Visualization of Cellobiohydrolase I from Trichoderma reesei Moving on Crystalline Cellulose Using High-Speed Atomic Force Microscopy

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Visualization of Cellobiohydrolase I from Trichoderma reesei Moving on Crystalline Cellulose Using High-Speed Atomic Force Microscopy Kiyohiko Igarashi,* Takayuki Uchihashi,† Anu Koivula,‡ Masahisa Wada,*,§ Satoshi Kimura,*,§ Merja Penttila¨,‡ Toshio Ando,† and Masahiro Samejima* Contents 1. Introduction 2. Sample Preparation for AFM Observations 2.1. Preparation of crystalline cellulose 2.2. Purification of cellulase 2.3. Highly oriented pyrolytic graphite disk 3. Observation of Cellulase Molecules on Crystalline Cellulose 3.1. Immobilization of crystalline cellulose on graphite surface 3.2. Observation of crystalline cellulose by HS-AFM 3.3. Observation of cellulase 4. Image Analysis 5. Conclusion Acknowledgments References

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Abstract Cellulases hydrolyze b-1,4-glucosidic linkages of insoluble cellulose at the solid/liquid interface, generating soluble cellooligosaccharides. We describe here our method for real-time observation of the behavior of cellulase molecules on the substrate, using high-speed atomic force microscopy (HS-AFM). When glycoside hydrolase family 7 cellobiohydrolase from Trichoderma reesei (TrCel7A) was incubated with crystalline cellulose, many enzyme molecules * Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Department of Physics, Kanazawa University, Kanazawa, Japan { VTT Technical Research Centre of Finland, P.O. Box 1000, VTT, Finland } College of Life Sciences, Kyung Hee University, Gyeonggi-do, Yongin-si, Republic of Korea {

Methods in Enzymology, Volume 510 ISSN 0076-6879, DOI: 10.1016/B978-0-12-415931-0.00009-4

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2012 Elsevier Inc. All rights reserved.

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were observed to move unidirectionally on the surface of the substrate by HSAFM. The velocity of the moving molecules of TrCel7A on cellulose I crystals was estimated by means of image analysis.

1. Introduction Cellulose is a linear polymer of b-1,4-linked glucose residues and is the major component of plant cell walls (Hon, 1994). The degree of polymerization in native celluloses ranges from thousands to tens of thousands of glucose units. In nature, cellulose chains are packed into ordered arrays to form insoluble microfibrils (Nishiyama et al., 2002, 2003; Wolfenden and Yuan, 2008). Microfibrils generally consist of a mixture of disordered amorphous cellulose and cellulose I, which forms highly ordered crystalline regions stabilized by intra- and intermolecular hydrogen bonds. To degrade cellulose, many organisms produce cellulases, which hydrolyze b-1,4-glycosidic linkages of the polymer. Cellulase is a generic term for enzymes hydrolyzing these linkages. However, if we consider the structure of microfibrils, cellulases can be subdivided into two categories, since all cellulases can hydrolyze amorphous cellulose, whereas only a limited number can hydrolyze crystalline cellulose (Teeri, 1997). The enzymes that hydrolyze crystalline cellulose are called cellobiohydrolases (CBHs) because the major product of the reaction is cellobiose, a soluble b-1,4-linked dimer (Teeri et al., 1998). Many CBHs share a two-domain structure, having a catalytic domain (CD) and a cellulose-binding domain (CBD) (Abuja et al., 1988a,b, 1989). As the initial step of the reaction, CBHs are adsorbed on the surface of crystalline cellulose via the CBD, and then glycosidic linkages are hydrolyzed by the CD ( Johansson et al., 1989; Sta˚hlberg et al., 1991). Since the reaction produces mainly soluble cellobiose from insoluble substrates, the hydrolysis of crystalline cellulose occurs at a solid/liquid interface. CBH belonging to glycoside hydrolase family 7 (Cel7) is the major secreted protein of many cellulolytic fungi, and Cel7A CBH from the industrially important cellulolytic ascomycete fungus Trichoderma reesei (TrCel7A) is one of the best-studied enzymes hydrolyzing crystalline cellulose to cellobiose (Tomme et al., 1988; http://www.cazy.org/). This enzyme has a two-domain structure: a 50 kDa CD and a small (3 kDa) CBD connected by a highly O-glycosylated linker region (Abuja et al., 1988b, 1989). Loss of the CBD causes a significant decrease of crystalline cellulose decomposition but has less effect on the hydrolysis of soluble or amorphous cellulose, suggesting that adsorption of the enzyme on the surface via the CBD is essential for the effective hydrolysis of crystalline cellulose (Sta˚hlberg et al., 1991). Presumably the two domains work cooperatively during the hydrolysis of solid substrates.

