Real-Time Imaging of Single-Molecule Fluorescence with a Zero

Jun 19, 2008 - Consolidated Research Institute for Advanced Science and Medical ... Kagami Memorial Laboratory for Materials Science and Technology, ...
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Anal. Chem. 2008, 80, 6018–6022

Real-Time Imaging of Single-Molecule Fluorescence with a Zero-Mode Waveguide for the Analysis of Protein-Protein Interaction Takeo Miyake,*,† Takashi Tanii,† Hironori Sonobe,† Rena Akahori,† Naonobu Shimamoto,‡,§ Taro Ueno,| Takashi Funatsu,| and Iwao Ohdomari†,§,⊥ Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku, Tokyo 169-8555, Japan, Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513 Waseda, Tsurumakicho, Shinjuku, Tokyo 162-0041, Japan, Nanotechnology Research Laboratory, Waseda University, 513 Waseda, Tsurumakicho, Shinjuku, Tokyo 162-0041, Japan, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan, and Kagami Memorial Laboratory for Materials Science and Technology, Waseda University, 2-8-26 Nishi-waseda, Shinjuku, Tokyo 169-0051, Japan Real-time imaging of single-molecule fluorescence with a zero-mode waveguide (ZMW) was achieved. With modification of the ZMW geometry, the signal-to-background ratio is twice that obtainable with a conventional ZMW. The improved signal-to-background ratio makes it possible to visualize individual binding-release events between chaperonin GroEL and cochaperonin GroES at a concentration of 5 µM. Two rate constants representing two-timer kinetics in the release of GroES from GroEL were measured with the ZMW, and the measurements agreed well with those made with a total internal reflection fluorescence microscopy. These results indicate that the novel ZMW makes feasible the direct observation of protein-protein interaction at an intracellular concentration in real time. Elucidation of protein-protein interaction is a central issue in proteomics. Single-molecule fluorescence imaging (SMFI) has become a powerful method for understanding the proteinprotein interaction because SMFI provides a time trajectory of a protein interacting with related molecules and allows researchers to obtain information about the elementary step of the reaction that has never been obtainable by conventional ensemble measurements.1–7 The major advantage of SMFI is * To whom correspondence should be addressed. Telephone: +81-3-52863380. Fax: +81-3-5272-5749. E-mail: [email protected]. † Faculty of Science and Engineering, Waseda University. ‡ Consolidated Research Institute for Advanced Science and Medical Care, Waseda University. § Nanotechnology Research Laboratory, Waseda University. | The University of Tokyo. ⊥ Kagami Memorial Laboratory for Materials Science and Technology, Waseda University. (1) Zander, Ch., Enderlein, J., Keller, R. A., Eds. Single Molecule Detection in Solution; Wiley-VCH: Berlin, 2002; pp 273-292. (2) Kumar, C. S. S. S. R.; Hormes, J.; Leuschner, C. Nanofabrication Towards Biomedical Applications; Wiley-VCH: Berlin, 2005; pp 197-225. (3) Ishijima, A.; Yanagida, T. Trends Biochem Sci 2001, 26, 438–444. (4) Shiroguchi, K.; Kinosita, K., Jr Science 2007, 316, 1208–1212. (5) Kumbhakar, M.; Nath, S.; Mukherjee, Tulsi; Mittal, Pal, J.; Pal, H. J. Photochem. Photobiol. C 2004, 5, 113–137. (6) Moerner, W. E.; Fromm, D. P. Rev. Sci. Instrum. 2003, 74, 3597–3619. (7) Weiss, S. Science 1999, 283, 1676–1683.

