Dynamic photo-control of kinesin on a photoisomerizable monolayer - Hydrolysis rate of ATP and motility of microtubules depending on the terminal group

Article abstract of DOI:10.1039/c2ob07167c

The reversibly and repeatedly altered gliding motility of microtubules driven by kinesin on the photoresponsive monolayer surface is studied. It was confirmed that an azobenzene monolayer surface needs to have free amino terminal groups for the successful dynamic control of the motility of microtubule. The surface of the azobenzene monolayer with terminal amino groups can dynamically control the ATP hydrolysis activity of kinesin which resulted in the change in motility of the microtubules.

Full text of DOI:10.1039/c2ob07167c

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PAPER
Dynamic photo-control of kinesin on a photoisomerizable
monolayer – hydrolysis rate of ATP and motility of microtubules
depending on the terminal group†
M. K. Abdul Rahim, Takashi Kamei and Nobuyuki Tamaoki*
Received 23rd December 2011, Accepted 19th February 2012
DOI: 10.1039/c2ob07167c
The reversibly and repeatedly altered gliding motility of microtubules driven by kinesin on the
photoresponsive monolayer surface is studied. It was confirmed that an azobenzene monolayer surface
needs to have free amino terminal groups for the successful dynamic control of the motility of
microtubule. The surface of the azobenzene monolayer with terminal amino groups can dynamically
control the ATP hydrolysis activity of kinesin which resulted in the change in motility of the
microtubules.
The ability to reversibly control the speed of microtubule
Introduction
gliding and the direction of movement on the surface of kinesin
by external stimuli would greatly improve the sophistication of
nano devices. Several methodologies have been used for the
artificial control of microtubule function which includes micro-
lithographic tracks,21,22 electric and magnetic fields,23 anti-
bodies24 and so on. When considering the biodevices of the
future, photochemical energy inputs offer advantages compared
to chemical energy inputs because (i) light does not generate
waste products; (ii) it can be switched on/off easily and rapidly;
(iii) photons, besides supplying the energy to the system, can
also be useful to “read” the state of the system and thus to
control and monitor the operation of the machine etc. Higuchi
et al.25 used the caged ATP as photo-controlled switch, because
their inactive caged states (OFF state of motility) can be con-
verted to active uncaged states (ON state of motility) by UV
light irradiation. Another group successfully used the photolysis
of caged ATP to develop molecular shuttles on engineered
kinesin tracks.26 On the other hand Tatsu et al. demonstrated the
switching of kinesin’s activity from the ON state to the OFF
state by the photolysis of caged peptide derived from the kinesin
C-terminus domain working as an inhibitor.27 In that study, they
successfully demonstrated an 80% reduction of the initial gliding
velocity of microtubules on the kinesin surface after the photo-
chemical deprotection of the o-nitrobenzyl protecting group on
the caged peptide. All these methods can either trigger the
initiation of the movement or cessation of the microtubule moti-
lity. To design the more useful nanoscale biodevices, it is impor-
tant that the system should provide ON and OFF switching of
gliding motility at any desired time and at any desired position
in space. To the best of our knowledge the light controlled
reversible and repeated switching of microtubule motility was
demonstrated only by our group.28
The regulation of nano assembly devices and molecular
machines using biomolecular motors as promising components
is one of the hot topics in nanotechnology and bio-
engineering.1–9 Kinesins are prominent motor proteins associated
with microtubules, which play a central role in both structural
and biological functions of the cells.10–12 The fundamental role
of kinesin is to actively distribute the intracellular materials like
organelles, vesicles, chromosomes along the microtubules13,14
and to drive the cell division process by converting the chemical
energy of adenosine triphosphate (ATP) into mechanical energy
with the positional movement towards the fast polymerizing plus
ends of microtubules.15–17 Each kinesin molecule has two
“heads”, a 50 nm long semi-flexible coiled-coil region which
binds to microtubules and hydrolyses ATP and a “tail”, which is
thought to bind to the cargo.18 Among the molecular motors, the
kinesin–microtubule system is used widely for developing
nanoscale biodevices, because the motility can be regenerated on
a surface comparatively easily. In the common in vitro construc-
tion of the kinesin–microtubule system, the kinesin are adsorbed
onto a surface such as glass and microtubules are able to glide
across the kinesin coated glass surface.14 The kinesin–micro-
tubule system has other advantages such as its small size, and
the linear movement of single kinesin along the microtubule
compared to other molecular motor proteins.19,20
Research Institute for Electronic Science, Hokkaido University, N20,
W10, Kita-ku, 001-0020, Sapporo, Hokkaido, Japan. E-mail:
tamaoki@es.hokudai.ac.jp; Fax: +81 11 706 9357; Tel: +81 11 706
9356
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob07167c
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General methods, instrumentation and measurements
We reported the reversible and repeated regulation of the moti-
lity of microtubule by the photoisomerization of the underlying
monolayer using two different wavelengths of light.28 For the
fabrication of such photoresponsive monolayer, we employed a
derivative of azobenzene; one of the most studied photochromic
compounds29–33 due to its strong photo-switching effect, reversi-
bility and simplicity of incorporation,34 with a triethoxy silane
group which react with the glass surface to be anchored and a
lysine group which can interact with motor proteins. Using this
approach, we described the reversible and repeated control of the
gliding velocity of microtubules driven by kinesin on the azo-
benzene monolayer (with a maximum of 15% difference in vel-
ocity) upon irradiation with UV and visible light.35 In this paper
we are reporting the generality of the system in the repeated
regulation of fast and slow modes of microtubule gliding moti-
lity by devising a series of surface of azobenzene monolayers
with various terminal groups. We also propose a mechanism that
accounts for the changes in the velocity of microtubule when the
photoresponsive monolayer surface is isomerized between E and
Z states upon irradiation of different wavelengths of light. We
believe that the method developed here should be applicable in
forthcoming nanoscale biodevices based on the kinesin–micro-
tubule system.