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TrCel7A is thought to hydrolyze crystalline cellulose chain in a processive manner, making consecutive cuts without releasing the chain (Kurasin and Va¨ljama¨e, 2011; von Ossowski et al., 2003). This is attributed to the long, tunnel-shaped active site topology of the CBH (Divne et al., 1994, 1998). Although the kinetics of crystalline cellulose hydrolysis by cellulases has been investigated intensively, the mechanism of crystalline cellulose degradation by CBHs is still not well understood. The main reason for the difficulty in elucidating the mechanism is the absence of analytical methods to monitor enzymatic reaction at a solid/liquid interface. However, in the last decade, methodology to visualize biomolecules using biological atomic force microscopy (AFM) has been developed (Ando et al., 2001, 2007, 2008a,b; Yamamoto et al., 2010), and dynamic protein behaviors have been observed using high-speed AFM (HS-AFM) (Kodera et al., 2010; Shibata et al., 2010; Uchihashi et al., 2011; Yokokawa et al., 2006). We have applied this methodology to track the movement of cellulase molecules on crystalline cellulose in order to better understand the mechanism of enzymatic degradation of cellulose (Igarashi et al., 2009, 2011).

2. Sample Preparation for AFM Observations 2.1. Preparation of crystalline cellulose In order to avoid the effect of heterogeneity due to amorphous regions, we prepared highly crystalline cellulose consisting mainly of cellulose Ia (>95% crystallinity) from the cell wall of green algae as a substrate for HS-AFM study (Araki et al., 1998; Igarashi et al., 2006, 2007). The green alga Cladophora sp. was harvested from the sea of Chikura, Chiba, Japan and the crystalline cellulose was purified by the following method. 1. Whole algae were immersed overnight in 5% KOH at room temperature. They were thoroughly washed in water and purified by bleaching in 0.3% NaClO2 at 70
C for 3 h. These treatments were repeated several times until the sample became perfectly white. 2. The purified cell wall was homogenized into small fragments using a double-cylinder-type homogenizer (US-150; Microtec Co., Ltd). 3. The sample was treated with 4 N HCl at 80
C for 6 h with continuous stirring. The samples were then washed with deionized water by successive dilution and centrifugation at 3200 g for 5 min until the supernatant became turbid. The obtained crystalline cellulose samples were examined by means of transmission electron microscopy (TEM) and FT-IR and X-ray diffraction analysis, as shown in Fig. 9.1.

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Figure 9.1 (A) Picture of green algae Cladophora spp. used in the experiments. Transmission electron microscopic picture (B), FT-IR spectrum (C), and X-ray diffraction (D) of crystalline cellulose purified from Cladophora and used in the present studies.

2.2. Purification of cellulase Cellulases typically interact with other cellulolytic enzymes in nature, showing a synergistic effect in cellulose hydrolysis. In the present experiment, highly purified TrCel7A was prepared, in order to avoid the effect of contaminating enzyme(s), by means of affinity column chromatography using a substrate/product analogue, 4-aminophenyl-1-thio-b-D-cellobioside, as described previously (Tilbeurgh et al., 1984). Since we are observing single molecules in the present experiment, purity of the protein preparation is of particular importance. Therefore, the experiments were carried out carefully using different protein batches, which should have different levels of contamination, to check reproducibility. Cel7A from T. reesei was purified from a commercial cellulase mixture, CelluclastÒ 1.5L (Novozyme, available from Sigma-Aldrich) as follows (Igarashi et al., 2006; Imai et al., 1998; Samejima et al., 1997): 1. CelluclastÒ (2.5 ml) was applied to a PD-10 column (GE Healthcare) equilibrated with 20 mM potassium phosphate buffer, pH 7.0, for desalting and buffer exchange. 2. After four repetitions of Step 1, crude enzyme ( 15 ml) was applied to a DEAE-Toyopearl 650S column (22  400 mm, column volume of 150 ml) equilibrated with 20 mM potassium phosphate buffer, pH 7.0. Cel7A was eluted with a linear gradient of KCl concentration from 0 to 0.5 M. 3. The fractions containing Cel7A from the anion-exchange column in Step 2 were pooled and further purified on a Phenyl-Toyopearl 650S (Tosoh) column (22  260 mm, column volume of 100 ml) equilibrated

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with 20 mM potassium phosphate buffer, pH 7.0, with a linear gradient of ammonium sulfate from 1.0 to 0 M. 4. The fractions from the Phenyl-Toyopearl column were dialyzed against 50 mM sodium acetate buffer, pH 5.0, and purified on an affinity column (Affigel-10; Biorad) with 4-aminophenyl-1-thio-b-D-cellobioside, as described previously. Purity of the enzyme was confirmed by SDSPAGE. No b-glucosidase or hydroxyethylcellulose-degrading activity was detectable in the Cel7A preparations.