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that it provides a way to directly observe the interactions between individual molecules in real time. In the past decade, protein function has been analyzed by directly observing the interaction between biomolecules with diffraction-limited optics and total internal reflection light in real time.8–11 By using epifluorescence microscopy and total internal reflection fluorescence microscopy (TIRFM), researchers have been able to observe the kinetics between the myosin and ATP, F1-ATPase, and ATP, as well as enzymatic reactions.12–14 However, because of the limitation of the focal volume defined by the diffraction limit of an objective lens, the concentration of fluorophore-labeled protein is limited to the nanomolar level; thus, there are only a few different proteins that can be measured.15 To observe single molecules under high concentration (micromolar concentration), Levene et al. have developed SMFI with zero-mode waveguides (ZMWs) consisting of subwavelength holes in a metal film, and they have analyzed DNA synthesis by fluorescence correlation spectroscopy using the ZMW.16 In their approach, under micromolar concentration, they were able to observe fluorophore-labeled proteins, but they were not able to directly observe the interaction of individual molecules. To make this observation, we have developed a fluorescence microscope system including a novel ZMW. The ZMW reduces the observation volume of the system and, as a result, suppresses background fluorescence from the nonbinding molecules within (8) Yamasaki, R.; Hoshino, M.; Wazawa, T.; Ishii, Y.; Yanagida, T.; Kawata, Y.; Higurashi, T.; Sakai, K.; Nagai, J.; Goto, Y. J. Mol. Biol. 1999, 292, 965– 972. (9) Oiwa, K.; Eccleston, J. F.; Anson, M.; Kikumoto, M.; Davis, C. T.; Reid, G. P.; Ferenczi, M. A.; Corrie, J. E. T.; Yamada, A.; Nakayama, H.; Trentham, D. R. Biophys. J. 2000, 78, 3048–3071. (10) Mehta, A. D.; Rief, M.; Spudich, J. A.; Smith, D. A.; Simmons, R. M. Science 1999, 283, 1689–1695. (11) Tinnefeld, P.; Sauer, M. Angew. Chem., Int. Ed. 2005, 44, 2642–2671. (12) Funatsu, T.; Harada, Y.; Tokunaga, M.; Saito, K.; Tanagida, T. Nature 1995, 374, 555–559. (13) Nishizaka, T.; Oiwa, K.; Noji, H.; Kimura, S.; Muneyuki, E.; Yoshida, M.; Kinosita, K., Jr Nat. Struct. Mol. Biol. 2004, 11 (2), 110–112. (14) Lu, L. H.;.; Xun, L. X.;.; Xie, S. Science 1998, 282, 1877–1882. (15) Samiee, K. T.; Foquet, M.; Guo, L.; Cox, E. C.; Craighead, H. G. Biophys. J. 2005, 88, 2145–2153. (16) Levene, M. J.; Korlach, J.; Turner, S. W.; Foquet, M.; Craighead, H. G.; Webb, W. W. Science 2003, 299, 682–686. 10.1021/ac800726g CCC: $40.75  2008 American Chemical Society Published on Web 06/19/2008

Figure 1. Schematic of a single-molecule fluorescence imaging system with a novel zero-mode waveguide for the detection and analysis of GroEL-GroES interaction.

the observation volume. Therefore, the ZMW makes it possible to clearly detect binding pairs of molecules. In this article, we describe how we have optimized the geometry of the ZMW to achieve a high signal-to-background ratio. We also discuss the observations made by this system of the interaction between chaperonin GroEL and cochaperonin GroES at a concentration of 5 µM. Finally, we evaluate the feasibility of our SMFI system with the novel ZMW. EXPERIMENTAL METHODOLOGY Experimental Design. Figure 1 shows the schematic of our real-time SMFI with a novel ZMW for the detection and analysis of GroEL-GroES interaction. The system was designed for our ongoing investigation into the “two-timer” kinetics present in the release of GroES from GroEL.17,18 In our system, a nanohole array is fabricated in a thin metal film deposited on a quartz slide. The slide with the nanohole array is mounted on a glass slide with a pair of rim spacers, which creates a simple flow cell in between the two slides. Molecules are introduced into the nanoholes by exchanging the solution in the flow cell. IC5-labeled GroEL is immobilized in a nanohole via biotinylated bovine serum albumin (biotin-BSA) and streptavidin. GroES is dissolved in the solution at a concentration of 5 µM, equivalent to the concentration in a living cell. We dissolve nonlabeled GroES at a concentration of 4.5 µM together with 500 nM Cy3-labeled GroES. If all the GroES molecules at 5 µM in solution are fluorescently labeled, the binding sites of an immobilized GroEL are always occupied by some of them, resulting in a continuous fluorescent signal. Mixing unlabeled (4.5 µM) and fluorescently labeled (0.5 µM) GroES makes it possible to obtain a continual binding signal and evaluate individual binding time. If the association of fluorescently labeled (20 nM) GroES with GroEL was observed by TIRFM in the presence of unlabeled (5 µM) GroES, the association event was very rare (∼10-5 s-1), and it was difficult to distinguish the association of GroES and GroEL from the nonspecific binding of GroES to the glass surface of the slide. Since the diameter of the (17) Taguchi, H.; Ueno, T.; Tadakuma, H.; Yoshida, M.; Funatsu, T. Nat. Biotechnol. 2001, 19, 861–865. (18) Ueno, T.; Taguchi, H.; Tadakuma, H.; Yoshida, M.; Funatsu, T. Mol. Cell 2004, 14, 423–434.