1H NMR spectra were recorded at 400 MHz using TMS as an
internal standard. Matrix-Assisted Laser Desorption Ionization
Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) was per-
formed on an applied Biosystems Voyager-DE pro instrument
with positive-ion mode. Absorption spectra were recorded on an
Agilent 8453 spectrophotometer and also a Hitachi U-3100
absorption spectrophotometer. Photo-isomerization studies were
conducted using a mercury–xenon lamp (Hamamatsu Photonics
K.K. 200 W) after passage through appropriate filters (366 or
440 or >500 nm).
Microtubules motility assay were carried out using fluor-
escence microscope (Olympus BX50 or Nikon TEi) equipped
with a high NA objective lens (100×, 1.30 NA for Olympus
BX50 and 100×, 1.45 NA for Nikon TEi) and the fluorescence
image was recorded with appropriate filters (Chroma) to remove
the excitation light, back-illuminate electron-cooled CCD camera
(EM9100-12, HAMAMATSU), and the image processing system
AQUACOSMOS (HAMAMATSU). Both were performed at
room temperature (22 °C).
Synthesis
The synthetic route to compounds 1b′–e′ and 2a are illustrated
in Schemes 1–3 and compound 2b was commercially purchased.
Synthetic details for 1a′ were reported in our previous paper.
Detailed synthetic procedures for 1b′–e′ and 2a are described
below. Compounds 4 and 5 were prepared according to the
literature.36
Experimental
Materials
Unless otherwise noted, all reagents including solvents were
obtained from major commercial suppliers such as TCI, Sigma-
Aldrich and Wako used directly without further purification.
DMF was routinely dried and/or distilled prior to use and stored
over molecular sieves (4A). Column chromatography was per-
formed with silica gel (63–210 μm).
Compound 7. Hydroxybenzotriazole (115 mg, 0.848 mmol)
and N,N′-dicyclohexylcarbodiimide (175 mg, 0.848 mmol) were
added under an argon atmosphere to a solution of Boc-Arg-
Scheme 1 (a) NaBO3, H3BO3, HOAc, HCl, MeOH, 60 °C. (b) NaOH. (c) For 7, 1b′ = Boc–Arg–(Boc)2–OH, for 8, 1c′ = Boc–Glu–(OtBu)–OH
and for 9, 1d′ = mono-tert-butyl malonate, DCC, HOBt, DMF. (d) OCN–(CH2)3–Si–(OEt)3, THF.
3322 | Org. Biomol. Chem., 2012, 10, 3321–3331
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Scheme 2 (a) HCl, H2O, NaNO2, NaOH, PhOH, 0–2 °C. (b) 10% NaOH, reflux 2 h. (c) Br–(CH2)5–COOEt, K2CO3, (CH3)2CO, 50–60 °C.
(d) Boc–Lys–(Boc)–OH·DCHA, DCC, HOBt, DMF. (e) Dioxane, 1 M aq. KOH. (f) OCN–(CH2)3–Si–(OEt)3, THF.
Scheme 3 Synthetic scheme of compound 2a.
(Boc)2-OH (268 mg, 0.565 mmol) in dry DMF (5 mL) at room
temperature. After 1 h, 4,4′-diaminoazobenzene (5) (150 mg,
0.707 mmol) was added and the mixture was stirred at room
temperature overnight. The mixture was then diluted with brine
and was extracted with ethyl acetate (EtOAc); the combined
extracts were dried (MgSO4) and the solvent was removed under
reduced pressure. The residue was purified by column chromato-
graphy (eluent: dichloromethane (DCM)–EtOAc). Yield: 41%.
1H NMR (400 MHz, CDCl3): δ = 1.40 (s, 9H), 1.51 (d, J = 4
Hz, 18H), 1.65–1.71 (m, 2H), 1.82–1.95 (m, 4H), 3.81 (m, 1H),
4.07 (br, 2H), 4.55 (m, 1H), 5.95 (q, J = 4 Hz, 1H), 6.72 (d, J =
8 Hz, 2H), 7.61 (d, J = 8 Hz, 2H), 7.77 (d, J = 8 Hz, 2H), 7.82
(d, J = 8 Hz, 2H), 9.19 (br, 1H), 9.29 (br, 1H). MS (MALDI):
Calculated for C33H48N8O7 [M + H]+ m/z 669.36; found m/z
669.66.
13C-NMR (400 MHz, CDCl3): δ = 171.10, 170.83, 161.17,
155.75, 155.15, 154.80, 149.29, 148.18, 141.84, 141.70, 124.07,
123.55, 120.14, 119.34, 82.96, 82.33, 80.00, 58.52, 55.63,
48.47, 42.70, 28.43, 28.09, 24.99, 23.47, 22.99, 18.31, 7.66. MS
(MALDI): Calculated for C43H69N9O11Si [M + H]+ m/z 917.48;
found m/z 917.43.
