A non-nucleoside triphosphate for powering kinesin-microtubule motility with photo-tunable velocity

Article abstract of DOI:10.1039/c3cc45933k

Three non-nucleoside triphosphates, one containing an azobenzene moiety, power a kinesin-microtubule system with high motile activity, with an observed maximum velocity of almost half of that obtained with ATP. The cis-trans photoisomerization of the azobenzene unit allows reversible and repeated control over the motile properties of kinesin.

Full text of DOI:10.1039/c3cc45933k

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A non-nucleoside triphosphate for powering kinesin-
microtubule motility with photo-tunable velocity†
Cite this: Chem. Commun., 2013,
49, 9935
Nishad Perur, Masao Yahara, Takashi Kamei and Nobuyuki Tamaoki*
Received 3rd August 2013,
Accepted 30th August 2013
DOI: 10.1039/c3cc45933k
www.rsc.org/chemcomm
Three non-nucleoside triphosphates, one containing an azobenzene
moiety, power a kinesin-microtubule system with high motile activity,
with an observed maximum velocity of almost half of that obtained
with ATP. The cis–trans photoisomerization of the azobenzene unit
allows reversible and repeated control over the motile properties of
kinesin.
Kinesin1 is a microtubule-based motor protein that plays
substantial roles in many cellular processes in eukaryotic cells.
The kinesin motor transports various cargos along the micro-
tubule filaments, employing adenosine triphosphate (ATP) as
its energy source.2–4 The kinesin-microtubule motility system is
an exquisite model of biologically derived nanoscale motion.
Among the several nucleosides and other ATP analogues have
been applied to study the motile activities of kinesin motor
protein,5–7 multifunctional species have been particularly use-
ful for in vitro kinetic studies of biological motor proteins.6,7
Azobenzene is one of the most studied photochromic com-
pounds8 because it undergoes cis–trans photoisomerization that
Fig. 1 Structures of adenosine triphosphate (ATP) and the non-nucleoside
triphosphates AzoTP, NPhAETP, and NPhAdMTP.
can be switched at particular wavelengths of light. The photoin- motility reversibly through alternating irradiation at two different
duced isomerization of azobenzene moieties has been applied to wavelengths.7
the switching functions of various biomolecules.9 It remains a
NPhAETP (Fig. 1) has been reported as a substrate for the
challenge, however, to control the motile activities of kinesin motor actomyosin motor protein system.12 Here, we synthesized
protein reversibly through the photoisomerization of azobenzene.10 NPhAdMTP (Fig. 1) as a new amide version of NPhAETP to
Recently, our group synthesized ATP analogues substituted with an investigate the effect of the linkage between the aromatic ring and
azobenzene unit at the ribose group and used them to drive and the triphosphate; we also synthesized AzoTP by exchanging the nitro
reversibly photocontrol the kinesin-microtubule motility system;11 group of NPhAdMTP with a photoresponsive phenylazo group
the observed change in the motility upon photoirradiation of the (Fig. 1). We characterized these compounds using 1H NMR spectro-
ATP analogue was, however, far from satisfactory.
scopy and electrospray ionization (ESI) mass spectrometry.
In this present study, we have used a novel photoresponsive
With these compounds in hand, we performed an in vitro
non-nucleoside triphosphate as a substrate to drive microtubule microtubule gliding assay13 using the flow cell method. In this
gliding on a kinesin-coated glass surface and to regulate its assay, we employed fluorescence optical microscopy to visualize the
gliding motility of fluorescently labeled microtubules14 on a kinesin-
coated glass surface. We calculated the gliding velocity by tracing
the leading point of microtubules over a period of 40 s.
The motility experiments revealed that all three of the tested
compounds—NPhAETP, NPhAdMTP, and AzoTP—are capable
Research Institute for Electronic Science, Hokkaido University, N20, W10, Kita-ku,
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: Synthesis and character-
of driving microtubule motility. NPhAETP and NPhAdMTP
provided saturated gliding velocities for the microtubules of
ization of compounds; experimental conditions for a kinesin-microtubule moti-
lity assay. See DOI: 10.1039/c3cc45933k
c
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Fig. 2 UV–Vis absorption spectra of AzoTP (5.85 Â 10À5 M) in water: (lack line)
before irradiation; (red line) photostationary state (PSS) at 366 nm; (blue line) PSS
at 436 nm. Inset: absorbance at 327 nm after alternating irradiation at 366 and
436 nm for 10 cycles.