nanohole is smaller than the wavelength of the excitation light, the light is confined within the nanohole; thus, the binding of the GroES specifically to the GroEL is fluorescently excited and preferentially detected. By estimating the elapsed times of the signal intensities of individual binding events, we can determine the binding time. Fabrication of ZMW. To fabricate the resist pattern on the quartz slide, a resist film of SAL601 (Roam and Haas) is coated on the quartz slide with a spin-coater, and then electron beam (EB) lithography is performed with an ELS-7500 scanning electron microscope (Elionix). The accelerating voltage is 50 kV, and the beam current is 100 pA. After EB patterning, the pattern is developed by immersing it in MFCD26 (Roam and Haas) for 7 min. An aluminum layer 100 nm thick is deposited on the resist pattern by an EB deposition system (EDX-6D, Ulvac). By immersing the slide in an aluminum etchant (2.3% H3PO4, 0.1% HNO3, 0.1% CH3COOH, and 97.5% H2O) for 3 min, the aluminum layer deposited on the sidewall of the resist pillars is removed. For patterning the aluminum layer, the resist film is removed by immersing the slide in Remover1165 (Roam and Haas) for 25 min and by subsequently exposing the slide to O2 plasma for 3 min. Fluorescence Microscopy. A fluorescence microscope (IX70, Olympus) is equipped with two oil immersion objectives (Plapon 60×, NA 1.49, Olympus and Uplsapo 100×, NA 1.4, Olympus) and a EMCCD camera (C9100-13, Hamamatsu Photonics). The illumination sources are 532- and 635-nm solid-state lasers (Compass 315M-100 and Radius 635-25, Coherent). Filter sets are as follows: a dichroic mirror (a custom-made item reflecting light at 532 and 633 nm, Asahi Spectra Co. Ltd.); an excitation filter (630DF30, Omega Optical) and emission filters (690DF55, Omega Optical and HQ700/75, Chroma Technology) for IC5 and Cy5 fluorescence; and an emission filter (FF01-593/40-25, Semrock) for Cy3 fluorescence. The images are digitally recorded at 5 frames/s on a hard disk drive for subsequent data analysis using a specially developed program as described previously.17 Measurement of Signal-to-Background Ratio. The signal intensity from a molecule immobilized on the ZMW is evaluated. Cy5-labeled BSA is used for the evaluation because BSA easily adsorbs on the surface of the glass. First, a buffer of 25 mM Analytical Chemistry, Vol. 80, No. 15, August 1, 2008