Compound 8. Hydroxybenzotriazole (115 mg, 0.848 mmol)
and N,N′-dicyclohexylcarbodiimide (175 mg, 0.848 mmol) were
added under an argon atmosphere to a solution of Boc-Glu-
(OtBu)-OH (172 mg, 0.565 mmol) in dry DMF (5 mL) at room
temperature. 4,4′-Diaminoazobenzene (5) (150 mg, 0.707 mmol)
was added after 1 h and the mixture was stirred at room tempera-
ture overnight. The mixture was then diluted with brine and was
extracted with ethyl acetate (EtOAc); the combined extracts were
dried (MgSO4) and the solvent was removed under reduced
pressure. The residue was purified by column chromatography
(eluent: DCM–EtOAc). Yield: 43%. 1H NMR (400 MHz,
CDCl3): δ = 1.45 (d, J = 8 Hz, 18H), 1.59–1.63 (m, 2H),
2.45–2.49 (t, J = 8 Hz, 2H), 4.09 (br, 2H), 4.24 (br, 1H), 5.4 (q,
J = 4 Hz, 1H), 6.71 (d, J = 8 Hz, 2H), 7.74 (d, J = 8 Hz, 2H),
7.77 (d, J = 8 Hz, 2H), 7.83 (d, J = 8 Hz, 2H), 9.12 (Br, 1H).
MS (MALDI): Calculated for C26H35N5O5 [M + H]+ m/z
498.26; found m/z 498.36.
Compound 1b′. Triethoxysilylpropyl isocyanate (62 mg,
0.251 mmol) was added to a solution of 7 (252 mg,
0.377 mmol) in anhydrous tetrahydrofuran (THF) (3 mL). The
mixture was refluxed under N2 atmosphere until the completion
of the reaction. The residue was washed with copious amounts
1
of hexane and was then dried under vacuum. Yield: 71%. H
NMR (400 MHz, CDCl3): δ = 0.61 (t, J = 8 Hz, 2H), 1.17 (t, J =
8 Hz, 9H), 1.43 (d, J = 4 Hz, 18H), 1.52 (s, 9H), 1.63 (m, 2H),
1.77 (m, 2H), 1.89 (m, 2H), 2.89 (q, J = 4 Hz, 2H), 3.23 (q, J =
4 Hz, 2H), 3.79 (q, J = 8 Hz, 6H), 3.96 (m, 1H), 4.34 (br, 1H),
5.97 (q, J = 4 Hz, 1H), 6.33 (m, 2H), 7.67 (d, J = 8 Hz, 2H),
7.81 (d, J = 4 Hz, 2H), 7.83 (d, J = 4 Hz, 2H), 7.85 (d, J = 4 Hz,
2H), 8.27 (s; urea H atom close to aryl, 1H), 9.26 (br, 1H).
Compound 1c′. Triethoxysilylpropyl isocyanate (76.6 mg,
0.31 mmol) was added to a solution of 8 (235 mg, 0.465 mmol)
in anhydrous tetrahydrofuran (THF) (3 mL). The mixture was
refluxed under N2 atmosphere until the reaction was completed.
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The residue was washed with copious amounts of hexane and
was then dried under vacuum. Yield: 79%. H NMR (400 MHz,
1.68 (m, 2H), 1.80 (m, 2H), 2.32 (t, J = 8 Hz, 2H), 3.98 (t, J = 8
Hz, 2H), 4.02 (br, 2H), 4.10 (q, J = 8 Hz, 2H), 6.72 (d, J = 8
Hz, 2H), 6.95 (d, J = 8 Hz, 2H), 7.75 (d, J = 8 Hz, 2H), 7.81
(d, J = 8 Hz, 2H). MS (MALDI): Calculated for C20H25N3O3
[M + H]+ m/z 356.20; found m/z 356.23.
1
CDCl3): δ = 0.65 (t, J = 8 Hz, 2H), 1.20 (t, J = 8 Hz, 9H), 1.46
(d, J = 8 Hz, 18H), 1.64 (m, 2H), 2.23–2.33 (m, 2H), 2.47–2.51
(t, J = 8 Hz, 2H), 3.26 (q, J = 4 Hz, 2H), 3.79 (q, J = 6 Hz, 6H),
4.24 (br, 1H), 5.39 (br, 1H), 5.42 (q, J = 4 Hz, 1H), 7.06 (br,
1H), 7.44 (d, J = 8 Hz, 2H), 7.73 (d, J = 8 Hz, 2H), 7.79 (d, J =
8 Hz, 2H), 7.84 (d, J = 8 Hz, 2H), 9.24 (s; urea H atom close to
aryl, 1H). 13C-NMR (400 MHz, CDCl3): δ = 171.21, 155.31,
149.63, 149.02, 142.22, 141.68, 124.00, 123.70, 119.94, 119.42,
82.94, 80.71, 58.53, 53.20, 42.68, 34.25, 31.00, 28.35, 27.96,
25.80, 23.48, 22.65, 18.33, 7.62. MS (MALDI): Calculated for
C36H56N6O9Si [M + H]+ m/z 745.39; found m/z 745.43.
