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Fast-scanning atomic force microscopy reveals the ATP/ADP-dependent conformational changes of GroEL

2006; Springer Nature; Volume: 25; Issue: 19 Linguagem: Inglês

10.1038/sj.emboj.7601326

1460-2075

Masatoshi Yokokawa, Chieko Wada, Toshio Ando, Nobuaki Sakai, Akira Yagi, Shige H. Yoshimura, Kunio Takeyasu,

Enzyme Structure and Function

Article14 September 2006free access Fast-scanning atomic force microscopy reveals the ATP/ADP-dependent conformational changes of GroEL Masatoshi Yokokawa Corresponding Author Masatoshi Yokokawa Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Chieko Wada Chieko Wada Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Toshio Ando Toshio Ando Department of Physics, Faculty of Science, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa, Japan Search for more papers by this author Nobuaki Sakai Nobuaki Sakai OLYMPUS Corporation, Hachioji, Tokyo, Japan Search for more papers by this author Akira Yagi Akira Yagi OLYMPUS Corporation, Hachioji, Tokyo, Japan Search for more papers by this author Shige H Yoshimura Shige H Yoshimura Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Kunio Takeyasu Kunio Takeyasu Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Masatoshi Yokokawa Corresponding Author Masatoshi Yokokawa Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Chieko Wada Chieko Wada Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Toshio Ando Toshio Ando Department of Physics, Faculty of Science, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa, Japan Search for more papers by this author Nobuaki Sakai Nobuaki Sakai OLYMPUS Corporation, Hachioji, Tokyo, Japan Search for more papers by this author Akira Yagi Akira Yagi OLYMPUS Corporation, Hachioji, Tokyo, Japan Search for more papers by this author Shige H Yoshimura Shige H Yoshimura Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Kunio Takeyasu Kunio Takeyasu Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan Search for more papers by this author Author Information Masatoshi Yokokawa 1, Chieko Wada1, Toshio Ando2, Nobuaki Sakai3, Akira Yagi3, Shige H Yoshimura1 and Kunio Takeyasu1 1Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan 2Department of Physics, Faculty of Science, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa, Japan 3OLYMPUS Corporation, Hachioji, Tokyo, Japan *Corresponding author. Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, Japan. Tel./Fax: +81 75 753 6852; E-mail: [email protected] The EMBO Journal (2006)25:4567-4576https://doi.org/10.1038/sj.emboj.7601326 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In order to fold non-native proteins, chaperonin GroEL undergoes numerous conformational changes and GroES binding in the ATP-dependent reaction cycle. We constructed the real-time three-dimensional-observation system at high resolution using a newly developed fast-scanning atomic force microscope. Using this system, we visualized the GroES binding to and dissociation from individual GroEL with a lifetime of 6 s (k=0.17 s−1). We also caught ATP/ADP-induced open–closed conformational changes of individual GroEL in the absence of qGroES and substrate proteins. Namely, the ATP/ADP-bound GroEL can change its conformation 'from closed to open' without additional ATP hydrolysis. Furthermore, the lifetime of open conformation in the presence of ADP (∼1.0 s) was apparently lower than those of ATP and ATP-analogs (2–3 s), meaning that ADP-bound open-form is structurally less stable than ATP-bound open-form. These results indicate that GroEL has at least two distinct open-conformations in the presence of nucleotide; ATP-bound prehydrolysis open-form and ADP-bound open-form, and the ATP hydrolysis in open-form destabilizes its open-conformation and induces the 'from open to closed' conformational change of GroEL. Introduction The atomic force microscopy (AFM) (Binnig et al, 1986; Hansma et al, 1988) is increasingly being used for studying biomolecules at the single molecule level. In particular, the AFM has been used to measure both structural and mechanical properties of individual DNA and proteins in physiologically relevant buffers (Oberhauser et al, 1999; Allison et al, 2002; Fotiadis et al, 2002; Sekiguchi et al, 2003; Yang et al, 2003). However, such investigations have been restricted by the inherent slow scan speed of AFM necessary for the nano-scale resolution. The scanning rates of commercially available AFMs are usually several seconds to minutes per frame, and therefore insufficient to monitor many physiological processes in cells that occur at much faster rates. Recently, this limitation has been challenged by using fast-scanning AFM (Viani et al, 2000; Ando