Article pubs.acs.org/JPCC
Resonant Excitation Effect on Optical Trapping of Myoglobin: The Important Role of a Heme Cofactor Tatsuya Shoji,† Noboru Kitamura,†,‡ and Yasuyuki Tsuboi*,†,‡,§ †
Department of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-0810, Japan § JST, PRESTO, Japan ‡
S Supporting Information *
ABSTRACT: We demonstrate the efficient trapping of myoglobin (Mb), which is a small protein, in aqueous solution using a 1064 nm laser beam. Conventional optical tweezers exert barely sufficient radiation force (RF) on nanometer-sized dielectric objects, such as small proteins and organic molecules, for manipulation of them. One possible candidate to enhance the RF is to use laser light with a wavelength that is electronically resonant with the electronic transition of the target object: resonant optical trapping (ROT). The near-infrared absorbance dependence of ROT of Mb and optical trapping of Mb without the heme cofactor (apomyoglobin) clearly indicates that the heme cofactor plays a crucial role in the ROT of Mb. Furthermore, confocal Raman microspectroscopy suggested the structural conformational change of trapped Mb. Such an ROT technique would open up new channels in the development of nanobioscience, such as transportation/crystallization techniques and a selective molecular sorting technique for biomolecules.
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INTRODUCTION Nowadays, optical tweezers using a focused laser beam have become excellent tools to three-dimensionally trap and manipulate a particle whose size ranges from a few tens of nanometers to tens of micrometers.1 Particularly, in the biosciences, optical tweezers have made great contributions to understanding the physical characteristics of biomolecules, as reviewed in the literature.2−5 For instance, by measuring the displacement of an optically trapped microsphere tethered to a biomolecule, several groups have revealed the movements of kinesin along microtubules,6 myosin along actin filaments,7 and RNA polymerase along a DNA chain during transcription.8 It is also noteworthy that the optical trapping of biomolecules molecular trappingprovides a powerful tool for handling/ micropatterning DNA,9,10 and assembling/crystallizing proteins and amino acids.11−17 Radiation force (RF), which is the driven force for optical tweezers, is proportional to the gradient of light intensity and polarizability of the target particle.18 Accordingly, a theoretical calculation of the RF strongly suggests that biomolecules smaller than ca. 10 nm in radius (a) are scarcely trapped, because RF decreases as the radius a of the target object decreases. Indeed, we demonstrated that hen egg-white lysozyme (a ∼ 2 nm) was optically trapped, not as individual molecules (because it is too small), but as larger lysozyme clusters (a ≥ 20 nm), in a supersaturated solution. Furthermore, we revealed that optical trapping (OT) of lysozyme frequently resulted in nucleation of a crystal.12 Such a molecular cluster trapping mechanism holds also for smaller molecules, such as amino acids.13−16 Beyond the simple © 2013 American Chemical Society
application of crystallization, molecular trapping can be potentially extended to nanoscience applications, such as molecular sorting and transporting techniques.19,20 However, as mentioned above, conventional optical tweezers hardly manipulate small molecules (a ≤ 10 nm) in solution unless the molecules aggregate each other to form larger molecular clusters. A powerful methodology to enhance the RF based on new physics, such as plasmon, is intriguing in the development of molecular trapping.21−23 One of possible candidates for a powerful technique based on novel physics is to use laser light whose wavelength is resonant with the electronic transition of the object to be trapped: the resonant effect. The origin of the RF enhancement under a resonant condition is an increase in the polarizability of the object at the resonant wavelength. On the basis of the resonant effect, researchers have demonstrated the cooling and trapping of atoms in vacuum.24−26 Furthermore, they have developed the methodology to realize Bose−Einstein condensation in the dilute gas phase of alkali atoms.27,28 Such manipulation of atoms is based on the resonant effect. In principle, such a resonant effect is applicable to molecular systems. However, in using resonant light to trap small particles, we frequently face several problems, such as a decrease in the RF due to broadening of the resonance absorbance at room temperature and photothermal damage of the objects. In the solution phase, Osborne et al. first reported that the translational diffusion of a Received: November 9, 2012 Revised: April 24, 2013 Published: April 25, 2013 10691
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dye molecule was slightly suppressed in the region around the focal point of a visible laser beam.29 This observation implies a potential application of “resonant optical trapping (ROT)” for molecular systems in the condensed phase. However, experimental demonstrations of ROT are limited to a few systems: gold nanoparticles,30−34 dye-doped polystyrene spheres,35−37 and fluorophore-labeled antibodies.38 Because manipulation of biomolecules is obviously important, it is fruitful if one can optically trap a small biomolecule. Along this line, we have made a preliminary exploration to show that heme proteins (cytochrome c and myoglobin) were efficiently trapped even though the proteins are as small as lysozymes (a = 2 nm).39 The heme proteins were trapped even in a nonsaturated solution where the formation of larger molecular clusters should be negligible. Because both cytochrome c and myoglobin commonly have a “heme” chromophore, it is implied that a resonant effect due to light absorption by a heme cofactor possibly plays an important role in the efficient trapping. In the present study, the effect of resonance on the OT of Mb was investigated in detail. Microscopic observations indicate that Mb was trapped with high efficiency in solution under a resonant excitation condition. Furthermore, we carried out the confocal Raman microspectroscopy for trapped Mb assemblies. The origin of such an efficient OT should be due to the resonant effect on the basis of the absorption of the incident laser light by the heme cofactor in Mb.