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HEPES-KOH, pH 7.4, 100 mM KCl, and 5 mM MgCl2 (hereafter called “buffer A”) containing 26 nM Cy5-labeled BSA is infused into the flow cell. After 5 min, the flow cell is washed five times with buffer A. Next, the flow cell is filled with buffer A containing an oxygen scavenger system (25 mM glucose, 2.5 µM glucose oxidase, 10 nM catalase, 10 mM dithiothreitol) that prolongs photobleaching time. Finally, fluorescent spots corresponding to the single BSA are observed by fluorescence microscopy, and then the time-averaged signal intensity is evaluated. The background intensity from the Cy5-labeled GroES in Brownian motion is evaluated. First, buffer A containing 30 µM nonlabeled BSA is infused into the flow cell. The BSA coated on the quartz surface prevents the nonspecific adsorption of GroES. After the nanohole with the buffer A is washed five times, the flow cell is filled with buffer A containing both 1 µM Cy5-labeled GroES and the oxygen scavenger system. Finally, fluorescent spots corresponding to the Cy5-labeled GroES are observed by fluorescence microscopy, and then the time-averaged background intensity is evaluated. Measurement of Dissociation Kinetics of GroES from GroEL. Buffer A containing 30 µM biotin-labeled BSA is infused into the flow cell. After the nanohole is washed one time with buffer A, a solution containing 17 µM streptavidin is introduced into the flow cell to bond the streptavidin with the biotin. After the nanohole is washed one time with buffer A, buffer A containing 30 nM GroEL labeled with both biotin and IC5 is infused into the flow cell to immobilize the GroEL on the streptavidin. After the nanohole is washed three times with buffer A, the flow cell is filled with buffer A containing 4.5 µM nonlabeled GroES, 500 nM Cy3GroES, the oxygen scavenger system, 2 mM ATP, and 75 µM pepsin as a nonnative protein. Next, IC5-labeled GroEL and Cy3labeled GroES are illuminated at 150 and 320 W/cm2, respectively. The binding-release events between the immobilized GroEL and the GroES are observed by fluorescence microscopy. DATA RESULTS AND DISCUSSION Measurement of Signal-to-Background Ratio. Achieving both high signal intensity and high signal-to-background ratio is a requirement for real-time SMFI. The fluorescence signal corresponding to the specific binding needs to be detected, preferably in the background fluorescence caused by the molecules in Brownian motion, as shown in Figure 1. For the signal intensity to be increased, the diameter of the nanohole needs to be increased, because the excitation light intensity decreases when confined within a smaller nanohole. In contrast, for the signal-tobackground ratio to be enhanced, the diameter of the nanohole needs to be decreased, because the decrease in the background intensity is more rapid than that in the signal intensity. (If we assume that individual molecules within the observation volume emit fluorescence with equivalent intensity S, the signal-tobackground ratio S/B is written as, S/B ) S/{C × V × S}, where C is the concentration of fluorophore and C × V is the number of molecules within the excitation volume V. C × V is less than one because V is extremely small.) Thus, the ZMW diameter should be optimally selected to achieve high signal-to-background ratio as well as high signal intensity. To achieve high signal intensity, we etched the quartz surface at the entrance of the waveguide, as shown in Supporting Information (SI) Figure 1. In this geometry, the volume of the 6020

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Figure 2. Signal and background intensities for different ZMW depths and diameters. (a) Distribution of fluorescence signal intensity from Cy5-labeled BSA adsorbed on a waveguide with both nanohole diameter and etching depth as parameters. (b) Comparison between average signal and average background intensity. The background intensity from the Cy5-labeled GroES in Brownian motion (1 µM) is evaluated by measuring the time-averaged fluorescence intensity of each nanohole. The extraordinary signals corresponding to the nonspecific adsorption are excluded.

etched region, not the effective volume of the near field, defines the observation volume, and the metal clad acts as a gate that allows the molecule to pass but prevents the excitation light from penetrating. Ideally, the metal layer on the quartz slide creates a boundary condition that the light intensity vanishes at the metal/ quartz interface. The point of the maximum intensity is located at a depth that is a quarter of a wavelength inside the quartz. Therefore, in the etched waveguides, the fluorescent dye is excited with much stronger light than it is in the conventional ZMW. Furthermore, the fluorescence from the dye smoothly propagates to the objective lens without being obstructed by the metal clad. To confirm that there is only one molecule in one nanohole, we measured the fluorescent signal from the Cy5-labeled BSA immobilized in the nanohole. Signal intensity is evaluated by integrating the brightness from the Cy5-labeled BSA of pixels (fluorescence) with reference to the brightness from the metal surface of pixels (see SI Figure 2). The quantized photobleach was observed at each spot, which enabled us to count the dye in the observation volume. When the signal of the single-step photobleach was detected, we judged that there was only one dye and, therefore, only one immobilized protein in the nanohole (see SI Figure 2b). To evaluate the signal-to-background ratio with both the nanohole diameter and the etching depth as parameters, we observed the fluorescence signal from the immobilized Cy5labeled BSA and the background from the Cy5-labeled GroES in Brownian motion, as described in Experimental Methodology. Figure 2a shows the distribution of the fluorescence signal intensity. The distribution depends on the nanohole diameter, and as the diameter increases, the peak becomes broad, shifting to a