et al, 2001), which can capture images in the subseconds order. In this work, we used a newly developed fast-scanning AFM to observe the behavior of individual GroEL at the single molecule level, and demonstrated the ability of fast-scanning AFM to analyze the millisecond-order reaction at nanometer resolution. Many newly synthesized proteins and denatured proteins in cells must undergo a series of folding processes to achieve their proper three-dimensional (3D) structures to become functionally active (Hightower, 1991; Bukau and Horwich, 1998; Hartl and Hayer-Hartl, 2002). The efficient folding of most proteins requires the assistance from molecular chaperones that prevent protein misfolding and aggregation in the crowded environment in the cells. One of the best-characterized chaperone systems is the Escherichia coli chaperonin GroEL–GroES system (Bukau and Horwich, 1998; Ranson et al, 1998; Grallert and Buchner, 2001). The detailed 3D structures of GroEL, GroES and the GroEL/ADP/GroES complex were resolved by X-ray crystallography (Braig et al, 1994; Hunt et al, 1996; Mande et al, 1996; Xu et al, 1997; Chaudhry et al, 2003) and electron microscopy (Chen et al, 1994; Schmidt et al, 1994; Ranson et al, 2006) (Figure 1). GroEL consists of two homo-heptameric rings that are stacked back-to-back, whereas GroES is a single homo-heptameric ring. The chaperonin reaction cycle begins with the binding of a polypeptide (unfolded protein) and GroES to one side of GroEL, which results in a formation of the GroEL/polypeptide/GroES complex in the presence of nucleotides. Once the GroEL/polypeptide/GroES complex is formed, the polypeptide chain is refolded toward its functional conformation in a large central cavity in the GroEL ring (in cis-conformation). Then, the GroEL/GroES complex decays with a lifetime of 8–15 s by the dissociation of GroES, followed by a release of the trapped protein from the cavity (Burston et al, 1995; Weissman et al, 1996; Rye et al, 1997, 1999). This final step, GroES dissociation, was triggered by the binding of ATP to the opposite ring (in trans-conformation) (Burston et al, 1995; Hayer-Hartl et al, 1995; Rye et al, 1997). Figure 1.3D structure of GroEL, GroEL–ATP complex and GroEL–GroES–ADP complex. (A) The crystal structure of GroEL (Braig et al, 1994); pdb 1GRL. (B) The cryo-EM map of GroEL(D398A)–ATP complex (Ranson et al, 2001); pdb 2C7E. (C) The crystal structure of GroEL–GroES–ADP complex (Xu et al, 1997); pdb 1AON. The side views (Upper) and the top views (Lower) of Van der Waals space-filled models highlighting the individual ring structures of GroEL (lower GroEL ring, red; upper GroEL ring, green) and GroES (yellow), are shown. Scale bar represents 5.0 nm. Figures were generated using RasMol (http://www.umass.edu/microbio/rasmol/). Download figure Download PowerPoint It is clear that ATP and GroES bindings play essential roles in chaperonin functions, but the details and the mechanisms of the ATP-dependent reaction cycle of GroEL–GroES system are still unclear although a number of reaction models, such as a two-timer model (Taguchi et al, 2001; Ueno et al, 2004) and a single-timer model (Rye et al, 1999), have been proposed. Especially, the structural consequences of ATP/ADP binding to GroEL remain poorly understood. To understand their structure and functional arrangement in 3Ds in physiological conditions, AFM observation has been useful (Radmacher et al, 1994; Thomson et al, 1996; Viani et al, 2000; Schiener et al, 2005). In this study, we used the prototype fast-scanning AFM, which is >100 times faster than conventional AFM, and constructed a real-time, single-molecule observation system to analyze molecular 3D-structures and dynamics (kinetics) at the same time. This technique allows us to visualize and analyze the association and dissociation reaction of individual GroEL–GroES complex in the presence of ATP, and the ATP/ADP dependent conformational changes of GroEL without GroES at the single molecule level in real time. Results Imaging GroEL in solution In order to detect the reactions and conformational changes of individual GroEL at the single molecule level in real time, the key techniques were the preparation of a flat and stable 2D GroEL crystal layer and the 3D observation by our fast-scanning AFM. GroEL was directly immobilized on a mica surface through electrostatics force without any modification of the GroEL molecule such as replacement of amino-acid sequence, fluorescent labeling or chemical treatments. This immobilization force was relatively loose, so that it would allow movements and flexible conformational changes of GroEL molecules on the mica surface. Fast-scanning AFM demonstrated that GroEL was highly packed