Figure 1. Size distributions of myoglobin (Mb) and apomyoglobin (apoMb) measured by a dynamic light-scattering method in buffer solutions: (a) Mb in pD 7.6, (b) pD 10, and (c) pD 12, and (d) apoMb in pD 7.6.
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EXPERIMENTAL SECTION Materials. Myoglobin (Mb, from equine skeletal muscle, Sigma, Bioultra M5696, m.w. ∼ 17 600), apomyoglobin (apoMb, from equine skeletal muscle, Sigma, protein sequencing standard A8673, m.w. ∼ 17 000), and D2O (Wako, 99.99%) were used without further purification. Each protein was dissolved in D2O buffer solutions: 0.1 mol/L phosphate buffer (pD 7.6 and 8.8) and 0.1 mol/L carbonate buffer (pD 10 and 12) solutions. The solutions were centrifuged at 13 900g for 5 min and then filtered through membrane filters (pore size = 0.20 μm) to remove microdusts. Dynamic light scattering (Figure 1) clearly indicates that Mb and apoMb are folded and homogeneously dissolved as individual molecules without aggregation.40,41 The concentrations of Mb and apoMb were precisely determined by measuring the absorbance at 409 and 280 nm, respectively: ε409(Mb) = 17.9 × 104 cm−1 L mol−1; ε280(apoMb) = 1.59 × 104 cm−1 L mol−1.42 A 30 μL droplet of the sample solution was added to inert paraffin oil in a microliter volume polydimethylsiloxane sample cell to prevent vaporization of solvent (liquid thickness ca. 2 mm). Although Marangoni convection should be taken into account in a trapping mechanism in a thin water membrane (thickness: 100 μm),14,15,43,44 such convection effects would be negligible in the present experimental system. The sample cell was covered by a cover glass for protecting from microdusts in the outside (Figure 2a). Confocal Raman Microspectroscopy for Chemical Analysis of Trapping Particle. Figure 2b shows a schematic illustration of our OT system combined with confocal Raman microspectroscopy. 45−47 We used two laser beams: a continuous-wave (cw) Nd3+:YAG laser beam (λ = 1064 nm, Spectron Laser System, SL-902T) as the light source for the optical tweezers and a cw Ar+ laser (λ = 488 nm, Coherent, Inova 70) as the excitation light source for Raman scattering. Raman microspectroscopy was carried out for analysis of the
Figure 2. Schematic illustrations of the experimental setup for optical trapping of Mb and apoMb: (a) a sample cell and (b) optical setup for the confocal Raman microspectroscopy system.
chemical structure of optically trapped Mb. The two laser beams were coaxially introduced into an inverted optical microscope (Nikon, ECLIPSE TE300). By using an oilimmersion objective lens (×100, N.A. = 1.30, CFI plan Fluor, working distance = 0.2 mm), two laser beams were tightly focused to the same position of sample solution and the focal positions were set to ∼100 μm far from a bottom of a cover glass surface. For observation of OT behavior of Mb, we carried out the traditional methods: the bright-field microscopy with a halogen lamp and the dark-field microscopy with backward Rayleigh scattering of the Ar+ laser (backscattered light).48,49 Using the backscattered light, we can observe a optically trapped nanoparticle consisting of Mb molecules, which is hardly observed with bright-field microscopy. Both microscopic 10692
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a buffer solution of Mb (pD 7.6, 0.1 mmol/L) during irradiation with the YAG laser light (Peff = 0.8 W). Although the size of the assembly did not become larger to be observable under bright field, the Mb assembly was also created during several minutes of YAG irradiation in lower concentration. Confocal Raman Microspectroscopy. To analyze the chemical structure of the assembly, confocal Raman microspectroscopy was performed on it, and the results are shown in Figure 4. Mb has a characteristic absorption band at around 500
images were obtained using a color charge-coupled-device (CCD) image camera system (Sony, DXC-200A) equipped with an optical filter for cutting near-infrared light, such as the YAG laser beam. Raman scattered light from the focal spot was spatially selected by passing through a pinhole (diameter: 100 μm) to ensure a confocal optical arrangement, and was detected by a cooled CCD camera (Andor Tec.) equipped with a polychromator (grating: 1200 grooves/mm). In this way, Raman spectra of trapped microparticle were recorded in 1300−1700 cm−1. The effective YAG laser power (Peff; laser power after passing through the objective lens) was measured using a power meter (Ophir, NOVA). Here, 0.1 W of Peff corresponds approximately to 13 MW/cm2. The effective Ar+ laser power was 10 mW for Raman excitation and 1.0 mW for backscattered observations, respectively. RF produced by these laser powers is negligible to achieve optical trapping of Mb (see the Supporting Information). All the measurements were carried out at room temperature (23 ± 0.5 °C).
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RESULTS AND DISCUSSION Optical Microscopic Observation. Figure 3a shows representative optical micrographs of a buffer solution of Mb
Figure 4. Confocal Raman spectra of a Mb assembly formed by a radiation force (red line) and nonheated (native Mb, black) and heated (40−80 °C, gray) Mb solutions. Each Raman spectrum was measured at room temperature (23 °C).