Fig. 3 Fluorescence images of the gliding microtubules driven by AzoTP (3 mM)
on the kinesin-adsorbed glass surface. (Top) Non-irradiated state; (bottom)
366 nm-irradiated state; (left) 0 s; (right) after 20 s in the flow cell. Red arrows:
leading points of the microtubules at 0 s; blue arrows: leading points of the same
microtubules after 20 s.
only 0.03 and 0.04 mm sÀ1, respectively; these values are 4 and
6%, respectively, of the saturated velocity driven by 1 mM of ATP
(0.67 mm sÀ1). In the case of kinesin, NPhAETP functioned, but with
limited efficiency compared with that of myosin. Our results
suggested that changing the linkage between the aromatic ring
and triphosphate unit from an amino to an amido group did not
affect the activity significantly. In contrast to the performance
induced by these two nitro derivatives, AzoTP provided high
microtubule gliding motility (0.33 mm sÀ1) at a concentration of
3 mM,15 corresponding to 49% of the velocity of the motility driven
by 1 mM ATP. These results indicate that the structure of the
aromatic group in the non-nucleotide has a significant effect on the
activity when used as a substrate of kinesin.
Fig. 2 displays the changes in the absorption spectra of an
aqueous solution of AzoTP upon irradiation with UV and visible
light. Under irradiation at 366 nm, the AzoTP solution reached
a cis-rich photostationary state (PSS); subsequent irradiation at
436 nm led to cis-to-trans conversion and re-establishment of
the trans-rich PSS. Accordingly, we examined the effect of the
Fig. 4 Repeatability of the changes in gliding velocity of microtubules in the
photoinduced isomerization of AzoTP on the microtubule glid- presence of AzoTP (3 mM) upon alternating irradiation with UV and visible light.
(1) Before irradiation; (2) after irradiation of sample (1) at 366 nm for 10 s;
ing velocity. Fig. 3 presents fluorescence images of gliding
microtubules after 0 and 20 s for both the non-irradiated state
and the 366 nm PSS in the same flow cell (videos are provided
(3) after irradiation of sample (2) at 436 nm for 30 s; (4) after irradiation of
sample (3) at 366 nm for 10 s; (5) after irradiation of sample (4) at 436 nm for
30 s. Error bars: standard deviations for 10 microtubules.
in the ESI†). Direct UV irradiation of the flow cell converted
trans-AzoTP to its cis form, thereby decreasing the gliding
velocity of the microtubules from 0.33 to 0.07 mm sÀ1. Subsequent
Fig. 5 presents the results of motility experiments performed
irradiation with visible light led to recovery of the initial velocity of with different concentrations of AzoTP in its non-irradiated and
the microtubule (to 0.28 mm sÀ1), due to cis-to-trans isomerization irradiated states. The red and blue circles indicate the gliding
of AzoTP. Fig. 4 displays the changes in motility over two cycles of velocities corresponding to non-irradiated (100% trans) and
1
alternating irradiation with UV and visible light. The maximum irradiated (cis-rich) states, respectively. From H NMR spectra,
decrease in velocity after photoirradiation of 3 mM AzoTP was 79% we estimated that the cis-rich PSS contained 92% of the cis
of the initial velocity of the trans form. The photocontrol over the isomer and 8% of the trans isomer (Fig. S4, ESI†). The black line
changes in velocity between the cis-rich and trans states was in Fig. 5 indicates the microtubule gliding velocity predicted
repeatable for up to 10 cycles of irradiation with UV and visible solely for the 8% content of the trans isomer; the measured
light (Fig. S7, ESI†). To test the effect of the irradiation on the gliding velocity for the cis-rich PSS was slightly higher than this
motility of the microtubules, we performed a control experiment line. Therefore, we estimated the contribution from the cis
using 1 mM ATP; we observed no significant changes in motility isomer (92% content) of AzoTP from the difference between the
upon alternating irradiation with UV and visible light (Fig. S6, ESI†). experimental velocities for the cis-rich PSS and the theoretical
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9936 Chem. Commun., 2013, 49, 9935--9937
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In summary, a new non-nucleoside triphosphate, AzoTP,
allows substantial control over kinesin-microtubule motility—
with a change in velocity of up to 79%—in a photoinduced and
reversible manner. We anticipate the application of AzoTP to
biomotor-based transportation systems and to the artificial
operation of natural motor proteins in living cells.