Figure 3. Single-molecule imaging of GroEL-GroES interaction. Cy3-labeled GroES and nonlabeled GroES are dissolved at a concentration of 500 nM and 4.5 µM, respectively. Because ATP is necessary for the binding of GroES to GroEL, the fluorescent images of Cy3-labeled GroES are then captured both in the presence and absence of ATP. (a) Fluorescence image of immobilized IC5-labeled GroEL. (b) Fluorescence image of Cy3-labeled GroES binding to immobilized GroEL in the presence of ATPsthe fluorescent signal corresponds to the binding of GroES to GroEL (Corresponding to (a), the position of the immobilized GroEL is pointed out with a yellow circle.). (c) Fluorescence image of Cy3labeled GroES in the absence of ATPsnote that no fluorescent signal is observed in the presence of Cy3-GroES because ATP is necessary for the binding of GroES to GroEL. (d) Elapsed time of fluorescence intensity corresponding to the binding-release of GroES.

stronger intensity. We have confirmed by direct observation of nanoholes that the fluctuation in the nanohole diameter is negligible and that the position and the orientation of the immobilized dye is the reason the signal intensity fluctuates in spite of being obtained from nanoholes with a same diameter. As shown in Figure 2b, the average signal intensity becomes stronger when the etched waveguides are used. The intensity obtained with an etched waveguide 60 nm in depth is twice as strong as the intensity obtained with a conventional waveguide. Figure 2b also shows the background intensity caused by the Cy5-labeled GroES in Brownian motion in the observation volume. The average signal intensity is increased ten times compared with the average background intensity, even at a concentration of 1 µM, indicating that the interaction of GroEL-GroES at an intracellular concentration can be observed. The highest signal-to-background ratio of 80 was achieved by using a waveguide 96 nm in diameter with an etched depth of 60 nm. Real-Time SMFI of GroEL-GroES Interaction. We also confirmed that the immobilized GroEL in the nanohole maintains its activity. Figure 3a show the fluorescent spots from the IC5labeled GroEL immobilized in the nanohole. We measured the

interaction between GroES and GroEL under the condition in which the number ratio of fluorescent spots of GroEL to nanoholes was less than 10%. The Poisson distribution of the number of GroEL in each nanohole assured us that more than 90% of the GroEL immobilized in nanoholes was single. The fluorescent images of Cy3-labeled GroES were then captured both in the presence and absence of ATP, because ATP is necessary for the binding of GroES to GroEL. As shown in Figure 3b, the fluorescent signals from the Cy3-labeled GroES are detected only in the presence of ATP. The feasibility of real-time SMFI with ZMW was investigated by testing direct observations of the interaction between GroEL and GroES at a concentration of 5 µM in real time. Figure 3d shows the elapsed time of the fluorescence intensity from Cy3labeled GroES. A fluorescence intensity corresponding to the binding-release of GroES is clearly identified, even at a high concentration of Cy3-labeled GroES in the solution. Figure 4 shows a histogram of the on-time duration corresponding to the binding time of the GroES. As reported by Taguchi et al.,17 the histogram is fitted by the following formula; Ckk′ [ exp(-kt) - exp(-k′t)]/ Analytical Chemistry, Vol. 80, No. 15, August 1, 2008

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Because of the limited concentration of fluorescently labeled molecules in the conventional fluorescence microscopy for SMFI, it has been unclear for more than a decade whether GroES binds to each ring of GroEL alternately or simultaneously.18,19 Although clarification of this issue was beyond the scope of our work, our fluorescence microscopy system opens up the possibility of understanding the reaction cycle between GroEL and GroES, because the system enables us to directly observe the interaction of individual molecules at an intracellular concentration in real time. In addition, our system can be used to analyze protein functions that are revealed only at an intracellular concentration,20 which means it has the potential to resolve the function of various proteins.

