Targeted Activation of Molecular Transportation by Visible Light Ammathnadu S. Amrutha,† K. R. Sunil Kumar,†,⊥ Takashi Kikukawa,‡,§ and Nobuyuki Tamaoki*,† †
Research Institute for Electronic Science, Hokkaido University, N20, W10, Kita-ku, Sapporo, Hokkaido 001-0020, Japan Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan § Global Station for Soft Matter, Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo 060-0810, Japan Downloaded via TUFTS UNIV on June 30, 2018 at 17:18:27 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
‡
S Supporting Information *
ABSTRACT: Regulated transportation of nanoscale objects with a high degree of spatiotemporal precision is a prerequisite for the development of targeted molecular delivery. In vitro integration of the kinesin-microtubule motor system with synthetic molecules offers opportunities to develop controllable molecular shuttles for lab-on-a-chip applications. We attempted a combination of the kinesinmicrotubule motor system with push−pull type azobenzene tethered inhibitory peptides (azo-peptides) through which reversible, spatiotemporal control over the kinesin motor activity was achieved locally by a single, visible wavelength. The fast thermal relaxation of the cis-isomers of azopeptides offered us quick and complete resetting of the trans-state in the dark, circumventing the requirement of two distinct wavelengths for two-way switching of kinesin-driven microtubule motility. Herein, we report the manipulation of selected, single microtubule movement while keeping other microtubules at complete rest. The photoresponsive inhibitors discussed herein would help in realizing complex bionanodevices. KEYWORDS: kinesin, microtubule, photoswitches, azo-peptides, push−pull azobenzene, bionanodevice
A
carbon grafted with thermoresponsive polymer chains. However, detachment of the microtubules from the adsorbed kinesin motors was observed, which is undesirable for the applications requiring site-specific delivery of the cargo.12 Furthermore, selective and electrical control over translocation of microtubules was reported using a microfabricated channel on functionalized graphene electrodes.13 Similarly, manipulation and organization of microtubules by applying an AC voltage was also realized.14 Further, taking advantage of an electric field, a single microtubule was steered in microchannels.15 The works involving electrical control required careful monitoring of the motility buffer pH and prevention of undesired electrolysis. The higher field strengths applied could result in significant heating of the buffer, influencing the gliding velocity.16 The microtubule movements were controlled by magnetic field through partial functionalization of microtubules with magnetic nanoparticles17,18 and microtubule movements were dynamically altered by applying programmable, micro-
ctive intracellular transportation of cellular cargos, at the expense of ATP hydrolysis energy, is an intriguing cellular function performed by the motor protein kinesin in association with cytoskeletal filaments called microtubules.1,2 The kinesin exhibits attractive force production abilities and high fuel efficiency. As a result, it is receiving great attention for potential applications in the development of hybrid bionanodevices.3,4 The gliding motility assay or the inverted geometry, where surface bound kinesin motors propel microtubules is convenient to actualize directional transport and offers opportunities to control the gliding movement of microtubules in the artificial milieu.5 Through traditional bioconjugation techniques,6 different cargos like DNA,7 quantum dots,8,9 viruses,10 catalysts,11 etc. can be loaded on the microtubules and such loaded molecular shuttles need to be guided to achieve site-specific delivery, separation, and sorting of the cargo. Complete and reversible spatiotemporal control over the movement of any such cargo carrying shuttle in an ensemble is an essential requirement for the development of fully functional, biomimetic nanotransportation systems. Kinesin activity was shown to be locally regulated by employing visible light in a “light-to-heat” converting layer of © 2017 American Chemical Society
Received: August 24, 2017 Accepted: November 10, 2017 Published: November 10, 2017 12292
DOI: 10.1021/acsnano.7b06059 ACS Nano 2017, 11, 12292−12301
Article
www.acsnano.org
Article
ACS Nano
Figure 1. Synthesized azo-peptides.