Compound 14. HOBt (200 mg, 1.48 mmol), EDC (284 mg,
1.48 mmol) and Et3N (0.196 μL, 1.4 mmol) were added to a
DMF (10 mL) solution of Boc-Lys-(Boc)·DCHA (520 mg,
1.4 mmol) at 0 °C under argon atmosphere. After keeping at
0 °C for 15 minutes and 30 minutes at room temperature, the
compound 13 (510 mg, 1.44 mmol) was added and the mixture
was stirred overnight at room temperature. The mixture was
diluted with brine and was extracted with ethyl acetate, dried
over MgSO4 and the solvent was removed under reduced
pressure. The residue was purified using column chromatography
(DCM–AcOEt mixture as eluents (8 : 2)) and 14 were obtained
Compound 9. Hydroxybenzotriazole (191 mg, 1.4 mmol) and
N,N′-dicyclohexylcarbodiimide (292 mg, 1.4 mmol) were added
under an argon atmosphere to a solution of mono-tert-butyl mal-
onate (151 mg, 0.94 mmol) in dry DMF (3 mL) at room temp-
erature. After 1 h, 4,4′-diaminoazobenzene (5) (250 mg,
1.18 mmol) was added and the mixture was stirred at room temp-
erature overnight. The mixture was then diluted with brine and
was extracted with ethyl acetate (EtOAc); the combined extracts
were dried (MgSO4) and the solvent was removed under reduced
pressure. The residue was purified through column chromato-
graphy (eluent: DCM–EtOAc). Yield: 46%. 1H NMR
(400 MHz, CDCl3): δ = 1.45 (s, 9H), 3.34 (s, 2H), 3.97 (br, 2H),
6.64 (d, J = 8 Hz, 2H), 7.62 (d, J = 8 Hz, 2H), 7.70 (d, J = 8 Hz,
2H), 7.76 (d, J = 8 Hz, 2H), 9.50 (br, 1H). MS (MALDI): Calcu-
lated for C19H22N4O3 [M + H]+ m/z 355.17; found m/z 355.08.
1
as yellow powder in 48% yield. H NMR (400 MHz, CDCl3): δ
= 1.19 (m, 2H), 1.24 (t, J = 6 Hz, 3H), 1.31 (m, 2H), 1.43 (d, J
= 8 Hz, 18H), 1.52 (m, 2H), 1.70 (m, 2H), 1.80 (m, 2H), 1.88
(m, 2H), 2.32 (t, J = 6 Hz, 2H), 3.15 (m, 2H), 3.79 (q, J = 8 Hz,
2H), 4.02 (t, J = 6 Hz, 2H), 4.17 (br, 1H), 4.61 (m, 1H), 5.21
(br, 1H), 6.96 (d, J = 12 Hz, 2H), 7.68 (d, J = 8 Hz, 2H), 7.85
(d, J = 4 Hz, 2H), 7.87 (d, J = 4 Hz, 2H), 8.64 (br, 1H). MS
(MALDI): Calculated for C36H53N5O8 [M + H]+ m/z 684.40;
found m/z 684.47.
Compound 15. 1 M KOH aq. solution (3 mL) was added to a
solution of 14 (200 mg, 0.29 mmol) in dioxane (6.0 mL). After
stirring for 2 h at room temperature the mixture was neutralized
+
Compound 1d′. Triethoxysilylpropyl isocyanate (104 mg,
0.42 mmol) was added to a solution of 9 (150 mg, 0.42 mmol)
in anhydrous tetrahydrofuran (THF) (3 mL). The mixture was
refluxed under N2 atmosphere until the completion of the reac-
tion. The residue was washed with copious amounts of hexane
and was then dried under vacuum. Yield: 83%. 1H NMR
(400 MHz, CDCl3): δ = 0.64 (t, J = 8 Hz, 2H), 1.19 (t, J = 8 Hz,
9H), 1.52 (s, 9H), 1.60–1.69 (m, 2H), 3.23 (q, J = 4 Hz, 2H),
3.44 (s, 2H), 3.78 (q, J = 6 Hz, 6H), 5.72 (m, 2H), 7.40 (d, J = 8
Hz, 2H), 7.55 (br, 1H), 7.63 (d, J = 4 Hz, 2H), 7.76 (d, J = 4
Hz, 2H), 7.80 (d, J = 4 Hz, 2H), 9.64 (s; urea H atom close to
aryl, 1H). 13C-NMR (400 MHz, CDCl3): δ = 169.08, 164.07,
155.60, 149.44, 147.87, 142.16, 139.24, 124.02, 123.65, 120.55,
119.13, 83.36, 58.48, 53.45, 42.68, 28.04, 18.31, 14.20, 7.66.
MS (MALDI): Calculated for C29H43N5O7Si [M + H]+ m/z
602.29; found m/z 602.39.
with 2 N NH4 Cl−, extracted with AcOEt and was dried over
MgSO4. The residue was purified by column chromatography
(AcOEt) and 15 was obtained in 47% yield. 1H NMR
(400 MHz, CDCl3): δ = 1.24 (m, 2H), 1.45 (d, J = 4 Hz, 18H),
1.50 (m, 2H), 1.68 (m, 2H), 1.81 (m, 2H), 1.86 (m, 2H), 2.03
(m, 2H), 2.33 (t, J = 8 Hz, 2H), 3.09 (m, 2H), 4.02 (t, J = 6 Hz,
2H), 4.05 (br, 1H), 4.65 (m, 1H), 5.21 (br, 1H), 6.96 (d, J = 12
Hz, 2H), 7.68 (d, J = 8 Hz, 2H), 7.85 (d, J = 4 Hz, 2H), 7.87 (d,
J = 4 Hz, 2H), 8.65 (br, 1H). MS (MALDI): Calculated for
C34H49N5O8 [M + H]+ m/z 656.34; found m/z 656.36.