Figure 3. Optical observations during YAG laser irradiation of Mb buffer solutions (pD 7.6): (a) 0.2 mmol/L, Peff = 0.6 W and (b) 0.1 mmol/L, Peff = 0.8 W; 0.0 min corresponds to the time at which the YAG laser was switched on. Optical micrographs in (a) 0.0−3.0 min and (b) 0.0−5.0 min were obtained from backscattered Ar+ laser light. The other micrographs in (a) 0.0 and 4.0−9.0 min and in (b) 10 min were obtained under bright-field illumination. The scale bar shows 5 μm.
nm, which is called the Q band of Fe protoporphyrin IX (a heme cofactor).50 Therefore, the electronic transition of this absorption band is resonant with the Ar+ laser light used here (λ = 488 nm; the absorption spectrum is shown in a later section). Thus, Figure 4 presumably corresponds to the resonant Raman spectra of Mb. The Raman bands of native Mb are in the figure as a reference (at 23 °C in the figure), and each band was safely assigned to the vibrational modes of the porphyrin ring in Mb, as summarized in Table 1.51−53 Before discussing the chemical structure of the assembly, denaturation of Mb should be examined here. A possible origin of such denaturation is thermal denaturation due to elevation of the temperature by a photothermal effect, although its possibility should be small since YAG laser light (1064 nm) is transparent to the D2O solvent (in detail, described later).54,55 The Raman spectra of thermally denatured Mb were also measured as references. The Mb solution was heated up to 40−80 °C and held at each temperature for 10 min, and then cooled to room temperature to measure the Raman spectra. As shown in Figure 4, the Raman spectra of Mb heated to 40−50 °C are similar to that of the original native form (bottom spectrum). In this temperature region, no micrometersized object was observed in the heated solution by bright-field microscopy. Upon further temperature elevation, thermally formed microparticles appeared in the solution, which were no longer dissolved in solution. This behavior is consistent with
(pD 7.6, 0.2 mmol/L) during irradiation with the YAG laser light (Peff = 0.6 W). Even during YAG laser irradiation for a few minutes, no sign of the molecular assembly of Mb was observed under bright-field illumination (0 and 4.0 min in the figure). By contrast, under dark-field observation using backscattered light, a single small particle that grew larger with irradiation time (0.5−3.0 min), was clearly observed. Upon irradiation over several minutes (5.0 and 9.0 min), the density of the particle increased, although the size appeared to be constant. On the basis of the observation that the particle grew with irradiation time, it could be ascribed to a molecular assembly of Mb. After switching off the YAG laser irradiation, the assembly was found to gradually dissolve in the solution. Such trapping behavior was observable and is qualitatively similar to that previously observed for artificial straight-chain polymer systems in D2O solution.45,46 These results strongly suggest that the assemblylike object in the figure was a Mb assembly formed by the RF, which was confirmed by Raman microspectroscopy (described in a later section). Figure 3b shows the backscattered images of 10693
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photothermal effect due to Mb should be considered. Assuming that heat produced by the photothermal effect is dissipated into the solvent by heat conduction, ΔT at the focal spot is estimated to be62 3P ΔT = α eff 4πκMb (1)
Table 1. Resonance Raman Frequencies and Their Assignments to Native, Thermally Denatured, and Radiation Force-Assembled Myoglobin in 1300−1700 cm−1a nature 1626 1612 1588 1563 1544 1516 1425 1398 1369 1340
(sh) (s) (w) (s) (s) (m) (w) (w) (s) (w)
denature 1640 (s) 1626 (sh)
assembly
1588 (m)
1626 1612 1588 1567 1544
(s) (sh) (w) (s) (w)
1398 (w) 1373 (s)
1398 (w) 1370 (s)
assignment ν(CC) ν(CC) ν10 ν37 ν2 ν11 ν38 δs(CH2) ν12 ν4 ν41
where α is the absorption coefficient of Mb, Peff the YAG laser power at the focal spot, and κMb the heat conductivity of Mb (2.0 mW cm−1 K−1).63 Using eq 1, we estimated ΔT to be 9.5 K (α ∼ 0.1 cm−1, Peff ∼ 0.8 W in Figure 3b) and 14 K (α ∼ 0.2 cm−1, Peff ∼ 0.6 W in Figure 3a). At room temperature (23 °C), optically trapped Mb would be transiently heated up to 37 °C, but not thermally aggregated. In such a photothermal effect, thermal convection is possibly induced,44 acting in favor of optical trapping by transporting Mb from outside of the irradiation area. This shows that the photothermal effect hardly contributes to the Mb assembly. Accordingly, we can safely consider the trapping mechanism based on the resonant effect to occur without the photothermal effect. Resonant Trapping Mechanism of Myoglobin. To reveal the resonant trapping mechanism, further experiments were focused on the role of the heme cofactor in Mb. For comparison, OT of apoMb was also examined. ApoMb is a myoglobin from which the heme cofactor has been removed and has a similar folding structure to that of myoglobin.64 Figure 5 shows dark-field images during YAG laser irradiation