2.3. Highly oriented pyrolytic graphite disk A bare mica surface is often used for AFM observations because one can easily obtain an atomically flat surface by cleavage of the top layer. However, crystalline celluloses are not immobilized on a mica surface under aqueous conditions because hydrophobic cellulose is poorly adsorbed on the hydrophilic mica surface in liquid environments. Only when a sample droplet containing cellulose is placed on the mica surface, and then naturally dried, can the polysaccharide be physically immobilized on the matrix. However, the AFM observations would then have to be carried out in air, not in a liquid environment, because the cellulose becomes detached from the mica when it is immersed in a liquid. Since further drying would affect the surface condition of cellulose, it is important to choose an immobilization method that does not involve drying the cellulose sample. In the present study, we used highly oriented pyrolytic graphite (HOPG) as a substrate to immobilize crystalline cellulose in a liquid environment because cellulose with its hydrophobic surface is readily adsorbed on the HOPG hydrophobic surface. Thus, when we use hydrophobic HOPG as a substrate, cellulose crystals are expected to be oriented with their hydrophobic surfaces in contact with the HOPG surface, and because of their symmetry, a hydrophobic surface is exposed at the top face as well. Cellulases typically bind on the hydrophobic surface of crystalline cellulose via the CBD. Therefore, we considered that HOPG would be preferable to mica as a support for observing cellulase molecules on cellulose. The HOPG substrate should be fixed on the z-scanner for HS-AFM observations. The size and flatness of the substrate are important for fast scanning because a large substrate decreases the z-piezo resonant frequency, and a rough surface destabilizes HS-AFM observations. HOPG disks are prepared from a HOPG plate with a sharp punch having a diameter of 1.5 mm. A HOPG disk thus obtained was fixed with glue to the AFM stage, and the top layer of the HOPG surface was cleaved to expose a clean surface. Splinters on the surface, which are often formed by partial cleavage, should be avoided because hydrodynamic turbulent flow around the rapidly scanning AFM tip would induce significant vibration of the substrate through partially cleaved layers. The following handling protocol is recommended:

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HOPG disk Drop of cellulose suspension Glue Exfoliation

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Figure 9.2 Schematic representation of fixation of crystalline cellulose on highly oriented pyrolytic graphite surface.

1. Cut out a HOPG plate with a thickness of 0.1 mm from a HOPG block (10  10  2 mm; SPI Supplies) with a cutter blade. 2. Punch out a HOPG disk (1.5 mm) with a punch. 3. Glue the HOPG disk onto a columnar glass stage (2 mm in height, 1.5 mm in diameter) with epoxy glue. After gluing, leave the sample for more than 1 h for the glue to set (see Fig. 9.2). 4. Fix the glass stage on the z-piezo with either nail polish or instant glue. Wait until the bond is tight, usually at least 10 min. 5. Press Scotch tape onto the HOPG surface. To obtain a flat area of the HOPG, ensure the tape is evenly stuck to the HOPG surface by wiping with a cotton bud. Then, peel off the top layer of the HOPG surface by removing the tape. 6. The flatness of the surface should be carefully inspected because a rough surface with many large terraces causes scattering of the laser light, resulting in serious interference between the incident and reflected lights.

3. Observation of Cellulase Molecules on Crystalline Cellulose 3.1. Immobilization of crystalline cellulose on graphite surface As shown in Fig. 9.2, 2 ml of crystalline cellulose suspension in water (0.1– 0.5%) was dropped on the freshly cleaved surface of a HOPG disk and incubated for 5–10 min. The disk was then rinsed three to five times with 18 ml of 20 mM sodium acetate buffer, pH 5.0. In the present experiment,

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we typically used 0.2% cellulose suspension, 5-min incubation, and rinsing three times by the same buffer. Changing these parameters greatly affects the number of crystals available for AFM observation. A higher concentration of cellulose suspension, a longer incubation period, and fewer washings increase the number of visible crystals. Since it is impossible to estimate how many cellulose crystals are fixed on the HOPG surface, only qualitative, but not quantitative, evaluation of the reaction products is possible in this experiment. The product analysis using HPLC has been described elsewhere (Igarashi et al., 2008).