Figure 4. Histogram of on-time duration. Solid line shows convolution of two exponentials, Ckk′ [ exp(-kt) - exp(-k’t) ]/(k′ s k), fit to the data by least-squares fitting. The two rate constants represent the two-timer kinetics of GroEL-GroES interaction. Closed circles indicate nonspecific adsorption events. (inset figure) Elapsed time of the number of the remaining GroES is shown. Solid line represents integration of the above formula.

(k′ s k), where C ) 1305 (the number of total events), k ) 0.47 s-1, and k′ ) 0.21 s-1. By an analysis of the remaining GroES as a function of time (inset of Figure 4), the two rate constants of rate-limiting steps were determined to be 0.44 and 0.23 s-1. These results indicate the existence of a kinetic intermediate in the release events of GroES from GroEL. To confirm that the influence of nonspecific adsorption is negligible, we evaluated the events of nonspecific adsorption of Cy3-labeled GroES on the surface of the nanoholes in which GroEL was absent. As shown in Figure 4, the frequency of the nonspecific adsorption is sufficiently low. To confirm that the on- and off-time duration measured with ZMW shows the binding release events precisely and to ascertain whether or not the two rate constants are equivalent to that measured by TIRFM, we directly observed the GroEL-GroES interaction at a concentration of 50 nM, which is equivalent to the concentration of Cy3-labeled GroES that can be observed with TIRFM. Because the ZMW is composed of a metal film, fluorescence may be quenched if the charge transfers to the metal film. The values of the two rate constants representing the two-timer kinetics of GroEL-GroES are in good agreement with those measured with TIRFM (data not shown), indicating that the influence of the quenching on the measurement is negligible. Offtime durations corresponding to the waiting time of the GroES binding to GroEL have also been measured. The histograms showed single-exponential curves (data not shown), which could be fitted with association rate constants (kon) of 2.1 × 106 M-1 s-1 for ZMW and 5.6 × 106 M-1 s-1 for TIRFM. These values were quite similar, suggesting that the nanohole structure does not affect the diffusion of GroES in the nanohole.

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CONCLUSION We have designed and developed a fluorescence microscope system including a novel ZMW, and we have investigated the feasibility of the system. The improved ZMW achieves a high signal-to-background ratio and enables us to visualize the interaction between GroEL and GroES at a micromolar concentration. We found that the two rate constants of rate-limiting steps representing two-timer kinetics in the release of GroES from GroEL were in good agreement with those measured with TIRFM, indicating that (i) the GroEL immobilized in the ZMW maintains the activity, and (ii) the two-timer kinetics can be detected and measured at an intracellular concentrationsprior to our system, the kinetics had been revealed only at a low concentration. In short, our SMFI with ZMW is effective for the direct observation of protein-protein interaction under micromolar concentrations in real time. ACKNOWLEDGMENT The authors thank Professor Keisaku Yamada and Professor Robert B. DiGiovanni of the School of Science and Engineering, Waseda University for their helpful advice. This work is supported by a Grant-in-Aid for Young Scientists (B), by a grant for Research Fellows awarded by the Japan Society for the Promotion of Science, by the Consolidated Research Institute for Advanced Science and Medical Care with funding from the Ministry of Education, Culture, Sports, Science and Technology, Japan, by Takayanagi Foundation for Electronics Science and Technology, and by the Mitsubishi Foundation. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review April 12, 2008. Accepted May 28, 2008. AC800726G (19) Grallert, H.; Buchner, J. J. Struct. Biol 2001, 135, 95–103. (20) Cross, R. A. Trends Biochem Sci 2004, 29, 301–309.