a
Abbreviations: s, strong; sh, shoulder; m, medium; w, weak intensities. The assignments are based on the published literature.51−53
the fact that thermal denaturation of Mb might take place above 60 °C.56,57 In this temperature region, new Raman bands appeared at around 1450 cm−1. In the Raman spectra of the solutions heated to 70 and 80 °C, a further new peak appeared at 1640 cm−1. Moreover, several Raman bands at 1425, 1516, 1544, and 1563 cm−1 became ambiguous or disappeared. Accordingly, all the Raman spectra are modified from that of the original native form. Thus, these Raman band changes can be ascribed to the characteristics of thermally denatured Mb. The structure of the RF-created Mb assembly is discussed on the basis of Raman spectroscopy. The Raman spectrum of the assembly is also shown in Figure 4 (top, red line). Although the intensity ratios of some peaks in the spectrum are different to those observed in the native form, a large part of the Raman peaks peculiar to the native form appear in the spectrum. Moreover, the spectrum of the assembly exhibited no signal around 1450 and 1640 cm−1. Considering the partially thermal denaturation in the RF-created assembly, we compared the Raman spectrum of the assembly to Raman spectra of native and denatured Mb with some ratios (see the Supporting Information). However, the change of the Raman spectrum of the Mb assembly could be hardly explained by partial thermal denaturation. Thus, we consider that the RF-created assembly is composed of Mb molecules without having undergone thermal denaturation. The Raman spectrum of the RF-created assembly implies that the RF slightly induces a change in the folding structure of Mb in the assembly. Such structural changes induced by the RF were also found in our previous work on OT of thermoresponsive polymers, such as poly(Nisopropyl acrylamide),45 poly(vinyl methyl ether),46 and amino acid clusters.13 Although the overall folding structure of trapped Mb still remains unclear and to reveal it is a challenging task, we can safely conclude that the Mb was successfully trapped without thermal denaturation. Experimental results that trapped Mb was not thermally denatured were reinforced by theoretical calculation of temperature elevation ΔT via the photothermal effect. We used D2O as the solvent for suppression of ΔT by absorbing YAG laser light. Ito et al. and Juodkazis et al. reported that ΔT is estimated to be 1−3 K at the focal point of the YAG laser (Peff = 1 W) into D2O solution, respectively.54,55 Thus, we safely ignored the photothermal effect due to D2O. As a matter of course, since Mb has an absorption band at 1064 nm (molar absorption coefficient ∼ 970 cm−1 L mol−1),58−61 the
Figure 5. Optical micrographs of 0.3 mmol/L apoMb buffer solution (pD 7.6) during YAG laser irradiation at Peff = 0.6 W under dark field with backscattered light of an Ar+ laser; 0.0 min corresponds to the time at which the YAG laser was switched on. The scale bar shows 5 μm.
to a buffer solution of apoMb. Despite intense laser irradiation (Peff ∼ 0.6 W) for several minutes (it was sufficient for assembling Mb (Figure 3)), apoMb was hardly assembled and trapped. The results clearly indicate that the heme cofactor plays a crucial role in the efficient trapping of Mb. To shed further light on the resonant effect on OT, we should examine the trapping behavior from the viewpoint of 1064 nm light absorption by Mb. If the resonant effect plays an important role in OT, Mb would be efficiently trapped with an increase in the absorbance of Mb at 1064 nm (YAG laser). Figure 6 shows the absorption spectra of Mb in the UV−vis and near-infrared (NIR) regions. In Figure 6b, the broad band at 900−1200 nm is assigned to the porphyrin-to-iron (ligandto-metal) charge transfer transition of the heme cofactor in Mb (molar absorption coefficient at 1064 nm ∼ 970 cm−1 L mol−1).58−61 Obviously, the absorption band overlaps the laser light wavelength (1064 nm). With the increasing pD of the solution, a D2O molecule coordinated to Fe(III) is substituted by a deuterated-hydroxyl ion (OD−) and the spin state of the Fe(III) is simultaneously switched from high spin (S = 5/2) to low spin (S = 1/2).65 As a result, the NIR absorption band is shifted to the shorter wavelength and the absorbance at around 10694
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Figure 6. Absorption spectra in the (a) UV−vis and (b) near-infrared regions: Mb in pD 7.6 (red line), pD 8.8 (orange shown only in (b)), pD 10 (blue), pD 11 (purple shown only in (b)), and pD 12 (green), and apoMb in pD 7.6 (gray shown only in (a)).