We thank Dr Tsuyoshi Fukaminato for his valuable contri-
butions to the kinesin-microtubule motility experiments. This
study was supported by the Cabinet Office, Government of
Japan, and the Japan Society for the Promotion of Science
(JSPS) through the Funding Program for World-Leading Inno-
vative R&D on Science and Technology (FIRST Program).
Notes and references
1 S. T. Brady, Nature, 1985, 317, 73; R. D. Vale, T. S. Reese and
M. P. Sheetz, Cell, 1985, 42, 39.
2 N. Hirokawa, Y. Noda and Y. Okada, Curr. Opin. Cell Biol., 1998, 10, 60.
3 N. Hirokawa and R. Takemura, Nat. Rev. Neurosci., 2005, 6, 201.
4 R. D. Vale, Cell, 2003, 112, 467.
5 T. Shimizu, K. Furusawa, S. Ohashi, Y. Y. Toyoshima, M. Okuno,
F. Malik and R. D. Vale, J. Cell Biol., 1991, 112, 1189.
6 T. Hiratsuka, Biochim. Biophys. Acta, 1983, 742, 496.
Fig. 5 Gliding velocities of microtubules plotted with respect to the concen-
tration of AzoTP. Red circles: gliding velocity in non-irradiated state; red line: curve
fitted using the Michaelis–Menten equation (Vmax = 0.61 mm sÀ1; Km = 2.6 mM).
Blue circles: observed gliding velocities in the cis-rich state after irradiation at
366 nm; black line: theoretical curve derived from the red line for 8% of trans-
AzoTP. Error bars: standard deviations for 10 microtubules.
7 For one-way control of the motility of kinesin through photochemi-
cal reactions, see: H. Higuchi, E. Muto, Y. Inoue and T. Yanagida,
Proc. Natl. Acad. Sci. U. S. A., 1997, 94, 4395; H. Hess, J. Clemmens,
D. Qin, J. Howard and V. Vogel, Nano Lett., 2001, 1, 235; A. Nomura,
T. Q. P. Uyeda, N. Yumoto and Y. Tatsu, Chem. Commun., 2006, 3588.
8 T. Ikeda and T. Ube, Mater. Today, 2011, 14, 480; F. Ercole,
T. P. Davis and R. A. Evans, Polym. Chem., 2010, 1, 37; N. Tamaoki
and T. Kamei, J. Photochem. Photobiol., C, 2010, 11, 47; T. Seki and
S. Nagano, Chem. Lett., 2008, 484; S. Yagai and A. Kitamura, Chem.
Soc. Rev., 2008, 37, 1520; K. G. Yager and C. J. Barrett, J. Photochem.
Photobiol., A, 2006, 182, 250.
velocities (black line) for the trans isomer (8% content) of
AzoTP. Accordingly, we found that the efficiency of the cis
isomer for microtubule motility was only 1/29 of that of the
trans isomer at an AzoTP concentration of 3 mM.