and aligned (an end-up orientation), forming a layer structure on the mica surface (two-dimensional (2D) protein crystal) in our experimental condition (Figure 2A). In this figure, the central channel of GroEL ring could be resolved. The diameters of the GroEL ring and the central channel measured from these images were 15.4±1.9 and 4.7±1.1 nm, respectively, in good agreement with previous AFM (Mou et al, 1996), EM (Chen et al, 1994) and X-ray crystallography (Braig et al, 1994) data (Figure 1A, top view), which show that the outer and inner diameters of GroEL are about 14.0 and 4.5 nm, respectively. Figure 2B shows the mechanically bisected GroEL layer. The height difference between the normal GroEL and the half GroEL was about 7.0 nm, precisely the same as a single heptameric ring of GroEL in trans based on X-ray crystallography (Braig et al, 1994). The surface of GroEL layer that is composed by GroEL 14-mers was basically flat in the absence of nucleotide. To quantify and compare the surface roughness in each image, we measured the root mean square (RMS) value of each image. In this case, RMS value was 0.60±0.02 nm (mean±s.d.). Figure 2.Topographic high-resolution image of GroEL by the fast-scanning AFM. The GroEL molecules were lightly absorbed on a mica surface in buffer solution. (A) This is an image of a series of fast-scanning AFM images. In this image, the central channels of the GroEL molecules are visible. The image (240 × 180 nm2, 192 × 144 pixels) was taken with a scan rate of 2 frames/s. Scale bar represents 50 nm. (B) In the lower left area of the image, the upper-heptameric ring of GroEL was removed, which had been scanned with higher tapping force (see Materials and methods for details). The height difference between the normal GroEL and the half GroEL is about 7.0 nm, in good agreement with the height of a single ring of GroEL (Braig et al, 1994). Scan size 400 × 300 nm2, 192 × 144 pixels. Scale bar represents 50 nm. The Z-scale is 20 nm. (C) Illustration of sample geometry. Download figure Download PowerPoint Our fast scanning AFM observation also demonstrated that when the density of the GroEL molecules on a mica surface is low and does not form a complete 2D protein layer, GroEL could randomly diffuse on the mica surface with the averaged 2D diffusion coefficient of 2.2±1.5 nm2/s (mean±s.d.). Namely, GroEL molecules frequently get out of and come in the scanning position (line) during the 1D (2D) observation by AFM (Thomson et al, 1996; Viani et al, 2000). This means that the previously demonstrated 1D or 2D observation techniques, which have much higher time resolution than the 3D-observation technique, are not suitable for a long-term (a few seconds) observation. Therefore, in this study, we utilized both 2D observation for short-time (<1 s) observations and 3D observation for long-term observations (several seconds). ATP/ADP mediated open–closed transition of GroEL To investigate the structural influence of nucleotides, a series of AFM images in Figure 3 (see Supplementary Movie 1) are taken in a buffer solution containing 50 μM ADP without GroES. In this experiment, the ATP contamination in ADP solution was removed by hexokinase (Motojima and Yoshida, 2003) to less than 0.1 nM in 50 μM ADP solution. Under these conditions, we observed some taller features (white spots in the figure) on GroEL layer (RMS value is 0.78±0.03 nm in the presence of 50 μM ADP). The most straightforward interpretation of our results is that the height fluctuation corresponds to the conformational change of GroEL during nucleotide binding to GroEL. These taller GroELs were 0.9±0.2 nm taller than the surrounding GroELs (Figure 4A). This elongation value is very similar to the height difference between the cis-ring and trans-ring conformations of GroEL estimated from 3D-model of the GroEL/ADP/GroES complex (Xu et al, 1997), and agrees well with cryo-EM studies (Roseman et al, 1996; Ranson et al, 2001). This height difference was also observed in the presence of ATP and nonhydrolysable ATP analogs (ATPγS, AMP-PNP and ADP+AlFx), but not observed in the absence of nucleotide (data not shown). Therefore, we concluded that, in the presence of nucleotides, GroEL molecules had at least two different conformations, one is open (elevated) conformation and another is closed (compact) conformation. Figure 3.Single-molecule imaging of chaperonin GroEL dynamics in the presence of ADP. Time-lapse sequential AFM images of GroEL in a buffer solution on mica surface were taken in the presence of 50 μM ADP alone with 192 × 144 pixels at a rate of 1 frame per second. Scale bars represent 50 nm. The Z-scale is 7.5 nm. In the AFM images, taller structure can be seen as the bright (white) features. As identified in the open white circle at 0 s, a 1 nm taller structure of GroEL (open conformation) than surrounding GroEL layer (closed conformation) had turned into closed conformation. Open–closed conformational changes of GroEL were repeatedly observed. To remove the effect of ATP contamination in ADP solution, we used hexokinase and reduced ATP concentration less than 0.1 nM in 50 μM ADP solution. Download figure Download PowerPoint Figure 4.Histograms of the height differences between the top of the taller structure of GroEL and top of the surrounding GroEL layer. Statistical section analyses of GroEL were performed using the data in the presence of (A) 100 μM ADP and (B) 2 μM ATP and 25 nM GroES. The lines are Gaussian fits of the height difference date for open-conformations of GroEL (solid line) and GroEL–GroES complex (broken line). Download figure Download PowerPoint In order to monitor the conformational changes of GroEL with higher time resolution, we used 2D-observation method (Viani et al, 2000), in which a single line was repeatedly scanned at a rate of 250–1000 Hz. This approach clearly demonstrated that both of the 'closed to open' and the 'open to closed' conformational changes were observed as 1 nm height difference, and completed within at least 10 ms in most GroEL molecules (more than 95%) (Figure 5). After this rapid 'closed to open' conformational change, GroEL kept an open conformation for a few seconds. These properties of the open–closed conformational changes were also observed in the presence of ADP, ATP and ATP analogs. Figure 5.Single-line-scan AFM imaging of chaperonin GroEL dynamics. Single-line-scan (2D observation) AFM images of GroEL in the buffer A on mica surface were taken in the absence (A) and presence of 50 μM ADP (B). (Scanning rate of 500 Hz, scan scale (Y-axis direction) of 162 nm and Z scale of 10.0 nm) In these AFM images, individual GroEL molecules can be seen as tube features (Viani et al, 2000). Lower panels indicate the X-axis cross-sections positioned at the white dotted lines indicated by the arrows in the AFM images, which represent typical height fluctuations under the conditions. One nanometer taller structures (open-conformations) of GroEL and height fluctuations can be seen as the bright (white) and long tube features in the presence of ADP (B), but not in the absence of nucleotide (A). These open–closed conformational changes of GroEL were repeatedly observed. Both the 'closed to open' and the 'open to closed' conformational changes in this image were completed within 5–10 ms. After this rapid 'closed to open' conformational change, GroEL kept an open conformation for a long time. Download figure Download PowerPoint We then examined the nucleotide concentration dependency of the GroEL conformational change (Figure 6). The ratio of the GroEL molecules in the 'open' conformation was counted in the presence of various concentrations (0–1000 μM) of nucleotides, and the data were fitted with the Hill equation. The Hill constants (nH) and K1/2 values are summarized in Figure 6A (inset). The K1/2 values of ATP and ATPγS are very similar (6–8 μM), whereas that of ADP is much higher (∼80 μM). At high concentrations of nucleotide, no more than 75% of GroEL had the open-form in our experiments (Figure 6A). Although we used purified proteins, a trace amount of the contamination of endogenous substrate protein could be contained and inhibit the conformational changes of some of GroEL molecules (up to 25%) in our experimental condition. Such decrement in the percentage of the open-form GroEL by contamination of substrate proteins has been previously demonstrated by biochemical experiments (Jackson et al, 1993). Figure 6.Occurrence frequency of the open-conformation of GroEL with increasing ATP/ADP/ATPγS concentration. Plot (A) shows the per cent occurrence of the open-form GroEL with increasing concentrations in the range of 0–1000 μM of ATP, ADP or ATPγS. (B) The same data in the range of 0–50 μM of ATP, ADP or ATPγS. The solid line in each case shows the optimal fitting curve of the data to the Hill equation. The parameters K1/2 and Hill constant (nH) are summarized in the inset table in (A). Download figure Download PowerPoint The lifetimes of the open conformation (open time) and closed conformation (closed time) were measured in the time-lapse 3D AFM images. The histograms of both open- and closed time in the presence of ADP, ATP or ATP analogs showed single exponential decays (Figure 7), indicating that the 'open to closed' and 'closed to open' conformational changes of GroEL in the presence of nucleotides were stochastic processes, which did not require ATP hydrolysis. The histograms were fitted to a