A photochromic ATP analogue driving a motor protein with reversible light-controlled motility: Controlling velocity and binding manner of a kinesin-microtubule system in an in vitro motility assay

Article abstract of DOI:10.1039/c2cc33552b

We synthesized two photochromic ATP analogues (ATP-Azos) featuring azobenzene derivatives tethered at the 2′ position of the ribose ring. In the presence of the ATP-Azo tethering p-tert-butylazobenzene, we observed reversible photo-control of the motility, velocity and binding manner, of a kinesin-microtubule system in an in vitro motility assay.

Full text of DOI:10.1039/c2cc33552b

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A photochromic ATP analogue driving a motor protein with reversible
light-controlled motility: controlling velocity and binding manner of a
kinesin–microtubule system in an in vitro motility assayw
Takashi Kamei, Tuyoshi Fukaminato and Nobuyuki Tamaoki*
Received 13th March 2012, Accepted 7th June 2012
DOI: 10.1039/c2cc33552b
We synthesized two photochromic ATP analogues (ATP-Azos)
featuring azobenzene derivatives tethered at the 20 position of
the ribose ring. In the presence of the ATP-Azo tethering p-tert-
butylazobenzene, we observed reversible photo-control of the
motility, velocity and binding manner, of a kinesin–microtubule
system in an in vitro motility assay.
Scheme 1 Photoresponsive ATP analogues 1a and 1b. Reversible trans
Adenosine 50-triphosphate (ATP) is a biogenic molecule that
(left)/cis (right) isomerizations of the azobenzene derivative moieties.
functions as an energy donor and interacts with various
a kind of ATPase motor protein, through an in vitro motility
proteins, ATPases. To investigate and artificially control the
assay performed by utilizing an ATP-Azo.
functions of ATPases, several photoresponsive ATP analogues,
such as fluorescent-labeled ATPs1 and caged-ATPs,2 have been
We designed our ATP analogues such that a single azobenzene
moiety would be appended to the ribose ring of the ATP unit, in
developed. The caged-ATP system is unique in that the hydro-
consideration of the fact that various ATP analogues modified at
lysis of ATP mediated by an ATPase is initiated by UV light
these positions can still interact with ATPases.1,10 We synthesized
irradiation to cleave the o-nitrophenyl protecting group of the
ATP analogues tethering an azobenzene or p-tert-butylazobenzene
g-phosphate unit of the ATP derivative. This photo-cleavage
moiety at the 20 position of the ribose ring (Scheme 1) using
process is irreversible, therefore it is impossible to modulate the
the approach shown in Schemes S1 and S2 (see ‘‘Syntheses of
hydrolysis rate with desired timing once the protecting group has
Compounds’’, ESIw).11 This synthetic pathway allows us to
been released. An ATP analogue having the potential to rever-
modify ATP only at the 20 position of the ribose ring, thereby
sibly photo-control the functions of ATPases would be a
avoiding any potential confusion, due to the presence of 20 and
promising molecule to control various biochemical phenomena.
30 isomers, when interpreting the experimental results12 and
In this regard, an ATP analogue possessing a photochromic unit
eliminating any possible contamination of ATP, in contrast to
that could undergo reversible conformational changes would be
other syntheses starting from ATP. The resultant ATP-Azos,
a candidate molecule for the reversible control of ATPase
1a and 1b, were obtained as their sodium salts.
activity under irradiation with light. Azobenzene, which exhibits
We examined the photoisomerization behavior of ATP-Azos
reversible conformational changes upon irradiation with light of
through alternating irradiation with UV light (l = 365 nm),
inducing trans-to-cis isomerizations of the azobenzene moieties
of the ATP-Azos, and visible light (l = 436 nm), inducing their
reverse cis-to-trans isomerizations [Scheme 1; Fig. S1 and S2
(presenting the examples for 1b), ESIw].
various wavelengths, is often applied as a photo-controllable
mediator of the functions of proteins3 and synthetic molecules.4–8
The photo-control of protein functions has been studied using
azobenzene derivatives conjugated to peptides or proteins.