9 M. Banghart, K. Borges, E. Isacoff, D. Trauner and R. H. Kramer, Nat.
Neurosci., 2004, 7, 1381; M. Volgraf, P. Gorostiza, S. Szobota, M. R. Helix,
E. Y. Isacoff and D. Trauner, J. Am. Chem. Soc., 2007, 129, 260;
S. Muramatsu, K. Kinbara, H. Taguchi, N. Ishii and T. Aida, J. Am. Chem.
Soc., 2007, 128, 3764; M. D. Yamada, Y. Nakajima, H. Maeda and
S. Maruta, J. Biochem., 2007, 142, 691; B. Schierling, A. J. Noel,
W. Wende, L. T. Hien, E. Volkov, E. Kubareva, T. Oretskaya,
M. Kokkinidis, A. Rompp, B. Spengler and A. Pingoud, Proc. Natl. Acad.
Sci. U. S. A., 2010, 107, 1361; F. Zhang, K. A. Timm, K. M. Arndt and
G. A. Woolley, Angew. Chem., Int. Ed., 2010, 49, 3943; F. Bonardi,
G. London, N. Nouwen, B. L. Feringa and A. J. M. Driessen, Angew.
Chem., Int. Ed., 2010, 49, 7234; C. Hoppmann, P. Schmieder, P. Domang,
G. Vogelreiter, J. Eichhorst, B. Wiesner, I. Morano, K. Ruck-Braun and
M. Beyermann, Angew. Chem., Int. Ed., 2011, 50, 7699; A. Mourot,
M. A. Kienzler, M. R. Banghart, T. Fehrentz, F. M. E. Huber, M. Stein,
R. H. Kramer and D. Trauner, ACS Chem. Neurosci., 2011, 2, 536.
10 M. K. A. Rahim, T. Fukaminato, T. Kamei and N. Tamaoki, Lang-
muir, 2011, 27, 10347; M. K. A. Rahim, T. Kamei and N. Tamaoki,
Org. Biomol. Chem., 2012, 10, 3321.
The kinesin-microtubule system can be driven by several
ATP analogues modified at its base or ribose unit5 and by
nucleoside triphosphates containing a variety of base units16
(e.g., guanine, thymine, uracil, cytosine, inosine). In this pre-
sent study, we have demonstrated that non-nucleosides can
also be suitable replacements for the base and ribose units
when preparing substrates for kinesin.
In sharp contrast to the performance of a photoresponsive
ATP derivative substituted at the ribose moiety with a 4-tert-
butylazobenzene unit,11 AzoTP exhibits quite a large change in
activity toward the kinesin-microtubule system upon photo-
isomerization. It has been established that the ribose group of
ATP does not interact specifically with any of the amino acid
residues of kinesin in the binding site, while the adenine unit is
sandwiched through a stacking interaction with a histidine 11 T. Kamei, T. Fukaminato and N. Tamaoki, Chem. Commun., 2012,
48, 7625.
residue from below and through a hydrophobic interaction
with a proline residue and the methylene group of an arginine
12 D. Wang, E. Pate, R. Cooke and R. Yount, J. Muscle Res. Cell Motil.,
1993, 14, 484.
residue from above.17 The azobenzene moiety of AzoTP pre- 13 M. E. Porter, J. M. Scholey, D. L. Stemple, G. P. A. Vigers, R. D. Vale,
M. P. Sheetz and J. R. McIntosh, J. Biol. Chem., 1987, 262, 2794;
J. Howard, A. J. Hudspeth and R. D. Vale, Nature, 1989, 342, 154.
14 M. Castoldi and A. V. Popov, Protein Expression Purif., 2003, 32, 83;
sumably plays a similar role to that of the adenine moiety of
ATP. In its trans form, an azobenzene unit is flat, allowing it to
undergo the stacking interaction found for ATP. In contrast, cis-
azobenzene has a round shape that would prevent it from being
sandwiched by the stacking and hydrophobic interactions. The
large difference in activity of AzoTP in its trans and cis states
can, therefore, be explained by considering these differently
shaped isomers experiencing different types of interactions in
the binding site.
J. Howard and A. A. Hyman, Methods Cell Biol., 1993, 39, 105.
15 The concentrations of AzoTP solutions were calculated based on the
molecular weight of tetrasodium triphosphate. Errors in the concen-
trations to within 10% can be expected due to the contamination of
lower order sodium species (mono-, di- and tri-) R. G. Yount,
D. Babcock, W. Ballantyne and D. Ojala, Biochemistry, 1971, 10, 2484.
16 S. A. Cohn, A. L. Ingold and J. M. Scholey, J. Biol. Chem., 1989, 264, 4290.
17 F. J. Kull, E. P. Sablin, R. Lau, R. J. Fletterick and R. D. Vale, Nature,
1996, 380, 550.
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This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 9935--9937 9937