Compound 1e′. The mixture solution of 100 mg (0.15 mmol)
of 15, 67 mg (0.31 mmol) of (3-aminopropyl) triethoxy silane
and 67 mg (0.31 mmol) of dicyclohexylcarbodiimide in DCM
(12 mL) was stirred for 2 h under ice cooling. The precipitated
dicyclohexylurea was removed by filtration. The filtrate was
evaporated under reduced pressure, washed with copious
amounts of hexane and was then dried under vacuum affording
the product as orange solid. The crude residue 1e′ was used
Compounds 11 and 12 were prepared according to the
literature.37
1
Compound 13. 4-(4′-Hydroxyphenylazo)aniline (12) (2.10 g,
10 mmol), 6-bromo-hexanoic acid ethyl ester (3.4 g, 15.2 mmol)
and potassium carbonate (2.8 g, 38 mmol) was dissolved in
acetone (60 mL) and the mixture was refluxed for 12 hours
under argon atmosphere. The mixture was then diluted with
brine and was extracted with ethyl acetate (EtOAc); the com-
bined extracts were dried (MgSO4) and the solvent was removed
under reduced pressure. The resulting mixture was submitted for
silica column chromatography using a mixed solvent (DCM–
EtOAc, 9 : 1) and 13 was obtained in 91% yield. 1H NMR
(400 MHz, CDCl3): δ = 1.24 (t, J = 6 Hz, 3H), 1.50 (m, 2H),
without any further purification. H NMR (400 MHz, CDCl3): δ
= 0.86 (t, J = 4 Hz, 2H), 1.24 (t, J = 6 Hz, 9H), 1.45 (d, J = 4
Hz, 18H), 1.51–1.61 (m, 10H), 1.68–17.76 (m, 2H), 1.80–1.88
(m, 2H), 1.95 (t, J = 8 Hz, 2H), 2.33 (t, J = 6 Hz, 2H), 3.09 (m,
2H), 3.79 (t, J = 4 Hz, 1H), 4.02 (t, J = 6 Hz, 2H), 4.11 (q, J = 4
Hz, 6H), 4.19 (br, 1H), 4.64 (m, 1H), 5.21 (br, 1H), 6.96 (d, J =
4 Hz, 2H), 7.67 (d, J = 12 Hz, 2H), 7.85 (d, J = 4 Hz, 2H), 7.87
(d, J = 4 Hz, 2H), 8.65 (br, 1H). Yield was 86%. 13C-NMR
(400 MHz, CDCl3): δ = 172.35, 169.57, 160.27, 155.22, 152.92,
148.04, 145.87, 138.77, 123.49, 122.47, 118.71, 113.58, 79.47,
78.25, 66.88, 58.81, 52.46, 48.60, 47.96, 34.52, 32.83, 31.64,
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29.88, 27.93, 27.37, 27.25, 25.25, 21.52, 18.35, 17.21, 8.59. MS
(MALDI): Calculated for C34H49N5O8 [M + H]+ m/z 859.49;
found m/z 859.62.
by adjusting the light and focus of the camera. To confirm the
deprotection of tert-Boc group, images were taken before and
after deprotection, and to study the reversible changes in surface
properties upon photoirradiation images were taken before and
after 366 nm wavelength light irradiation.
Compound 2a. Triethoxysilylpropyl isocyanate (17) (2.5 g,
10.1 mmol) was added to a solution of hexylamine (16) (1.53 g,
15.18 mmol) in THF (6 mL) and was refluxed for 36 hours
under argon atmosphere. After the completion of reaction, the
residue was washed with copious amount of hexane and was
Proteins preparations
Tubulins were purified from porcine brains through two cycles
of polymerization–depolymeraization processes in the presence
of a high-molarity PIPES buffer.40 Microtubules were polymer-
ized using the purified tubulins and labelled with tetramethylrho-
damine succinimidyl ester. Kinesin utilized in this research was
a recombinant kinesin consist of 573 amino acid residues from
N-terminus of a conventional human kinesin. This recombinant
kinesin fused with His-tag in the N-terminus (plasmid; pET30b)
was expressed in Escherichia coli Rosetta (DE3) pLysS and
purified by the general method utilizing Ni–NTA–agarose.
1
dried under vacuum. Yield: 78%. H NMR (400 MHz, CDCl3):
δ = 0.52 (t, J = 12 Hz, 2H), 0.79 (t, J = 4 Hz, 3H), 1.11 (t, J = 8
Hz, 9H), 1.20 (m, 4H), 1.37 (m, 2H), 1.50 (m, 2H), 1.78 (m,
2H), 3.05 (m, 4H), 3.71 (q, J = 6 Hz, 6H), 5.23 (m, 2H). MS
(MALDI): Calculated for C16H36N2O4Si [M + H]+ m/z 349.24;
found m/z 349.36.
Preparation of monolayer
Preparation of monolayer was done by a modification of the
methods described elsewhere38 through the formation of siloxane
linkages between 1a–d or 2a and the substrate. Glass or quartz
plates were purified ultrasonically in acetone, in concentrated
nitric acid and aqueous solution of saturated sodium bicarbonate
in sequence. Each ultra-sonication was carried out for 10 min
and followed by washing with Milli-Q water. After drying at
120 °C for 30 minutes, the plates were dipped in a mixed THF
solution containing compound 1a′–e′ along with compound 2a
or 2b (total concentration: 2 mM) at room temperature for
30 minutes. The modified plates were dried at 120 °C for
30 minutes and washed ultrasonically in THF for 10 minutes
and dried again at 120 °C for 30 minutes. The tert-Boc protect-
ing groups of 1a′–e′ were removed by dipping the monolayer in
a 30% trifluoroacetic acid (TFA) in DCM solution39 which
yielded the target compounds 1a–e. The quantitative removal of
the tert-Boc protecting group was verified by the measurements
of contact angle before and after deprotection.
Flow cells
The flow cells were prepared by placing two strips of double-
stick tape on a glass slide ca. 6–9 mm apart and covered with an
18 × 18 or 22 × 22 mm cover slip consisting of monolayer. The
inner volume of the obtained flow cell was ca. 10–12 μL. Sol-
utions were pipetted on one side and sucked out on the other
side by capillary action using Whatman filter paper or a Kim
wipe as shown elsewhere.41,42
Motility assays
For these experiments, a simple protocol using the standard
capillary flow technique reported elsewhere for kinesin motility
assay with polarity marked taxol microtubules11 was employed.