However, there have been few studies of the development of
small-ligand nucleotides connected to azobenzene derivatives.9
In this study, we synthesized azobenzene derivatives of ATP,
ATP-Azos, as a novel class of ATP analogues. Herein, we demon-
strate the reversible photo-control of a motile property of kinesin,
We used an in vitro motility assay of kinesin13 to assess the
properties of the ATP-Azos in their different isomeric states as
substrates of ATPase, thereby allowing the ATPase activity to
be microscopically visualized in terms of dynamic movements.
In the assay, we confirmed that fluorescent-labeled micro-
tubules (MTs)14 were driven by recombinant kinesin (kinesin-1)
molecules coated on the glass surface in the presence of ATP-
Azos added instead of ATP, and evaluated the motility
behaviors of the MTs (see ‘‘Observation of Motility of
Kinesin–Microtubule System’’, Fig. S3, ESIw).
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
w Electronic supplementary information (ESI) available: Syntheses of
compounds, observation of motility of kinesin–microtubule system.
See DOI: 10.1039/c2cc33552b
Table 1 and Fig. 1 display the sliding velocities of the MTs
driven by the kinesins at various concentrations of the ATP-Azos
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Table 1 Sliding velocity of MTs in the presence of each substrate
induced the reverse cis-to-trans isomerization of 1b, resulting
in a decrease in the velocity to 76 nm sꢁ1—almost the same
value as that obtained initially prior to UV irradiation
(Fig. 1a). We also observed this tendency—an increase in
velocity after irradiation with UV light and a decrease with
visible light—at other concentrations of 1b. The NMR data
shown in Fig. S2 (ESIw) suggest that about 10% of trans-1b
remained at the photostationary state under UV irradiation.
Complete isomerization would have achieved larger difference
in the velocity change upon irradiation. When the concen-
tration of trans-1b was too high (1 mM and ‘‘Vis’’ for 650 mM,
Fig. 1a), we could not measure the velocities of the MTs
because they were not bound to the kinesin-coated glass
surface and drifted in the assay buffer. Therefore we could
not measure or determine Km for trans-1b, by the same token,
or compare Km for trans-1b to that for cis-1b.
Sliding velocity of MTs
Average ꢀ s.e.m. (nm sꢁ1), n = 15–50
Concentration trans-1a cis-1a
trans-1b cis-1b
ATP
1 mM
100 mM
372 ꢀ 7 383 ꢀ 7 nd 236 ꢀ 14 656 ꢀ 26
179 ꢀ 4 175 ꢀ 3 75 ꢀ 3 117 ꢀ 3
B390a
a
Estimation (see Fig. S4, ESI).
To confirm the reproducibility and repeatability of the
changes in the sliding velocities of the MTs upon irradiation
with light, we inverted the order of the irradiation sequence.
We observed the same tendency in the velocity changes as
mentioned above when subjecting cis-1b (‘‘UV 1st’’, Fig. 1b),
itself obtained after irradiation at 365 nm, to irradiation at 436
nm, converting it into trans-1b (‘‘Vis’’) with an accompanying
decrease in the MT velocity, followed by irradiation at 365 nm
to reform cis-1b (‘‘UV 2nd’’), accompanied by an increase. We
could not measure the velocity of the MTs at 1 mM trans-1b
(Fig. 1b, ‘‘Vis’’) for the same reason mentioned above.
Fig. 1 Photoresponsive sliding velocities of MTs in the presence of 1b
(error bars; s.e.m.). The concentration of 1b corresponding to each
curve is presented, except for 1 mM [circles for (a), squares for (b)].