single-exponential model to obtain the rate constant of the nucleotide-induced conformational change; F(t)=C1 k1 exp(−k1t), where F(t) is the number of open form (or closed form) that had an open (or closed) time t, C1 is the number of the total events and k1 is the rate constant (kopen → closed and kclosed → open). The obtained rate constants at various concentrations of nucleotides are summarized in Table I. At the low concentration of nucleotide, the rate constant of the open to closed conformational change did not depend on the nucleotide concentration, whereas that of the closed to open conformational change varied depending on the concentrations of ADP, ATP or ATPγS. This result indicates that the % occurrence of the open-form GroEL is strongly dominated by the rate constant of the 'closed to open' conformational change. Therefore, the result in Figure 6 indicates that the 'from closed to open' conformational change is a cooperative process for consumption of nucleotides. Figure 7.Statistical analysis of conformational changes of GroEL in the presence of nucleotides. Histograms in (A) and (C) show the typical distributions of the lifetime of open-conformation of GroEL in the presence of 50 μM ADP and 5 μM ATP, respectively. Histograms in (B) and (D) show the typical distributions of the lifetime of closed-conformation of GroEL in the presence of 50 μM ADP and 5 μM ATP, respectively. Some GroELs that do not undergo conformational changes at all over the period of observation were excluded from the analyses. The histograms were fitted with a single–exponential function as the following equation: F(t)=C1k1 exp(−k1t), where F(t) is the number of open-form (or closed-form) GroEL that had an on (or off) time t, C1 is the number of the total events and k1 is the rate constant. The rate constants (k1) were obtained by the nonlinear least-squares curve fitting method. The same analyses were performed in the presence of different concentrations of nucleotides. The obtained fitted curves were summarized in the inset figures. The obtained rate constants in each nucleotide condition are averaged and summarized in Table I. Download figure Download PowerPoint Table 1. Rate constants of 'open to closed' and 'closed to open' conformational changes of GroEL with increasing nucleotide concentration Nucleotide Concentration (μM) kopen → closed (s−1, mean±s.d.) kclosed → open (s−1, mean±s.d.) Open conformation (%, mean±s.d.) ADP 10 1.05±0.20 <0.1 1±1 30 1.11±0.25 0.10±0.03 7±1 50 0.96±0.08 0.42±0.02 18±3 ATP 1 0.71±0.21 <0.1 1±1 3 0.68±0.06 0.12±0.05 14±1 5 0.52±0.06 0.37±0.23 31±6 7 — 0.63±0.16 40±9 ATPγS 3 0.45±0.12 <0.1 1±0 5 0.41±0.06 <0.1 11±3 7 0.30±0.05 0.26±0.08 30±4 ADP+AlFx 50 0.52±0.02 0.35±0.04 19±4 AMP-PNP 400 0.34±0.02 0.12±0.02 32±4 The values of the rate constants are the mean±s.d. of 3–5 independent experiments. Dynamics of the GroEL–GroES interactions with ADP/ATP An addition of GroES to the imaging solution without nucleotide did not cause any apparent changes in the surface feature of the GroEL layer (RMS value is 0.58±0.03 nm), which is consistent with the previous experiments (Chandrasekhar et al, 1986). On the other hand, in the presence of 2 μM ATP and 500 nM GroES, much taller features than the open conformation of GroEL were occasionally observed (RMS value of 1.10±0.08 nm) (Figure 8A). The histogram of the height difference from the surface of the GroEL layer (Figure 4B) has two peaks near 3.9±0.6 and 1.3±0.5 nm. The mean value of the taller one (3.9±0.6 nm) well corresponds with the height difference between the GroEL/ADP/GroES complex (Xu et al, 1997) and GroEL (Braig et al, 1994), and the shorter one (1.3±0.5 nm) corresponds to the height difference between the open- and the closed conformations. In the series of AFM images shown in Figure 8A (see Supplementary Movie 2), GroES molecules repeatedly associated with and dissociated from GroEL. Immediately after the dissociation of GroES, GroEL was found to be in either open (10% of the total) or closed (90% of the total) conformation, suggesting that GroEL switches back to the closed conformation immediately (within 1 s) after the dissociation from GroES (Figure 9). Figure 8.Single-molecule imaging of chaperonin GroEL–GroES dynamics in the presence of nucleotides. Time-lapse sequence AFM images of GroEL on mica surface in a buffer solution were taken in the presence of (A) 2 μM ATP and 500 nM GroES, (B) 20 μM ADP and 25 nM GroES or (C) 10 μM Caged ATP and 25 nM GroES with 192 × 144 pixels at a rate of 1 frames pre second. Scale bars represent 100 nm. The Z-scale is 10 nm. (A) GroES molecules were repeatedly found to bind to and dissociate from GroEL. ATP concentration was kept low (unsaturated condition) in