patterned magnetic fields.19 The methods employing magnetic fields use complicated microfabrication techniques and require prefunctionalization of the microtubules. Caged ATP20,21 and caged inhibitors22 were used to switch the microtubule motility ON and OFF, respectively upon photoirradiation. Still, localizing their activities into a particular region of interest was not possible, due to the one-way switching properties of these photoswitches and the associated fast diffusion of the active molecules in the solution. It is found that two-way regulation of biological functions23 or material properties24,25 could be realized by incorporating reversible photoswitches into biological molecules or molecular assemblies, respectively. Recently, we have reported some significant research findings on the photoregulation of kinesindriven microtubule motility. We chose azobenzene, a reversible, robust photoswitch, and attempted to couple its photoisomerization event to optically manipulate the kinesin activity with a high degree of spatiotemporal control. A non-nucleoside ATP analog containing azobenzene (AzoTP) reported by our group, reversibly regulated the microtubule velocity with a change up to 79% in its two isomeric states.26 Also, azobenzene tethered inhibitory peptides (azo-peptides) earlier designed by us stopped the microtubule motility completely in their trans-state and allowed their motility in the cis-rich state, which was generated upon the UV light irradiation.27,28 With these two photoswitches, we achieved the local regulation of kinesin motor activity by simultaneous irradiation with UV and visible light using a simple, in-house built optical setup.29 By reducing the light beam size, we were able to select a microtubule and manipulate its movement specifically. Though our method involved no surface patterning and prefunctionalization, optimization of the two light intensities to achieve an appropriate isomer ratio at the specific region of interest was challenging. Also, while moving a specific, single microtubule, slow movements of neighboring microtubules were observed. Moreover, continuous UV light irradiation used could be detrimental to proteins. Herein, we report a range of azo-peptides whose photoresponsive azobenzene units are designed to show a reduced
HOMO−LUMO energy gap by the introduction of electron releasing (push) and electron withdrawing (pull) substituent pairs at the p and p’ positions. These substitutions not only enhanced the inhibitory activities of the azo-peptides but also shifted the π−π* transition band into the visible wavelength region and reduced the lifetime of cis azo-peptides. By taking advantage of the quick thermal relaxation behavior of the cis azo-peptides, we successfully achieved the complete and reversible spatiotemporal control over the kinesin motor activity by a single, visible wavelength. The high degree of precision obtained, allowed the manipulation of a particular, single microtubule movement without disturbing the adjacent filaments. We emphasize that employing visible wavelength in the local regulation of kinesin activity is advantageous over the UV light, being innocuous to proteins and offers easy implementation into the fluorescence optical microscope.
RESULTS Synthesis of Azo-peptides. The general protocol for solid phase peptide synthesis based on the Fmoc strategy was adopted in the synthesis of azo-peptides.27 Synthesized azopeptides are presented in Figure 1. Azo-peptides 1a, 2a, and 3a bear -Ile-Pro-Lys-Ala-Ile-Arg-OH and 1b, 2b, and 3b bear -ArgIle-Pro-Lys-Ala-Ile-Arg-OH peptide units. These two peptides have been reported to possess the vital amino acids responsible for the complete inhibition of kinesin motor activity.28 Azopeptides 2a and 2b were obtained by coupling the azobenzene carboxylic acid derivatives at the N-termini of the peptides. Azo-peptides 1a, 1b, 3a, and 3b were procured by coupling the intermediates formed by the reaction between azobenzene amine derivatives and N,N′-carbonyldiimidazole at the Ntermini of the peptides. Synthesized azo-peptides were purified by reversed-phase HPLC and characterized by ESI-TOF mass spectra. Mass spectra (Figures S1−S5) and HPLC chromatograms (Figure S6) affirming the purity of azo-peptides are displayed in the Supporting Information. The mass spectra and HPLC chromatogram of the azo-peptide 2a are excluded from the Supporting Information since they are presented in our 12293
DOI: 10.1021/acsnano.7b06059 ACS Nano 2017, 11, 12292−12301
Article
ACS Nano Scheme 1. Reversible Isomerization of Azo-peptides
Figure 2. (a) UV−visible absorption spectra of the azo-peptides 1b (red line), 2b (blue line), and 3b (black line) measured in BRB-80 buffer at 25 °C before irradiation. (b) Change in the optical density (OD) profile of azo-peptide 1b at 461 nm after laser photoflash (λ = 532 nm). The solid red line is a fit with the exponential form of the first order rate equation [y = m1∗exp(−m2∗t); t = time (ms); fitting parameters m1 = −6.89, m2 = 1.702; R = 0.99].