3.2. Observation of crystalline cellulose by HS-AFM The washing procedure does not completely remove crystalline cellulose weakly adsorbed on the HOPG surface, and consequently, the amplitude of the cantilever often starts to reduce at a point where the tip still seems to be far from the substrate, as the tip is moved toward the surface. A large cantilever amplitude (> 10 nm) should be used for the approach because then the AFM tip can punch through the weakly bound cellulose layer. After the tip approaches the surface, we sometimes clean the surface by scanning it with the tip at a small set-point amplitude over a wide area (normally 1 mm  1 mm) to remove weakly bound cellulose. This cellulose sometimes sticks to the tip and/or the cantilever, causing fluctuation of the amplitude, preventing AFM images. In this case, the tip is withdrawn from the substrate and a new approach should be tried. After removal of the weakly bound cellulose, we set the cantilever amplitude at less than 1–3 nm and start imaging cellulose firmly immobilized on the substrate, as shown in Fig. 9.3A–C. Use of the small amplitude is essential not only to avoid

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Figure 9.3 (A) AFM pictures of crystalline cellulose using our modified HS-AFM apparatus, 1500  1500 nm in 200  200 pixels. Bar indicates 300 nm. (B) Close-up view of the white square in A, 500  500 nm, 200  200 pixels. Bar indicates 100 nm. (C) Close-up view of the white rectangle in B, 300  150 nm, 200  100 pixels. Bar indicates 50 nm. (D) The same crystalline cellulose observed in C after addition of 2.0 mM TrCel7A. Bar indicates 50 nm.

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removal of crystalline cellulose from the substrate but also to prevent disturbance of cellulase binding to the cellulose.

3.3. Observation of cellulase Crystalline cellulose on the HOPG surface was first visualized as described in Section 3.2. For fast- and less-invasive imaging of cellulases on the crystalline cellulose surface, it is desirable that the fast-scanning direction of the AFM tip along the x direction is parallel to the long axis of the cellulose crystal. After identifying crystalline cellulose tightly immobilized in an appropriate orientation on the HOPG surface, without interference from other crystals, images of the crystal were monitored for 10–20 sec. Typically, crystalline cellulose that is not tightly bound to the surface is easily lost into solution during scanning. On the other hand, crystals that survive the initial observation can be observed again for at least 30 min after addition of enzyme. Highly purified TrCel7A, prepared as described above, was added from the scanner inlet to give a final concentration of 0.2–20 mM (Figs. 9.3D and 9.4A); 2 mM TrCel7A (final concentration) was used routinely. After injection of the cellulase into the observation solution, the solution was slowly and carefully stirred by pipetting. In general, floating

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Figure 9.4 (A) Time-lapse images of TrCel7A molecules on crystalline cellulose observed by HS-AFM. Red and white arrows indicate individual mobile and immobile molecules, respectively. Bar indicates 50 nm. Schematic representation of the interaction of TrCel7A and the hydrophobic surface of crystalline cellulose (B) and slice image of crystalline cellulose with TrCel7A adsorbed at the 110 face of the crystal (C).

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molecules in the buffer solution during scanning significantly reduce the AFM imaging quality. This is because the sensitivity of the optical beam deflection is altered when the refractive index of the solution is changed. To minimize such disturbance of imaging quality, we used automatic drift compensation, in which the free oscillation amplitude is kept constant by using a higher harmonic oscillation amplitude with slow feedback control. During AFM observations, it is difficult to identify the features in the images because AFM just shows the shape of the molecule. We therefore compared the AFM images with TEM images.

4. Image Analysis For statistical analysis of the velocities of individual cellulase molecules linearly moving on crystalline cellulose, we developed analysis software in IGOR Pro (WaveMetrics Inc.), which semiautomatically tracks the masscenter position of molecules on the cellulose in successive AFM frames. Since AFM images sometimes contain large noise spikes, probably due to adsorption of cellulase molecules on the cellulose, it is difficult to achieve completely automatic tracking, so we used a semiautomatic tracking method. The calculation is carried out using the following procedures. 1. To reduce noise spikes in the AFM images, all raw images (Fig. 9.5A) were processed with an averaging filter of 2  2 pixels (Fig. 9.5B) before the analysis. 2. Choose a single cellulase molecule on the first frame and set the region of interest (ROI) by enclosing the molecule within a rectangle (Fig. 9.5C). 3. Within the ROI, the slope of the image is compensated by first-order plane fitting and the mass-center position is calculated (Fig. 9.5C).