1100 nm decreases with increasing pD value.58,65,66 That is, we can control the absorption at the laser wavelength and, hence, possibly control the resonantly enhanced RF. We also examined OT of Mb in buffer solutions with pD = 8.8, 10, and 12 and compared them with that in the pD = 7.6 buffer solution (Figure 3). Figure 7 shows optical micrographs (dark-field microscopy) of the Mb buffer solutions during YAG laser irradiation. Considering that RF is proportional to the gradient of light
intensity (YAG laser Peff) and polarizability of Mb (NIR absorbance), YAG laser intensity required for OT would become higher with the increase in pD due to the decrease in the NIR absorbance (Figure 6b). In the pD 8.8 solution where the NIR absorbance is the same as that in the pD 7.6, the Mb assembly (Peff = 0.7 W, Figure 7a) was successfully formed, similarly to that in the pD 7.6 solution (Peff = 0.6−0.8 W, Figure 3). In the pD 10 buffer solution (Figure 7b), the intense YAG laser irradiation (Peff = 2.0 W) was necessary for Mb assembly formation. The threshold of the YAG laser power Pth to trap Mb in pD 10 was determined to be 1.2 W, which was obviously higher than that in the pD 7.6 (Pth = 0.6 W), indicating that the NIR absorption band of Mb plays an important role in Mb trapping (resonant effect). In the pD 12 solution (Figure 7c), only blinking of the backscattered light (488 nm) was observed at Peff = 2.0 W, meaning that trapping and escaping of Mb at the focal point was repeated, independent of the irradiation time. Stable OT was no longer achieved at this pD value. The pD dependence of the OT behavior is clearly consistent with that of the NIR absorbance. Thus, it is strongly implied that resonant excitation of heme at 1064 nm enhances the RF, resulting in efficient Mb trapping. Although this may be fundamentally understood on the basis of the classic oscillator model of the dielectric constant (see the Supporting Information), a recent theoretical calculation indicates that the mechanism of ROT is not straightforward. The recent theoretical calculation indicates that the RF under ROT is significantly enhanced with intensely focused laser light, which induces nonlinear optical effects, such as absorption saturation, as suggested by Kudo and Ishihara et al.67 Such nonlinear optical effects should be considered to theoretically clarify the present trapping behavior. Although conventional optical tweezers exert an insufficient RF on smaller molecules for molecular manipulation, optical tweezers under resonant conditions would open a new channel for manipulating molecules.
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CONCLUSIONS In conclusion, we investigated the optical trapping of Mb with focused near-infrared laser light. The trapping efficiency of Mb increased with an increase in the near-infrared absorbance of a heme cofactor. By contrast, apoMb (Mb without a heme cofactor) was scarcely trapped. These results indicate that the heme cofactor is indispensable to the efficient optical trapping
Figure 7. Backscattered light images of Mb solutions: (a) 0.6 mmol/L, pD 8.8, Peff = 0.7 W, (b) 0.4 mmol/L, pD 10, Peff = 2.0 W, and (c) 0.4 mmol/L, pD 12, Peff = 2.0 W. The times shown in (a) and (b) correspond to the time elapsed after switching on the YAG laser. Only the optical micrographs in (c) were obtained in the steady state. Scale bar is 5 μm. 10695
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(10) Ichikawa, M.; Matsuzawa, Y.; Koyama, Y.; Yoshikawa, K. Molecular Fabrication: Aligning DNA Molecules As Building Blocks. Langmuir 2003, 19, 5444−5447. (11) Kita, S.; Hachuda, S.; Otsuka, S.; Endo, T.; Imai, Y.; Nishijima, Y.; Misawa, H.; Baba, T. Super-Sensitivity in Label-Free Protein Sensing Using a Nanoslot Nanolaser. Opt. Express 2011, 19, 17683. (12) Tsuboi, Y.; Shoji, T.; Kitamura, N. Crystallization of Lysozyme Based on Molecular Assembling by Photon Pressure. Jpn. J. Appl. Phys. 2007, 46, L1234−L1236. (13) Tsuboi, Y.; Shoji, T.; Kitamura, N. Optical Trapping of Amino Acids in Aqueous Solutions. J. Phys. Chem. C 2010, 114, 5589−5593. (14) Sugiyama, T.; Adachi, T.; Masuhara, H. Crystallization of Glycine by Photon Pressure of a Focused CW Laser Beam. Chem. Lett. 2007, 36, 1480−1481. (15) Masuhara, H.; Sugiyama, T.; Rungsimanon, T.; Yuyama, K.; Miura, A.; Tu, J. Laser-Trapping Assembling Dynamics of Molecules and Proteins at Surface and Interface. Pure Appl. Chem. 2011, 83, 869− 883. (16) Usman, A.; Uwada, T.; Masuhara, H. Optical Reorientation and Trapping of Nematic Liquid Crystals Leading to the Formation of Micrometer-Sized Domain. J. Phys. Chem. C 2011, 115, 11906−11913. (17) Sugiyama, T.; Yuyama, K.; Masuhara, H. Laser Trapping Chemistry: From Polymer Assembly to Amino Acid Crystallization. Acc. Chem. Res. 2012, 45, 1946−1954. (18) Harada, Y.; Asakura, T. Radiation Forces on a Dielectric Sphere in the Rayleigh Scattering Regime. Opt. Commun. 