previous report.28 Azobenzene amine derivatives were synthesized by following the earlier reported procedures.30,31 Reversible Isomerization of Azo-peptides. The transisomers of azo-peptides presented here undergo photoisomerization by absorbing visible light forming cis-isomers whereas the cis-isomers formed showed quick thermal relaxation behavior forming the trans-isomers as depicted in the Scheme 1. Figure 2a presents the UV−visible absorption spectra of azo-peptides 1b, 2b, and 3b in BRB-80 buffer at 25 °C before visible light (510 nm) irradiation. The absorption maxima (λmax) corresponding to the π−π* transition band were observed in the visible wavelength region. Upon visible light irradiation, significant changes in the spectral profiles corresponding to the cis-isomer formation were not detected with our conventional spectrophotometer, indicating the quick thermal relaxation property of the cis-isomer generated by photoirradiation with the rate exceeding the acquisition speed of the spectrophotometer which is ca. 1 s. The different azobenzene chromophores presented here are “push−pull” type azobenzenes. It is well-known that in “push−pull” type azobenzenes, the cis (Z)- to trans (E)-thermal isomerization occurs faster than in regular azobenzenes.32 Therefore, we adopted a laser flash photolysis technique to determine the thermal relaxation rates and lifetimes of the cis-isomers. The flash photolysis experiments were carried out in BRB-80 buffer at 25 °C. The change in optical density (OD) at 461 nm when the solution of azo-peptide 1b was flashed with the laser (λ= 532 nm) is presented in Figure 2b. Upon laser flash for 7 ns, we observed a sudden decrement in the OD. Within ca. 2 ms the change in OD was recovered. The flash photolysis
experiment indicated the generation of cis-isomer by visible light irradiation and fast thermal relaxation of the photogenerated cis-isomer into the trans-isomer in the dark. Thermal relaxation rate constants and the lifetimes of cis-isomers were determined by fitting the curve to the exponential form of the first order rate equation. UV−visible absorption spectra and flash photolysis curves of all the remaining azo-peptides are presented in the Supporting Information (Figures S7−S12). Table 1 shows the thermal relaxation rate constant (k) and lifetime (τ) values of cis-isomers obtained from the flash photolysis experiments. Table 1. Thermal Relaxation Rate and Lifetime of cis Azopeptides azo-peptide 1a 1b 2a 2b 3a 3b
thermal relaxation rate constant (k) (s−1)
lifetime of cis azo-peptide (τ) (ms)
× × × × × ×
0.652 0.587 0.058 0.056 0.026 0.028
1.53 1.70 1.71 1.79 3.79 3.72
103 103 104 104 104 104
In vitro gliding motility assays were performed to investigate the concentration dependent inhibitory activities of the azopeptides in BRB-80 buffer at 25 °C in the presence of 1 mM ATP. Figure 3 shows the gliding velocities of microtubules at different concentrations of the azo-peptides in dark and under continuous visible light (488 nm; intensity ca. 8 × 10−1 W/ 12294
DOI: 10.1021/acsnano.7b06059 ACS Nano 2017, 11, 12292−12301
Article
ACS Nano
Figure 3. Gliding velocities of microtubules plotted against the concentrations of the azo-peptides (black circles, gliding velocity under dark condition; red circles, gliding velocity under continuous visible light irradiated condition). Error bars present the standard deviation for five microtubules in one flow cell.
cm2) irradiated conditions. In the trans-state of the azo-peptide 1a (Figure 3a), the gliding velocity sharply decreased at a concentration of 0.5 mM and became zero at a 0.75 mM concentration. Motility was observed only under continuous 488 nm light irradiation and stopped immediately after the removal of light irradiation. At a 0.75 mM concentration, the gliding motility was switched between ON and OFF states completely and reversibly for several cycles by a single, visible wavelength of light (Movie S1 demonstrates the ON/OFF switching of gliding motility by 488 nm light in the presence of a 0.3 mM concentration of the azo-peptide 1b). The IC50 value calculated for the trans-isomer of azo-peptide 1a is 0.29 mM.
Table 2 presents the IC50 values calculated for the different trans azo-peptides and the minimum azo-peptide concentration displaying complete ON/OFF switching of the microtubule movement. Dependence of Gliding Velocity on the Actinic Light Intensity. Further, we studied the effect of irradiated light intensity on the gliding velocity of microtubules with azopeptides 1b (0.3 mM), 2b (0.75 mM), and 3b (0.3 mM) in the presence of 1 mM ATP by measuring the gliding velocities of the microtubules at different intensities of the irradiated light (488 nm). The intensity of the laser beam at the sample ca. 8 × 10−1 W/cm2 was considered as 100%. ND50, ND25, ND12, 12295
DOI: 10.1021/acsnano.7b06059 ACS Nano 2017, 11, 12292−12301
Article
ACS Nano
μM), casein, oxygen scavengers, ATP (1 mM) and azo-peptide 1b (0.3 mM). The flow cell was analyzed under fluorescence optical microscope. Initially, all the microtubules were in the resting state. We irradiated a selected microtubule with the 488 nm laser light (diameter of the circular light beam = 5 μm), which initiated the microtubule movement. When the microtubule started moving, the position of the light beam was slowly changed, and the microtubule was translocated to the other region of the imaging area (see Movie S2). The microtubule moved with the velocity equal to 0.47 ± 0.01 μm/s. Figure 5 presents the selected single microtubule movement under continuous 488 nm light irradiation (intensity ca. 3 × 10 W/ cm2). Adjacent microtubules remained in the same position throughout the experiment. In another experiment, we prepared the flow cell as mentioned above. A selected microtubule was irradiated at its trailing end with the 488 nm laser light (intensity ca. 3 × 10 W/ cm2; diameter of the circular light beam = 5 μm). Irradiation at the trailing end induced bending of the microtubule initially and prolonged irradiation at the trailing end finally resulted in breaking of the microtubule at the position of bending (see Movie S3). Figure 6 presents the sequential images showing bending and breaking of the selected single microtubule and translocation of the newly formed two microtubules by light.