For all experiment the starting buffer was BRB80 (80 mM
PIPES, 2 mM MgCl2, 1 mM EGTA, pH 6.95 with KOH). The
azobenzene embedded flow cells were sequentially filled with a
standard kinesin solution (0.3069 mg mL−1, which is just suffi-
cient for the movement of microtubules) and kept in moist con-
ditions for five minutes. Unbound kinesins were washed out
with a solution containing 20 μl of BRB80 assay buffer + 1 mM
DTT + 1 mM MgATP + 10 μM taxol. And then a motility buffer
consisting of BRB80 and microtubules labelled by tetramethyl-
rhodamine succinimidyl ester were stabilized with taxol
(5–10 μm in length). After keeping like this for five minutes, the
unbound microtubules were washed out by the assay buffer con-
taining taxol with an added oxygen scavenger system (2-mercap-
Optical measurements and photoisomerization
For photo-switching the azobenzene 1a′–d′, samples (in aceto-
nitrile, 22 °C) were irradiated at 366 nm (trans to cis) and
>500 nm (cis to trans), and 1e′ sample (in DCM, 22 °C) were
irradiated with 366 nm (trans to cis) and 440 nm (cis to trans)
respectively, using a mercury–xenon lamp (Hamamatsu Photo-
nics K.K. 200 W) and band-pass filters (366, >500 and 440 nm).
Thermal relaxation of the cis to the trans isomer was monitored
with the same spectrophotometer under dark condition. The
absorption spectra of mixed monolayer of 1a–e incorporated on
a quartz plate were recorded with same method and time as in
solution, before and after photoirradiation using appropriate
band-pass filters (366, >500 and 440 nm) on a Hitachi U-3100
absorption spectrophotometer.
toethanol: 0.14 M, glucose 20 mM, catalase: 20 μg ml−1
,
glucose oxidase: 100 μg ml−1). The flow cells were sealed with
immersion oil to prevent evaporation and were transferred to the
fluorescence microscope. The translocation of microtubules were
monitored and recorded after UV and visible light irradiation.
Contact angle measurements
ATPase assays
The sample consisting of the monolayer was placed on a flat
surface and water, which is used as the fluid, was dropped on the
sample using a syringe. The image of the sample was obtained
In the presence of azobenzene monolayer and microtubules, the
kinesin ATPase activity was measured via a simple and sensitive
colorimetric assay based on the determination of released
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inorganic phosphate ion by an improved malachite green pro-
cedure.43,44 In short, at fixed time intervals, aliquots were
removed from the motility assay system and quickly mixed with
an equal volume of ice-chilled perchloric acid (PCA) and mala-
chite green reagent. The mixture was kept at 25 °C for
40 minutes and then the absorbance was measured at 630 nm
with a Hitachi U-3100 absorption spectrophotometer and con-
centration of inorganic phosphate (Pi) was determined. Then the
same flow cell was irradiated with 366 nm light until the sample
reached its photostationary state (PSS). The aliquots at fixed time
intervals were removed from the motility assay system and
mixed with equal volumes of ice-chilled PCA and malachite
green reagent and the same procedure was repeated. In order to
know the reversibility, the above procedure was repeated by ir-
radiating the flow cells with visible light.
on the basis of HPLC, we estimated 93% purity with unreacted
azobenzene starting material as impurity. Since the azobenzene
starting material is not reactive towards the glass surface, we did
not consider its presence as a problem for monolayer formation.
One of the important parameters for fabrication of dynamic
monolayer surfaces is reversibility of switching. Pace et al.45
already reported that steric constraints of tightly packed matrixes
can restrict conformational changes, thus hindering isomerization
in azobenzene. In order to avoid such hindrance, we prepared a
mixed monolayer which can reduce the steric constraints. For
compound 1a–d, we used 90% of non-photo isomerizable com-
pound 2a for preparing mixed monolayer which forms hydrogen
bonding with each other, and for 1e we used 90% of 2b. The
removal of tert-Boc groups on the surface upon TFA treatment is
confirmed by the change in the contact angle. The contact angle
of monolayer, before release of tert-Boc group of 1b′ was 74°
and after 4 h treatment with TFA, it is reduced to 66°. The other
compounds also show a similar trend for the water contact angle,
which is summarized in the table (ESI Table S1†).
Fig. 1 shows the absorption spectra of azobenzene monolayer
before and after photoirradiation on a quartz plate and also in
solution. The details of absorption spectra of 1a on quartz
surface and in solution are described in our previous report.28
The absorption spectra of monolayer of 1b on quartz surface
before the irradiation of 366 nm light shows a strong absorption
band at 375 nm which corresponds to the π–π* transition of the
substituted E-azobenzene. The intensity of the band is decreased
upon 366 nm light irradiation and the n–π* transition band
around 500 nm was increased. The film reaches its PSS within
40 seconds. On further irradiation with >500 nm wavelength
light led to the recovery of the π–π* transition; the system
reached another PSS within 240 seconds. These spectral changes
can be repeated for many cycles (inset). The similar behaviour of
absorption spectra was observed for 1b′ maintaining tert-Boc
groups in CH3CN solution which is the typical phenomenon
shown between the E and Z isomerization states of azobenzene
derivatives.46,47 The thermal stability of Z isomer of 1b′ in
CH3CN at 22 °C (the temperature at which microtubule motility
assay has been done) was also confirmed under dark conditions
(inset). Only a very small percentage of Z isomer of 1b′ converts
into E isomer after 1 h and it takes more than 24 h for a complete
recovery (Fig. 1, right). Therefore, the contribution of thermal-
back reaction can be excluded from our few hour long exper-
iments of the motility assay. Similar photoisomerization changes
and thermal stabilities are observed for 10% 1c or 1d along with
90% 2a and 1e with 2b on quartz surface and also in correspond-
ing solution of 1b′–e′ upon alternate irradiation of corresponding
UV and visible light. After 366 nm light irradiation, the film
reaches its PSS within 40 seconds for 1c, 1d and 1e. On further
irradiation with corresponding visible light, the film reaches
another PSS within 300 seconds for 1c and 1d and for 1e it is
270 seconds. From the spectra, conversion to the Z isomer is
estimated to be about 40–50% under 366 nm light photostation-
ary state.