(a) ‘‘No irra’’: prior to irradiation; ‘‘UV’’: irradiation with UV light
after ‘‘No irra’’; ‘‘Vis’’: irradiation with visible light after ‘‘UV’’. MTs
bound to the glass surface were not observed in the following cases:
Vis for 650 mM; no irra for 1 mM (n = 50 for 30 mM to 300 mM;
n = 35 for 650 mM; n = 11 for 1 mM). (b) ‘‘UV 1st’’: irradiation with
UV light; ‘‘Vis’’: irradiation with visible light after ‘‘UV 1st’’; ‘‘UV 2nd’’:
irradiation with UV light after ‘‘Vis’’. MTs bound to the glass surface
were not observed in the following case: 1 mM, ‘‘Vis’’ [n = 50 for 30 mM
to 300 mM; n = 35 (UV 1st), 12 (Vis), 18 (UV 2nd) for 650 mM; n = 17
(UV 1st) or 8 (UV 2nd) for 1 mM].
Irradiation with UV light had no effect on the kinesin-
induced motilities of the MTs in the presence of ATP (i.e. not
the ATP-Azos) (Fig. S4, ESIw). Therefore, our observation of
a different sliding velocity for each isomeric state of 1b clearly
reveals that the action of ATPase could be controlled dyna-
mically through irradiation with light in the presence of the
photochromic ATP analogue. We did not observe any signifi-
cant changes in the sliding velocities of the MTs before or after
UV light irradiation of 1a, which lacks the tert-butyl group
(Table 1). Thus, the nature of the substituent on the azo-
benzene unit of the ATP-Azo is a crucial factor affecting the
photo-controlled functions of ATPases. The hydrophobic tert-
butyl group presumably enhanced the difference in one or
more of the properties (e.g. binding affinity, hydrolysis rate) of
the trans and cis states of 1b as substrates, relative to that of
1a, ultimately resulting in observable changes in the motile
velocities of the kinesin–MT–1b system.
in their trans and cis forms. The average velocities of the MTs at
1 mM of trans-1a and cis-1a were 372 ꢀ 7 and 383 ꢀ 7 nm sꢁ1
,
respectively—approximately 60% of the value (ca. 660 nm sꢁ1
)
obtained for ATP at 1 mM, which was the sufficient concen-
tration for saturating the sliding velocity of the MTs (Table 1).
At 100 mM of 1a, a concentration close to the value of Km for
ATP (85 mM, obtained through our motility assay; Fig. S3,
ESIw), we recorded velocities of 179 ꢀ 4 and 175 ꢀ 3 nm sꢁ1
for trans-1a and cis-1a, respectively—approximately 45% of
the value for ATP (ca. 390 nm sꢁ1; Table 1). Thus, trans–cis
isomerization of 1a did not significantly affect the sliding
velocity of the MTs. In contrast, for 1b, which features a
tert-butyl group at the azobenzene terminus, we observed a
significant difference in the sliding velocities of the MTs before
and after irradiation with UV light. The average velocity of the
MTs at 100 mM trans-1b was 75 nm sꢁ1 [ca. 20% of the
velocity at 100 mM ATP (ca. 390 nm sꢁ1); Table 1 and Fig. 1a].
After converting trans-1b to cis-1b upon irradiation with UV
light, the velocity reached 117 nm sꢁ1 (an increase of 56%;
Table 1 and Fig. 1a). Subsequent irradiation with visible light
As mentioned above, the sliding velocity of the MTs was
clearly photo-controllable in the presence of 1b at or below
300 mM (Fig. 1). At high concentrations of 1b, however, we
observed interesting behavior for the interactions between the
MTs and the kinesin units coated on the glass surface,
providing another approach toward photo-controlling the
motility of the kinesin–MT system, as described below.
At 650 mM of trans-1b, the number of MTs attached to the
glass surface was less than that at or below 300 mM; these MTs
tended to glide on the kinesin-coated glass surface in a
discontinuous manner or detach from the surface. In the
presence of 1 mM of trans-1b, we observed only drifting
MTs—that is, none were bound to the kinesin units coated
on the surface (Fig. 2a). After converting trans-1b into cis-1b
through UV light irradiation, the MTs were bound to and slid
c
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Notes and references
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c
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Chem. Commun., 2012, 48, 7625–7627 7627