1996, 124, 529−541. (19) Ashok, P. C.; Dholakia, K. Optical Trapping for Analytical Biotechnology. Curr. Opin. Biotechnol. 2012, 23, 16−21. (20) Chen, Y.-F.; Serey, X.; Sarkar, R.; Chen, P.; Erickson, D. Controlled Photonic Manipulation of Proteins and Other Nanomaterials. Nano Lett. 2012, 12, 1633−1637. (21) Pang, Y.; Gordon, R. Optical Trapping of 12 nm Dielectric Spheres Using Double-Nanoholes in a Gold Film. Nano Lett. 2011, 11, 3763−3767. (22) Pang, Y.; Gordon, R. Optical Trapping of a Single Protein. Nano Lett. 2012, 12, 402−406. (23) Toshimitsu, M.; Matsumura, Y.; Shoji, T.; Kitamura, N.; Takase, M.; Murakoshi, K.; Yamauchi, H.; Ito, S.; Miyasaka, H.; Nobuhiro, A.; Mizumoto, Y.; Ishihara, H.; Tsuboi, Y. Metallic-NanostructureEnhanced Optical Trapping of Flexible Polymer Chains in Aqueous Solution as Revealed by Confocal Fluorescence Microspectroscopy. J. Phys. Chem. C 2012, 116, 14610−14618. (24) Chu, S.; Hollberg, L.; Bjorkholm, J.; Cable, A.; Ashkin, A. Three-Dimensional Viscous Confinement and Cooling of Atoms by Resonance Radiation Pressure. Phys. Rev. Lett. 1985, 55, 48−51. (25) Phillips, W. D.; Prodan, J. V.; Metcalf, H. J. Laser Cooling and Electromagnetic Trapping of Neutral Atoms. J. Opt. Soc. Am. B 1985, 2, 1751. (26) Chu, S. Nobel Lecture: The Manipulation of Neutral Particles. Rev. Mod. Phys. 1998, 70, 685−706. (27) Anderson, M. H.; Ensher, J. R.; Matthews, M. R.; Wieman, C. E.; Cornell, E. A. Observation of Bose-Einstein Condensation in a Dilute Atomic Vapor. Science 1995, 269, 198−201. (28) Davis, K.; Mewes, M.; Andrews, M.; Druten, N.; van Durfee, D.; Kurn, D.; Ketterle, W. Bose-Einstein Condensation in a Gas of Sodium Atoms. Phys. Rev. Lett. 1995, 75, 3969−3973. (29) Osborne, M. A.; Balasubramanian, S.; Furey, W. S.; Klenerman, D. Optically Biased Diffusion of Single Molecules Studied by Confocal Fluorescence Microscopy. J. Phys. Chem. B 1998, 102, 3160−3167. (30) Toussaint, K. C.; Liu, M.; Pelton, M.; Pesic, J.; Guffey, M. J.; Guyot-Sionnest, P.; Scherer, N. F. Plasmon Resonance-Based Optical Trapping of Single and Multiple Au Nanoparticles. Opt. Express 2007, 15, 12017. (31) Seol, Y.; Carpenter, A. E.; Perkins, T. T. Gold Nanoparticles: Enhanced Optical Trapping and Sensitivity Coupled with Significant Heating. Opt. Lett. 2006, 31, 2429−2431. (32) Pelton, M.; Liu, M.; Kim, H. Y.; Smith, G.; Guyot-Sionnest, P.; Scherer, N. F. Optical Trapping and Alignment of Single Gold
of Mb: a resonant effect of the heme cofactor on optical trapping. Furthermore, a result of confocal Raman microspectroscopy possibly suggests that the RF induced a structural deformation of the trapped Mb, whose structure is different from that of both native and thermally denatured Mb. These results demonstrate that resonant optical trapping of biomolecules was successful, making it possible to manipulate other smaller proteins.
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ASSOCIATED CONTENT
S Supporting Information *
An Ar+ laser irradiation effect and a theoretical calculation of the elevation in temperature by the photothermal effect is available in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Prof. Hajime Ishihara, Mr. Tetsuhiro Kudo (Osaka Prefecture University), Prof. Hiroshi Miyasaka, Dr. Tetsuro Katayama, Dr. Yukihide Ishibashi, and Dr. Syoji Ito (Osaka University) for valuable discussions. This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan for the Priority Area “Strong Photons-Molecules Coupling Fields (470)” (No. 19049004), No. 20550002, and the Global COE Program (Project No. B01: Catalysis as the Basis for Innovation in Materials Science). T.S. was financially supported by a Research Fellowship from the Japanese Society for the Promotion of Science (JSPS Research Fellowships for Young Scientists). Y.T. and T.S. are very grateful to Laser System Ltd. for financial support.