Results and discussion
The dynamic control of the motility of microtubule driven by
kinesin, upon photo-isomerization of azobenzene monolayer was
studied with alkyl silane compounds shown in Chart 1, namely,
3-(triethoxysilyl)propyl urea attached to azobenzene moiety with
one lysine (1a), arginine (1b), glutamic acid (1c) or malonic acid
residue (1d) at the end and a 3-(triethoxysilyl)propyl 6-hydroxy-
hexanoic amide attached to azobenzene moiety with lysine
residue (1e) at the end. One of the issues in the synthesis of
silane functionalized azobenzene was the difficulty in purifying
the final product. Without the purification of the final products,
In addition to UV-visible absorption spectroscopy measure-
ments, the reversible conformation changes of azobenzene
monolayer surface were confirmed by means of contact angle
measurements (Fig. 2). The contact angle of monolayer surface
Chart 1 Chemical structures of photoresponsive azobenzene silane
compounds (1a–e) along with alkyl silane chains (2a and 2b).
of 1a before irradiation was 65
1°. After UV irradiation it
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Fig. 1 Absorption spectral changes of 10% of 1b–e and 90% of 2a or 2b in quartz surface after deprotection of the tert-Boc group [left (1), (3), (5),
(7)] and that of 1b′–d′ in acetonitrile and 1e′ in DCM before deprotection of the tert-Boc group [right (2), (4), (6), (8)]. (a) Before irradiation. (b)
Photostationary state (PSS) at 366 nm. (c) PSS at visible light. The inset in the left side shows the absorbance changes at 376 nm after alternating
irradiation at 366 nm and visible light over five cycles. The inset in the right side shows the thermal-back relaxation at 22 °C in the dark with the indi-
cated time as well as spectra of pure E-isomer (a) and photostationary state under 366 nm light irradiation (b). Smoothing was done to the original
spectra of the monolayer to remove spike like noise coming from the instrument.
increased to 69
1°. Regardless of the terminal group, the
The velocity of rhodamine-labelled microtubules on a kinesin-
containing surface functionalized with azobenzene derivatives
was evaluated with the inverted motility assay.11 It is already
known that in order to get the proper orientation and to avoid
inactivation of kinesin, blocking proteins such as casein,
contact angle was increased after irradiation with 366 nm light
irradiation. Monolayers of 1b–e also showed good reversible
photo switching of surface wettability by UV and visible
irradiation (data not shown).
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streptavidin, bovine serum albumin are first adsorbed on the
substrate.48–51 But in the present study we systematically
removed the blocking proteins and introduced kinesin directly on
the photoresponsive azobenzene monolayer. Since there is no
blocking protein, we first confirmed the kinesin activity with
observable microtubule speed by measuring the average speed of
a large population of microtubules. We found that, even though
the length of time that the kinesin was catalytically active on the
azobenzene monolayers was shorter than that on the correspond-
ing surfaces having blocking proteins, it was sufficiently long
enough to perform all of the motility experiments prior to a
serious decrease in activity. Next, we investigated the effect of
UV light irradiation on the motility of microtubules driven by
kinesin in the presence of ATP without functionalized surface.
The result shows that the gliding velocities of microtubules with
(black circle) and without (triangle) UV irradiation was almost
identical (ESI Fig. S2†). This suggested that UV irradiation on a
flow cell did not have any detectable effect on the microtubule
gliding motility.
irradiation of flow cell, showed a higher velocity of the micro-
tubule than in the E state in 1a–c and 1e, making it possible to
induce
a statistically significant velocity change. Further
irradiation of the flow cell with appropriate visible light, the
motility of microtubule almost reached its initial state and the
velocity increased again upon further irradiation with 366 nm
wavelength of light. In contrast, upon alternate irradiation of
flow cell of 1d with UV and visible light, no change was
observed in the motility of microtubule. Fig. 3 shows variable
and reversible microtubule speeds observed in the gliding assay
in four states: prior to irradiation (100% E), after irradiation at
366 nm (Z-rich), after subsequent irradiation with visible light
(E-rich) and after subsequent irradiation at 366 nm (Z-rich).
Starting with the flow cell in its E isomer rich state and the
subsequent irradiation with 366 nm wavelength light, the con-
trollable changes in the velocity of 1a, 1b and 1e was 13–15%
of the initial velocity in the E form. In the case of 1c with a glu-
tamic acid residue the speed difference between the E and Z rich
state was found to be about 9%. At the same time 1d with a
malonic acid substituent shows the identical microtubule velocity
in E and Z isomeric forms. In contrast to the results of photo-
regulation of motility with the deprotected amino acid substi-
tuted moieties; no change in the velocity of the microtubules
upon photoirradiation was detected for the azobenzene contain-
ing the protected amino acid terminal group (Fig. 4).
