con-
Scientific Exactive mass spectrometer with electrospray ioniza- taining paclitaxel (10 µM) and casein (0.1 mg mL−1) they were
tion. A FDU-2200 (EYELA) lyophilization system was used for incubated for 1 min. Then BRB80 buffer containing paclitaxel
freeze-drying. UV-Visible spectrophotometry was carried out (10 µM), β-mercaptoethanol (0.14 M), glucose oxidase
on a UV-1800 (Shimadzu) UV-Visible spectrophotometer, in (0.1 mg mL−1), catalase (20 µg mL−1), glucose (20 mM), casein
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(0.5 mg mL−1) and energy molecule (AzoTP derivatives, Computational docking simulation
0.05–2.0 mM) was perfused into the flow chamber. Then the
The computational docking simulation was carried out with
flow cell was subjected to microtubule gliding motility obser-
vation under a fluorescence microscope. Microtubule filament
gliding was imaged using an epifluorescence microscope
based on an IX 70 (Olympus) equipped with a 60×/NA 1.45 oil
immersion objective lens (PlanApo, Olympus). Images
obtained under the fluorescence microscope were projected
onto an EMCCD camera (DL-604M-0EM-H1, Andor
Technology). The camera was controlled by the andor solis
software platform ver. 4.23.30008.0. The typical frame rate was
1 s and the observation duration was 40–120 s depending on
the faster/slower movement of the microtubules. The position
of the microtubules was determined manually by mouse click-
ing on the tip of the filament in the Fiji image processing soft-
ware package based on ImageJ and tracked typically first and
last frames of the observation. The gliding velocity of at least
10 microtubules in randomly chosen microscopic fields was
measured in each experiment. The velocity of each filament
was determined by a linear least-squares fit, assuming that the
filaments move in a straight line. To study whether cis–trans
rich state AzoTP derivatives induced microtubule gliding vel-
ocity, the flow cell was irradiated with UV 365 nm (0.68 W
cm−2) light for 8 s and visible 436 nm (21.7 mW cm−2) light
for 25 s, respectively.
the program of CLC Drug Discovery Workbench (QIAGEN,
Denmark) using the parameters and scoring function of
PLANTS(PLP) score.49 We manually inspected the docking
modes with high-scored results.
Kinesin–myosin composite motility assay
The flow cell chamber for microscopic observation was pre-
pared by taping a nitrocellulose coated cover slip (18 × 18 mm)
and a glass slide (76 × 26 mm) together with the double-sided
tape to make a flow path having a volume of 3–3.5 µL with a
height of ca. 100 µm. The photocontrollable study was per-
formed by direct irradiation of UV (365 nm, 0.68 W cm−2) and
visible (436 nm, 21.7 mW cm−2) light to the flow chamber. To
achieve the kinesin–myosin composite motility assay, the per-
fusion protocol described below was followed, 3.5 µL HMM
(300 µg mL−1) solution in assay buffer (pH 7.4; HEPES,
100 mM; MgCl2, 4 mM; EGTA, 1 mM; NaCl, 25 mM; DTT,
1 mM) was added to the flow chamber and it was incubated
for 60 seconds. After 60 seconds, 3.5 µL of kinesin solution
containing BSA (kinesin: ca. 300 nM; BSA: 5.0 mg mL−1) in the
assay buffer was perfused into the chamber and it was also
incubated for 3 min. Next, 3.5 µL solution of fluorescent dye
labelled microtubules (0.25 µM as tubulin dimer) containing
paclitaxel (10 µM) and BSA (0.5 mg mL−1) in the assay buffer
was perfused to the chamber and it was incubated for 60
seconds. Then, 3.5 µL of fluorescent dye labelled actin (1.2 µg
ml−1) containing paclitaxel (10 µM) and BSA (0.5 mg mL−1) in
assay buffer was perfused to the chamber and it was incubated
for 60 seconds. After the incubation, 3.5 µL solution of a sca-
venger mixture [paclitaxel (10 µM), glucose oxidase (0.1 mg
mL−1), catalase (20 µg mL−1), glucose (20 mM), BSA (0.5 mg
ml−1), and 1e/ATP (desired concentration)] in the assay buffer
was perfused to the flow cell. The flow cell was subjected to
microtubule and actin filament motility observation under an
epifluorescence microscope (IX 70, Olympus) equipped with
an EMCCD camera (DL-604M-0EM-H1, Andor Technology) at
25 °C.
In vitro actin–myosin gliding motility assay
Actin filament gliding on nitrocellulose coated surface bound
HMM motors in a flow cell was observed using fluorescence
microcopy at 23.5 °C, as described previously.29 In brief, 3.5 µL
HMM (300 µg ml−1) solution in assay buffer (pH 7.4; HEPES,
100 mM; MgCl2, 4 mM; EGTA, 1 mM; NaCl, 25 mM; DTT,
10 mM) was added to the flow chamber and incubated for 60
seconds. After 60 seconds, unbound HMM molecules were
washed by using 3.5 µL assay buffer with BSA (0.5 mg ml−1
)
and incubated for 3 minutes. Then 3.5 µL of CF®633 phalloi-
din labelled actin (1.2 µg ml−1) was perfused to the chamber
and incubated for 60 seconds. After the incubation 3.5 µL solu-
tion of a scavenger cocktail [glucose oxidase (0.1 mg mL−1),
catalase (20 µg mL−1), glucose (20 mM)], BSA (0.5 mg ml−1
)
and AzoTPs/NTPs (0.05–2.0 mM) in assay buffer was perfused
to the flow cell. Then the flow cell was subjected to actin fila-
ment gliding motility observation under an epifluorescence
microscope (IX 70, Olympus) equipped with an EMCCD
camera (DL-604M-0EM-H1, Andor Technology). The typical
frame rate was 0.5 s and the observation duration was 10–120 s
depending on the faster/slower movement of the actin fila-
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
ments. The gliding velocity of at least 10 actin filaments in ran- We thank Ms Miho Yamada for the HR-MS analyses at the
domly chosen microscopic fields was measured in each experi- OPEN FACILITY in Hokkaido University. This work was sup-
ment; however, stationary filaments that did not move at all ported by Grant-in-Aid for Scientific Research (B) 18H02042 to
were excluded from velocity measurements. The gliding vel- N. T. and a Grant-in-Aid for Young Scientists (B) KAKENHI
ocities were measured using MTrackJ plugin in the Fiji image Grant Number 16K16635 to K. M. A part of support was also
processing software package based on ImageJ; typically each provided by the Akiyama Life Science Foundation, Grants-in-
actin filament was tracked every 0.5 s or 10 s depending on the Aid for Regional R&D Proposal-Based Program from Northern
faster/slower movement of actin filaments.
Advancement Center for Science & Technology of Hokkaido
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Japan and Takeda Science Foundation to K. M. We also 24 R. Tucker, P. Katira and H. Hess, Nano Lett., 2008, 8, 221–
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45 Usually at higher concentration of AzoTPs (>3.5 mM), the
microtubules were detached form kinesin and floating in
the flow cell. In case of 1e, the detachment of microtubules
was observed at >2.0 mM concentration. Therefore, the
microtubule gliding velocity was shown up to 3.0 mM for
all AzoTP derivatives except for 1e (up to 2.0 mM).
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