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REFERENCES
(1) Ashkin, A.; Dziedzic, J. M.; Bjorkholm, J. E.; Chu, S. Observation of a Single-Beam Gradient Force Optical Trap for Dielectric Particles. Opt. Lett. 1986, 11, 288−290. (2) Bustamante, C.; Bryant, Z.; Smith, S. B. Ten Years of Tension: Single-Molecule DNA Mechanics. Nature 2003, 421, 423−427. (3) Moffitt, J. R.; Chemla, Y. R.; Smith, S. B.; Bustamante, C. Recent Advances in Optical Tweezers. Annu. Rev. Biochem. 2008, 77, 205− 228. (4) Stevenson, D. J.; Gunn-Moore, F.; Dholakia, K. Light Forces the Pace: Optical Manipulation for Biophotonics. J. Biomed. Opt. 2010, 15, 041503. (5) Veigel, C.; Schmidt, C. F. Moving into the Cell: Single-Molecule Studies of Molecular Motors in Complex Environments. Nat. Rev. Mol. Cell Bio. 2011, 12, 163−176. (6) Block, S. M.; Goldstein, L. S.; Schnapp, B. J. Bead Movement by Single Kinesin Molecules Studied with Optical Tweezers. Nature 1990, 348, 348−352. (7) Finer, J. T.; Simmons, R. M.; Spudich, J. A. Single Myosin Molecule Mechanics: Piconewton Forces and Nanometre Steps. Nature 1994, 368, 113−119. (8) Abbondanzieri, E. A.; Greenleaf, W. J.; Shaevitz, J. W.; Landick, R.; Block, S. M. Direct Observation of Base-Pair Stepping by RNA Polymerase. Nature 2005, 438, 460−465. (9) Yang, A. H. J.; Moore, S. D.; Schmidt, B. S.; Klug, M.; Lipson, M.; Erickson, D. Optical Manipulation of Nanoparticles and Biomolecules in Sub-Wavelength Slot Waveguides. Nature 2009, 457, 71−75. 10696
dx.doi.org/10.1021/jp311091u | J. Phys. Chem. C 2013, 117, 10691−10697
The Journal of Physical Chemistry C
Article
Nanorods by Using Plasmon Resonances. Opt. Lett. 2006, 31, 2075− 2077. (33) Dienerowitz, M.; Mazilu, M.; Reece, P. J.; Krauss, T. F.; Dholakia, K. Optical Vortex Trap for Resonant Confinement of Metal Nanoparticles. Opt. Express 2008, 16, 4991. (34) Zelenina, A. S.; Quidant, R.; Nieto-Vesperinas, M. Enhanced Optical Forces between Coupled Resonant Metal Nanoparticles. Opt. Lett. 2007, 32, 1156−1158. (35) Chirico, G.; Fumagalli, C.; Baldini, G. Trapped Brownian Motion in Single- and Two-Photon Excitation Fluorescence Correlation Experiments. J. Phys. Chem. B 2002, 106, 2508−2519. (36) Hosokawa, C.; Yoshikawa, H.; Masuhara, H. Enhancement of Biased Diffusion of Dye-Doped Nanoparticles by Simultaneous Irradiation with Resonance and Nonresonance Laser Beams. Jpn. J. Appl. Phys. 2006, 45, L453−L456. (37) Kendrick, M. J.; McIntyre, D. H.; Ostroverkhova, O. Wavelength Dependence of Optical Tweezer Trapping Forces on Dye-Doped Polystyrene Microspheres. J. Opt. Soc. Am. B 2009, 26, 2189−2198. (38) Li, H.; Zhou, D.; Browne, H.; Klenerman, D. Evidence for Resonance Optical Trapping of Individual Fluorophore-Labeled Antibodies Using Single Molecule Fluorescence Spectroscopy. J. Am. Chem. Soc. 2006, 128, 5711−5717. (39) Tsuboi, Y.; Shoji, T.; Nishino, M.; Masuda, S.; Ishimori, K.; Kitamura, N. Optical Manipulation of Proteins in Aqueous Solution. Appl. Surf. Sci. 2009, 255, 9906−9908. (40) Kataoka, M.; Nishii, I.; Fujisawa, T.; Ueki, T.; Tokunaga, F.; Goto, Y. Structural Characterization of the Molten Globule and Native States of Apomyoglobin by Solution X-ray Scattering. J. Mol. Biol. 1995, 249, 215−228. (41) Wilkins, D. K.; Grimshaw, S. B.; Receveur, V.; Dobson, C. M.; Jones, J. A.; Smith, L. J. Hydrodynamic Radii of Native and Denatured Proteins Measured by Pulse Field Gradient NMR Techniques. Biochemistry 1999, 38, 16424−16431. (42) Harrison, S. C.; Blout, E. R. Reversible Conformational Changes of Myoglobin and Apomyoglobin. J. Biol. Chem. 1965, 240, 299−303. (43) Yuyama, K.; Rungsimanon, T.; Sugiyama, T.; Masuhara, H. Selective Fabrication of α- and γ-Polymorphs of Glycine by Intense Polarized Continuous Wave Laser Beams. Cryst. Growth Des. 2012, 12, 2427−2434. (44) Louchev, O. A.; Juodkazis, S.; Murazawa, N.; Wada, S.; Misawa, H. Coupled Laser Molecular Trapping, Cluster Assembly, and Deposition Fed by Laser-Induced Marangoni Convection. Opt. Express 2008, 16, 5673−5680. (45) Tsuboi, Y.; Nishino, M.; Sasaki, T.; Kitamura, N. Poly(Nisopropylacrylamide) Microparticles Produced by Radiation Pressure of a Focused Laser Beam: A Structural Analysis by Confocal Raman Microspectroscopy Combined with a Laser-Trapping Technique. J. Phys. Chem. B 2005, 109, 7033−7039. (46) Tsuboi, Y.; Nishino, M.; Matsuo, Y.; Ijiro, K.; Kitamura, N. Phase Separation of Aqueous Poly(vinyl methyl ether) Solutions Induced by the Photon Pressure of a Focused Near-Infrared Laser Beam. Bull. Chem. Soc. Jpn. 2007, 80, 1926−1931. (47) Tsuboi, Y.; Nishino, M.; Kitamura, N. Laser-Induced Reversible Volume Phase Transition of a Poly(N-isopropylacrylamide) Gel Explored by Raman Microspectroscopy. Polym. J. 2008, 40, 367−374. (48) Smith, T. A.; Hotta, J.; Sasaki, K.; Masuhara, H.; Itoh, Y. Photon Pressure-Induced Association of Nanometer-Sized Polymer Chains in Solution. J. Phys. Chem. B 1999, 103, 1660−1663. (49) Singer, W.; Nieminen, T.; Heckenberg, N.; Rubinsztein-Dunlop, H. Collecting Single Molecules with Conventional Optical Tweezers. Phys. Rev. E 2007, 75, 1−5. (50) Schomacker, K. T.; Champion, P. M. Investigations of the Temperature Dependence of Resonance Raman Cross Sections: Applications to Heme Proteins. J. Chem. Phys. 1989, 90, 5982−5993. (51) Abe, M.; Kitagawa, T.; Kyogoku, Y. Resonance Raman Spectra of Octaethylporphyrinato-Ni(II) and meso-Deuterated and 15N Substituted Derivatives. II. A Normal Coordinate analysis. J. Chem. Phys. 1978, 69, 4526−4535.
