Purified and Cy5-labeled Trichoderma reesei Cel7A and its variants (Cel7A catalytic domain, catalytically deficient E212Q mutant and the catalytic domain of E212Q) interacting at the surface of crystalline cellulose fibrils were imaged by Total Internal Reflectance Fluorescence Microscopy (TIRFM).  

Enzymes 

T. reesei Cel7A was purified from a commercial Trichoderma reesei cellulase preparation (Sigma Aldrich Catalog number C2720).  

The E212Q gene was constructed with the pGEM-T easy vector (Promega Corporation, WI, USA) and transformed into Escherichia coli DH5α + Amp+. The gene was extracted and ligated to the pTrEno vector with the eno promoter. The pTrEno/E212Q vector, containing eno promoter and an E212Q fragment, was transformed into the T. reesei strain AST1116 via electroporation. The pTreno plasmid expressed the E212Q in T. reesei in glucose-rich media without contamination from the CBHI wild-type. The transformed T. reesei AST1116 was spread onto potato-dextrose agar plates with hygromycin B as a selection marker at 30 °C until the sporulating lawn was observed. The colonies from the sporulating lawns were transferred to Mandels and Andreotti Medium (MA) with 1 M glucose and hygromycin B at 30 °C with 200 rpm for 3 days. The E212Q enzyme (E212Qintact) was purified by Fast Protein Liquid Chromatography (FPLC) through a multistep process. Briefly, the protein was isolated using hydrophobic interaction chromatography followed by anion exchange chromatography using Resource Q column, a second round of hydrophobic interaction chromatography Resource ISO column and finally size exclusion chromatography.  The purified product was confirmed as E212Q by Western blot analysis and protein sequencing. The loss of cellulolytic activity of E212Q compared to the wildtype Cel7A was confirmed on the recalcitrant algal cellulose and highly digestible phosphoric acid swollen cellulose. 

Isolated catalytic domains of TrCel7A and E212Q (Cel7ACD and E212QCD) were obtained by limited proteolysis of the purified Cel7A and E212Q. Cel7A or E212Q was incubated with immobilized papain (ThermoFischerScientific, cat# 20341), equilibrated in the digestion buffer (20mM sodium phosphate, 10mM ethylenediamine tetraacetic acid, 20mM cysteine HCl) for 7 hours at 37°C and gentle agitation. The supernatant containing the cleaved fractions were separated from the papain by centrifugation, then concentrated and separated by size exclusion chromatography (Superdex 200, GE healthcare) in 5mM NaOAc and 100 mM NaCl, pH 5. The fractions containing the isolated catalytic domain as confirmed by SDS Page were pooled, concentrated, and stored for further use. 

All enzymes were labeled with Cy5 fluorophore (GE Healthcare Life Sciences, Amersham) per manufacturer procedures and separated from excess dye using Zeba Desalting columns (Thermo Fisher Scientific). This procedure resulted in a degree of labeling (moles of Cy5/mole protein) of 0.71 and 0.25 for WTintact and WTcore, respectively, and 0.63 and 0.79 for E212Qintact and E212Q core, respectively. 

Substrate 

Algal cellulose (AC) was purified from Cladophora aegagropila by sequential and repeated alkali and acidified hypochlorite treatments. Isolated algal cellulose fibrils were further treated with 5 M hydrochloric acid at 70 oC overnight. The residual AC fibrils were washed thoroughly with water to remove excess acid, then stored at 4 ^oC with 0.02 % sodium azide until further use.   

Never-dried cellulose fibrils were deposited onto hydrophobically-silanized glass coverslips by gravity-aided settling in the imaging channels. Imaging channels of 10 μL working volume were built onto the hydrophobized glass coverslips. The channels were filled with cellulose suspensions (0.025 – 0.25 mg/mL), sealed, and allowed to settle until ready for use (minimum settling time was overnight in the refrigerator). Prior to use, unbound cellulose was rinsed off the surface with buffer and the channels were incubated with 10 mg/ml of Bovine Serum Albumin (BSA) for > 10 minutes to passivate the surface. 

Total internal reflection fluorescence microscopy for imaging cellulases  

A through-objective total internal reflection excitation fluorescence microscopy (TIRFM) setup was used to collect single-molecule fluorescence images of individual Cy5-labeled enzyme molecules bound to algal cellulose fibrils. Excitation laser beam at 637 nm was reflected by a multiline dichroic mirror (FF500/646-Di01, Semrock) and focused at the back aperture of a 1.49 NA 60X oil-immersion objective (Olympus) to provide total-internal-reflection (TIR) excitation at the cover glass / water interface across a ~50 µm diameter area in the object plane. Sample emission was collected and imaged by the same objective onto the 512 ´ 512-pixel sensor of an electron multiplying CCD (EMCCD) camera (Photonmax, Princeton Instruments). The overall magnification of the imaging system (150´) mapped each EMCCD pixel to a 106 ´ 106 nm area in the object plane. A 37 nm wide bandpass filter centered at 676 nm was used to isolate Cy5 fluorescence excited at 637 nm.  

In a typical experiment, the Cy5-labeled enzymes in an oxygen scavenging buffer at a concentration of 50-100 pM were loaded into the 10 μL channel and imaged over the course of 40 - 50 min at 1 frame per second to generate movies consisting of a series of sequential images (image stacks). The oxygen scavenging buffer (glucose oxidase, catalase, 2% glucose and trolox in 50 mM sodium acetate buffer pH 5) was used to enhance Cy5 photostability and reduce fluorophore blinking. Under the imaging conditions used in this study, Cy5 exhibited two decay lifetimes of 196 s and 1100 s when illuminated in buffer with the oxygen scavenging system. When the oxygen scavenging buffer was not used, Cy5 decay lifetimes were 5 s and 20 s. 

Each data set has three files: 

*.spe are raw data that can be opened in ImageJ 

*_ReadMe.txt contain the metadata 

*_FRAMDC.txt contain drift-corrected positions of all spots located by the DAOSTORM analysis 

Miscellaneous notes

• File names start with the name of the enzyme followed by an identifier (time at which acquisition of this dataset was initiated).
• All data sets were collected using 0.2 mW laser power EXCEPT the TrCel7A101907, which was collected using 2 mW laser power.
• Note the EMD gain in each data set (included in the ReadMe file).