Table 2. Inhibitory Activity and Photoswitchability of Azopeptides azo-peptide
IC50 trans (mM)
minimum concentration showing complete ON/ OFF switching of motility (mM)
1a 1b 2a 2b 3a 3b
0.29 0.21 0.86 0.27 0.25 0.17
0.75 0.30 1.25 0.75 0.60 0.30
ND6, and ND1 are the filters used which transmitted 50% (ca. 4 × 10−1 W/cm2), 25% (ca. 2 × 10−1 W/cm2), 12% (ca. 1 × 10−1 W/cm2), 6% (ca. 5 × 10−2 W/cm2), and 1% (ca. 8 × 10−3 W/cm2) of the source light intensity, respectively. Figure 4 presents the changes in gliding velocity upon changing the light intensity.
DISCUSSION We recently reported the local regulation of kinesin motor activity through azobenzene-based photoswitches.29 There, we selectively induced motility into a particular microtubule by irradiating the selected microtubule with one wavelength and the entire imaging area with the other wavelength. There, the irradiation of the entire imaging area with the second wavelength was essential to avoid the activation of motors present in the surrounding region by diffused active molecules. In this case, optimizing the intensities of two wavelengths to obtain clearly distinguishable kinesin motor activity between the locally irradiated area and the surrounding was important and challenging. Between the trans- and cis-forms of azobenzene, the cis-form is thermally less stable than the trans- and the lifetime of cis-isomer largely depends upon the substitution pattern of the azobenzene.32 This property prompted us to go for the push−pull type azobenzenes. The short lifetime for cis-isomers observed in the case of push−pull type azobenzenes was beneficial for us with which, use of two distinct wavelengths for two-way switching can be circum-
Figure 4. Gliding velocities of microtubules plotted against the percentage intensity of 488 nm light. Error bars show the standard deviation for five microtubules in one flow cell. Note: 100% intensity is ca. 8 × 10−1 W/cm2.
Local Regulation of the Microtubule Movement. We used the same optical setup developed by our group earlier to translocate the microtubule and to manipulate its movement selectively.29 Here we used only one wavelength of light, unlike our previous report where we used UV and visible wavelengths simultaneously. The schematic representation of the optical setup used is provided in the Supporting Information (Figure S13). We prepared a flow cell with surface adsorbed kinesin (0.125 mg/mL), fluorescent dye-labeled microtubules (0.025
Figure 5. Sequential fluorescence images presenting the translocation of the selected, single microtubule by light in the presence of azopeptide 1b (0.3 mM) and ATP (1 mM). The images display the locations of the microtubule at 0 s, 1 min and 26 s, 2 min and 31 s, and 4 min and 37 s. White spots, 488 nm light beam; arrow marks, positions of the moving microtubule. 12296
DOI: 10.1021/acsnano.7b06059 ACS Nano 2017, 11, 12292−12301
Article
ACS Nano
Figure 6. Sequential fluorescence images presenting the bending and breaking of the selected microtubule by light in the presence of azopeptide 1b (0.3 mM) and ATP (1 mM). (1) Initial state; (2) irradiation at the trailing end (inset: bending of the microtubule filament); (3) after 4 s of irradiation at the trailing end (inset: breaking of the microtubule at the position of bending); (4) formation of two new filaments; (5)−(8) translocation of the newly formed microtubules by light one by one. White spots -488 nm light beam.