order to observe and analyze single molecule events. In the AFM images at 0 s, for example, a 4 nm taller structure (GroEL–GroES complex) can be seen in the right open white circle. (B) In the presence of ADP, extremely stable association of GroES with GroEL (GroEL–ADP–GroES complex) was achieved. Same results were observed with the wide range of concentration (25–500 nM). This result indicates that tip-sample interaction in our experiments is basically week enough to keep the GroEL–ADP–GroES complex stable. (C) After UV illumination at time 1 s, GroES quickly began to associate to and dissociate from individual GroEL with the generation of ATP. Download figure Download PowerPoint Figure 9.Single-molecule imaging of GroES dissociation from GroEL in the presence of ATP. (A) Time-lapse sequence AFM images of GroEL on mica surface in a buffer solution were taken in the presence of 2 μM ATP and 500 nM GroES at a rate of 1 frames per second. Scale bars represent 50 nm. Time 0 was set at the top of the first scan. Initially, some GroEL–GroES complex can be seen as white spots (white arrows, at time 0). Then, the GroES molecules were dissociated from GroEL during the observation (1–2 s for the lower complex and 2–3 s for the upper complex). In the case of the lower one, after GroES dissociation, open conformation was observed. (B) Time-lapse sequence cross-sections positioned by the black arrows (i, ii). Download figure Download PowerPoint The duration time of GroES-unbound form of GroEL in the presence of a certain concentration of GroES (25 nM) and ATP (100 μM) ('off time') was measured (Figure 10A). The histogram of the 'off-time' was well fitted to a single exponential curve, indicating that the GroES binding was also a stochastic process. The association rate constant of GroES to GroEL (kon) (2.3 × 107 M−1 s−1) was calculated by curve fitting, which is half of the value measured by the previous bulk-phase experiments (4.6 × 107 M−1 s−1) (Rye et al, 1999; Taguchi et al, 2001; Taniguchi et al, 2004). This is reasonable because one side of GroEL ring is inaccessible to GroES in our experimental condition. Thus, the semiimmobilized GroEL sample does not lose the activity for GroES binding and the effect of probe scanning to the GroES binding reaction is low enough to be ignored. Figure 10.Statistical analyses of GroES binding and dissociation events of GroEL in the presence of ATP. Histogram in (A) shows the distribution of the duration time between the GroES dissociation from and the next GroES binding to individual GroEL molecule in the presence of 25 nM GroES and 100 μM ATP. Some GroELs that do not undergo conformational changes at all over the period of observation in the presence of ATP was excluded from the analyses. The histogram was fitted with a single-exponential function as the following equation: H(t)=C3k3 exp(−k3t), where H(t) is the number of GroEL that had an off time t, C3 is the number of the total events and k3 is the rate constant. The rate constant (k3) was obtained by the nonlinear least-squares curve fitting method. Then, the association rate constant for GroES binding (kon) was calculated. Histogram in (B) shows the distribution of the lifetime of GroEL–GroES complex. Individual GroES molecules that attached to and dissociated from GroEL molecules repeatedly were used for the analyses over the observation time of ∼10 min. The solid line was obtained by the least-square fitting to the equation: C1k2k2′[exp(−k2t)−exp(−k2′t)]/(k2−k2′) derived from the two-step reaction of scheme (1). Download figure Download PowerPoint The duration time of GroES-bound GroEL ('on time') in the presence of 500 nM GroES and 2 μM ATP was measured and are summarized in Figure 10B. This histogram has a maximum peak at 4 s (the average lifetime is 6 s) and does not show a single exponential decay, indicating that the dissociation reaction of GroES from GroEL is probably not governed by a single rate-determining step, rather obeys two sequential transitions (two timer model), as previously proposed by Taguchi et al (2001) and Ueno et al (2004). The histogram data were well fitted to the equation deduced from scheme (1): G(t)=C2k2k2′[exp(−k2t)−exp(−k2′t)]/(k2′−k2), where G(t) is the number of GroEL/GroES complex that had a lifetime t, k2=0.37 s−1, k2′=0.35 s−1 and C2=295 (the number of the total events). We also analyzed the ATP concentration dependency on GroES-retained time (Table II). The time constants of the lifetimes of two independent states were 3 and 2–3 s, respectively, and the sum was 5–6 s, which well agrees with the previous value (7–15 s) estimated in bulk-phase kinetics (Burston et al, 1995; Weissman et al, 1996; Rye et al, 1997, 1999; Taguchi et al, 2001).