(52) Sitter, A.; Reczek, C.; Terner, J. Observation of the FeIVO Stretching Vibration of Ferryl Myoglobin by Resonance Raman Spectroscopy. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1985, 828, 229−235. (53) Hu, S.; Smith, K. M.; Spiro, T. G. Assignment of Protoheme Resonance Raman Spectrum by Heme Labeling in Myoglobin. J. Am. Chem. Soc. 1996, 118, 12638−12646. (54) Juodkazis, S.; Mukai, N.; Wakaki, R.; Yamaguchi, A.; Matsuo, S.; Misawa, H. Reversible Phase Transitions in Polymer Gels Induced by Radiation Forces. Nature 2000, 408, 178−181. (55) Ito, S.; Sugiyama, T.; Toitani, N.; Katayama, G.; Miyasaka, H. Application of Fluorescence Correlation Spectroscopy to the Measurement of Local Temperature in Solutions under Optical Trapping Condition. J. Phys. Chem. B 2007, 111, 2365−2371. (56) Yan, Y.-B.; Wang, Q.; He, H.-W.; Zhou, H.-M. Protein Thermal Aggregation Involves Distinct Regions: Sequential Events in the HeatInduced Unfolding and Aggregation of Hemoglobin. Biophys. J. 2004, 86, 1682−1690. (57) Meersman, F.; Smeller, L.; Heremans, K. Comparative Fourier Transform Infrared Spectroscopy Study of Cold-, Pressure-, and HeatInduced Unfolding and Aggregation of Myoglobin. Biophys. J. 2002, 82, 2635−2644. (58) Hanania, G. I.; Yeghiayan, A.; Cameron, B. F. Absorption Spectra of Sperm-Whale Ferrimyoglobin. Biochem. J. 1966, 98, 189− 192. (59) Day, P.; Smith, D. W.; Williams, R. J. P. Crystal Spectra of a Heme and Some Heme−Protein Complexes. Biochemistry 1967, 6, 1563−1566. (60) Thomson, A. J.; Gadsby, P. M. A. A Theoretical Model of the Intensity of the Near-Infrared Porphyrin-to-Iron Charge-Transfer Transitions in Low-Spin Iron(III) Haemoproteins. A Correlation between the Intensity of the Magnetic Circular Dichroism Bands and the Rhombic Distortion Parameter. J. Chem. Soc., Dalton. Trans 1990, 1921−1928. (61) Gadsby, P. M. A.; Thomson, A. J. Assignment of the Axial Ligands of Ferric Ion in Low-Spin Hemoproteins by Near-Infrared Magnetic Circular Dichroism and Electron Paramagnetic Resonance Spectroscopy. J. Am. Chem. Soc. 1990, 112, 5003−5011. (62) Wurlitzer, S.; Lautz, C.; Liley, M.; Duschl, C.; Fischer, T. M. Micromanipulation of Langmuir-Monolayers with Optical Tweezers. J. Phys. Chem. B 2001, 105, 182−187. (63) Yu, X.; Leitner, D. M. Vibrational Energy Transfer and Heat Conduction in a Protein. J. Phys. Chem. B 2003, 107, 1698−1707. (64) Abbruzzetti, S.; Crema, E.; Masino, L.; Vecli, A.; Viappiani, C.; Small, J. R.; Libertini, L. J.; Small, E. W. Fast Events in Protein Folding: Structural Volume Changes Accompanying the Early Events in the N→I Transition of Apomyoglobin Induced by Ultrafast pH Jump. Biophys. J. 2000, 78, 405−415. (65) Nozawa, T.; Yamamoto, T.; Hatano, M. Infrared Magnetic Circular Dichroism of Myoglobin Derivatives. Biochim. Biophys. Acta, Protein Struct. 1976, 427, 28−37. (66) Bowen, W. J. The Absorption Spectra and Extinction Coefficients of Myoglobin. J. Biol. Chem. 1949, 179, 235−245. (67) Kudo, T.; Ishihara, H. Proposed Nonlinear Resonance Laser Technique for Manipulating Nanoparticles. Phys. Rev. Lett. 2012, 109, 087402.
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