with the kinesin pocket through H-bonding interactions. This enhanced inhibition property was observed with the azopeptide 3b also when compared with the azo-peptides 1b and 2b (Table 2 and Figure 3b, d, and f). Between 1a and 2a (Figure 3a and c), 1a showed more affinity toward kinesin owing to the presence of an additional -NH moiety along with the dimethylamino group, with which multiple H-bonding interactions are expected between the azobenzene unit and the kinesin pocket. Similar behavior was observed with the azopeptides 1b and 2b (Figure 3b and d), where 1b showed stronger inhibition property than 2b. The introduction of push−pull substituent pair into the azobenzene unit shifted the π−π* transition band into the visible wavelength region (Figure 2a). Therefore, a visible wavelength (488 nm) was chosen to elicit the photochemical conversion of trans azo-peptide into cis azo-peptide. The kinesin-driven microtubule movement was observed only under the continuous irradiation of 488 nm light. The microtubule movement stopped immediately after removing the light irradiation due to the spontaneous conversion of cis azopeptide into trans azo-peptide in the dark driven by the fast thermal relaxation. Thus, we were able to regulate the kinesindriven microtubule movement reversibly by the single wavelength of light. We believe that the gliding velocity of microtubules observed under the continuous light irradiation is the resultant of two factors. The first factor is the affinity of the cis form of azo-peptide for the kinesin motor domain. The higher the affinity the lower is the velocity observed. The second factor is the intensity of the irradiated light beam used for the trans- to cis-isomerization. Quick thermal relaxation obviously leads to a small steady state fraction of the cis-isomers and the high-intensity light is required to generate a large enough fraction of cis-isomers to observe the kinesin-driven microtubule movement. The experiment performed to examine the dependence of gliding velocity over the intensity of irradiated beam (Figure 4) justified our hypothesis.
vented by the fast thermal relaxation process offering quick and complete resetting of the trans-state in the dark. In the flash photolysis experiments, azo-peptides 3a and 3b showed the shortest lifetime values for cis-isomers (Table 1) which are attributed to the presence of a strong push−pull substituent pair (electron-donor N′-ethyl-1,2-ethanediamino and electron-acceptor nitro groups) causing the asymmetric electron distribution. It is observed that the azobenzenes with large asymmetric electron distribution possess a low HOMO− LUMO energy gap which facilitates the fast thermal relaxation of the cis-isomer into trans.33 Azo-peptides 2a and 2b showed longer lifetime than the azo-peptides 3a and 3b due to the presence of relatively weaker push−pull substituent pair (electron-donor dimethylamino group and electron acceptor carbonyl group). Azo-peptides 1a and 1b showed the longest lifetime for cis isomers because; the electron-acceptor carbonyl group was connected to the azobenzene ring through the amine, NH. Hence, the asymmetry in the electron distribution was reduced. Structure−property relationship studies of various azopeptides performed by us earlier revealed that an additional Arg unit at the N-terminus of the peptide interacts through electrostatic interaction with the acidic amino acid residue of the kinesin motor domain and improves the affinity of the azopeptide toward kinesin.28 Evidently, azo-peptides with the ArgIle-Pro-Lys-Ala-Ile-Arg-OH peptide unit (1b, 2b, and 3b) showed enhanced inhibition compared to the respective azopeptides having a -Ile-Pro-Lys-Ala-Ile-Arg-OH peptide unit (1a, 2a, and 3a) in the in vitro gliding motility assays (Table 2 and Figure 3). On the other hand, substituent groups present on azobenzene also significantly affected the inhibitory activities of the azo-peptides. Among azo-peptides 1a, 2a, and 3a, azopeptide 3a showed complete inhibition at relatively lower concentration than the others (Table 2 and Figure 3a, 3c, and 3e). Here the N′-ethyl-1,2-ethanediamine substituent on azobenzene acted as an additional moiety capable of interacting 12297
DOI: 10.1021/acsnano.7b06059 ACS Nano 2017, 11, 12292−12301
Article
ACS Nano
bleaching (Figure S14). In our earlier report we had used a 365 nm light (intensity ca. 2 × 102 W/cm2) to locally irradiate a selected microtubule and the entire flow cell area was irradiated with the 510 nm light (intensity ca. 2.6 × 10−2 W/cm2). Though the cis azo-peptide used in our earlier report possessed longer lifetimes (in the order of hours), considerably high concentration (2 mM) of the azo-peptide used and rapid diffusion of inhibitory trans-isomers generated under 510 nm light irradiation from the surrounding prompted us to make use of high intensity UV light to selectively drive the single microtubule.29 It must be beneficial to compare the needed light intensity in the present experiment to regulate the motility of single microtubules with those used in other related optical methods. In optogenetics34 - a tool used to optically regulate the functioning of genetically modified cells, the photoactive units are covalently bound to the proteins and therefore diffusion of the other isomer is not observed. In addition, the cis-isomers of the chromophores present (e.g. cis-p-coumaric acid present in photoactive yellow protein35) exhibited lifetime in the order of seconds. As a result a lower intensity light (