In order to know the effect of isomerization of the azobenzene
moieties on the motility of rhodamine-labelled microtubules
driven by the kinesin-mediated ATP hydrolysis, starting with the
azobenzene monolayer in the E state, the average gliding vel-
ocity of the microtubules was measured. The Z rich state of the
azobenzene monolayer obtained after 366 nm wavelength light
In our previous report28 two possible mechanisms were
suggested to explain the change in the motility of kinesin–micro-
tubule system upon the photoisomerization of azobenzene mono-
layer surface. One is that the photoisomerization induced a
change in the interaction between the monolayer surface with
positively charged amino groups and microtubules being rich in
the negatively charged carboxylate groups.52 The other one is
that the photoresponsive monolayer affects the activity of
kinesin.
Fig. 2 Reversible switching of contact angle of photoresponsive com-
pound 1a for water before irradiation (left) and after 366 nm light
irradiation (right).
To investigate the influence of azobenzene functionalized
monolayer on the activity of kinesin, a microtubule activated
Fig. 3 Fast and slow mode of microtubule motility upon photoirradiation. (1): 1b, (2): 1c, (3): 1d, (4): 1e. State 1: Prior to irradiation. State 2: After
irradiation at 366 nm. State 3: After subsequent irradiation at visible light. State 4: After subsequent irradiation at 366 nm. The number of microtubules
measured was 38, 44, 35, and 30 for 1b; 40, 35, 37, and 25 for 1c; 32, 32, 30, and 25 for 1d; and 46, 41, 43, and 37 for 1e; for states 1–4, respectively.
The error bars indicate the standard error of the mean.
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Fig. 4 Changes in microtubule motility upon photoirradiation of tert-Boc protected compounds. (1): 1b′, (2): 1c′, (3): 1d′, (4): 1e′. The velocity was
calculated with the shortest distance of the terminal positions of the microtubule before and after 10 seconds motility. State 1: Prior to irradiation. State
2: After irradiation at 366 nm. State 3: After subsequent irradiation with visible light. The error bars indicate the standard error of the mean.
ATPase assay was performed in the same buffer condition as the
motility assay. We determined the rate at which ATP hydrolysis
products are formed by using malachite green colorimetric
method as explained in the Experimental section. Fig. 5 shows
the time dependent inorganic phosphate release from hydrolysis
of ATP by kinesin at different states of 1a. When the azobenzene
moiety of 1a was in E form, the initial steady state phosphate
release rate was 5.05 × 10−3 nmol min−1 μl−1. Upon UV
irradiation of flow cell with 366 nm light about 30 seconds,
phosphate release rate was found to be increased to 5.74 × 10−3
nmol min−1 μl−1. The difference in the release rates of phosphate
ion between E and Z isomer was 14% which shows comparable
correlation with the gliding velocity of microtubule at two differ-
ent isomeric surfaces. On further irradiation with >500 nm, the
inorganic phosphate release rate reduced to 5.14 × 10−3 nmol
min−1 μl−1 and the difference in the release rates between E and
Z isomer was 12%. Similarly, the rate of inorganic phosphate
release was switched for 1b–c and 1e monolayer surfaces, with
similar behaviour as seen in the gliding velocity of microtubule
at two different isomeric surfaces (ESI Fig. S3–S6†). In contrast,
the release rate of inorganic phosphate in protected azobenzene
derivatives upon alternate irradiation with UV and visible light is
the same, a striking exact correlation with the microtubule
Fig. 5 Time dependent inorganic phosphate released from hydrolysis
of ATP by kinesin in different environments of azobenzene functiona-
lized surface of 1a before irradiation [(1) squares], after 366 nm
speeds on the two, E and Z, isomeric states (data not shown).
From the results it is clear that the change in motility of micro-
tubule depends on terminal group of the surface monolayer.
When our photoresponsive azobenzene monolayer 1a, 1b or 1e
presenting two or more free amino groups with a net positive
charge in the buffer solution was used for the inverted motility
assay experiment, the gliding velocity of microtubule in the Z
state was 13–15% higher than the gliding velocity in the E state.
The azobenzene monolayer surface of 1c offering one negatively
charged carboxyl group and one positively charged amino group
gave an increase in microtubule velocity of 9% in the Z state
than the gliding velocity in the E state. In contrast negatively
charged surface of 1d and non-polar surface of protected
irradiation [(1) stars] and after visible light irradiation [(1) triangles].
Enlarged figure of phosphate release before and after 366 nm irradiation
(2) and after 366 nm and >500 nm irradiation (3).
monolayer didn’t show any change in the gliding velocity
between two isomeric forms.
The striking exact correlation between the difference in the
gliding velocity of microtubule driven by kinesin and the differ-
ence in the rate at which the ATP hydrolysis products formed at
two isomeric surfaces, E and Z, allows us to conclude that the
change in the microtubule gliding motility is mainly due to the
change in activity of kinesin. It has already been reported that
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ATP hydrolysis activity of kinesin which further induces the
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Acknowledgements
This work was supported by a grant-in-aid for science research
in a priority area “New Frontiers in Photochromism (no. 471)”
from the Ministry of Education, Culture, Sports, Science,
and Technology (MEXT), Japan. M. K. A. R. acknowledges
The Ushio Foundation for
a scholarship. We thank to
Dr Y. Hiratsuka, Dr Y. Hachikubo and Dr T. Q. P. Uyeda for
kindly giving recombinant kinesin construct and Dr A. Kakugo,
Y. Tamura and Prof. J. P. Gong for help in purification of protein
specimen.
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