Figure 9.5 Image analysis of HS-AFM pictures using IGOR Pro (version 6.1.2). Pictures A–F are representatives of the steps described in Section 4.

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4. At the last frame of the successive images, the same molecule is indicated by enclosing the molecule within a rectangle and the mass-center position is calculated by slope compensation as before (Fig. 9.5D). Then, the positions of the target molecule in the first and last frames are determined. The expected trajectory is calculated as a linear function of the first and the last positions (Fig. 9.5E). 5. At the second frame, the rectangular ROI is moved to the second position along the expected trajectory. The two-dimensional correlation coefficient r for the ROI of the second frame is calculated, using the ROI of the first frame as the reference image. The two-dimensional correlation coefficient r is defined as follows; P P   m n ðImn  I ÞðRmn  RÞ r ¼ rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi P P P P  2 2   ð Þ ð Þ I  I R  R mn m n mn m n Here, Imn and Rmn are pixel intensities at a point (m, n) in the analyzing  are mean values of the and the reference images, respectively. I and R intensity matrices I and R, respectively. In the present case, the analyzing and the reference images correspond to the second and the first images, respectively. The ROI of the second frame is moved around the expected position with a tolerance of 5  5 pixels. The position of the ROI in the second frame is fixed to give the maximum value. Then, the mass-center position is calculated within the ROI; this represents the position of the molecule in the second frame. 6. The procedures described above are repeated up to the last frame to track the molecular position for all frames (Fig. 9.5E). 7. After the positions of the molecule in all frames have been calculated, we check that the calculated positions are consistent with the images because coincidental large noise signals sometimes produce errors. If that appears to be the case, we manually choose the ROI in the relevant frames and the mass-center position is recalculated in the corrected ROI. The movement from the initial position and the off-axis movement of the molecule (distance between the actual center of molecule and the line in Fig. 9.5E) analyzed in Fig. 9.5 are plotted in Fig. 9.6. Since CBH molecules move linearly on a crystalline surface, as mentioned above, off-axis movement was quite small (0.83 nm), suggesting that this analysis can provide a reasonable estimate of the velocity. The distance from the initial position clearly increased with time, indicating that this image analysis technique can track enzyme molecules on the substrate.

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Figure 9.6 Time courses of mobility (black filled circles) and off-axis movement (red filled circles) obtained from the image analysis, as described in Section 4 and Fig. 9.5.

5. Conclusion We have successfully visualized CBH (TrCel7A) molecules moving on crystalline cellulose, using HS-AFM. The key features of the technique are the use of fine crystalline substrates, a highly purified enzyme preparation, and a flat hydrophobic surface suitable for adhesion of the substrates, in addition to the development of the image analysis technique and customization of the HS-AFM itself. We have shown in our previous reports (Igarashi et al., 2009, 2011) that the observed movement reflects the catalytic reaction and processivity of the enzyme. Therefore, detailed analysis of the movement should throw light on the mechanism of enzymatic decomposition of crystalline cellulose. This technique is expected to be applicable not only to cellulases but also to other types of glycoside hydrolases, that is, hemicellulases, chitinases, and amylases, and should lead to a deeper mechanistic understanding of how these enzymes act at a solid/liquid interface and how biomass degradation occurs in nature. Moreover, this type of enzymatic reaction should be involved in the degradation of many insoluble materials, such as biodegradable plastics, so HS-AFM is expected to be a powerful tool for analysis of the reactions of various biomacromolecules on surfaces.

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ACKNOWLEDGMENTS This research was supported by Grants-in-Aid for Scientific Research to K. I. (19688016 and 21688023), T. U. (21023010 and 21681017), and T. A. (20221006) from the Japanese Ministry of Education, Culture, Sports, and Technology; by a grant of the Knowledge Cluster Initiative to T. A.; by a Grant for Development of Technology for High Efficiency Bioenergy Conversion Project to M. S. (07003004-0) from the New Energy and Industrial Technology Development Organization; and by an Advanced Low Carbon Technology Research and Development Program from the Japan Science and Technology Agency to K. I. and T. U.

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