Specificity of O-glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

Significance

Plant biomass decomposition has broad implications for the global carbon cycle, agriculture, and ecology, and it is primarily accomplished by fungi. Recently, research into fungal biomass degradation mechanisms has been driven by the growing biofuels industry, because enzymes from fungi are among the primary catalysts being investigated for industrial processes to convert polysaccharides into upgradeable sugars. Understanding the mechanisms used by polysaccharide-degrading enzymes and identifying means to improve their performance is of paramount importance because of the scale of enzyme production for biofuels processes. Here, we use glycoprotein synthesis and biophysical measurements to characterize the specific effects of glycosylation on ubiquitous fungal carbohydrate-binding modules for biomass degradation, which reveal key features of the importance of posttranslational modifications on enzyme function.

Abstract

The majority of biological turnover of lignocellulosic biomass in nature is conducted by fungi, which commonly use Family 1 carbohydrate-binding modules (CBMs) for targeting enzymes to cellulose. Family 1 CBMs are glycosylated, but the effects of glycosylation on CBM function remain unknown. Here, the effects of O-mannosylation are examined on the Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase at three glycosylation sites. To enable this work, a procedure to synthesize glycosylated Family 1 CBMs was developed. Subsequently, a library of 20 CBMs was synthesized with mono-, di-, or trisaccharides at each site for comparison of binding affinity, proteolytic stability, and thermostability. The results show that, although CBM mannosylation does not induce major conformational changes, it can increase the thermolysin cleavage resistance up to 50-fold depending on the number of mannose units on the CBM and the attachment site. O-Mannosylation also increases the thermostability of CBM glycoforms up to 16 °C, and a mannose disaccharide at Ser3 seems to have the largest themostabilizing effect. Interestingly, the glycoforms with small glycans at each site displayed higher binding affinities for crystalline cellulose, and the glycoform with a single mannose at each of three positions conferred the highest affinity enhancement of 7.4-fold. Overall, by combining chemical glycoprotein synthesis and functional studies, we show that specific glycosylation events confer multiple beneficial properties on Family 1 CBMs.

Terrestrial plant biomass is primarily degraded in nature by fungi and bacteria, which secrete synergistic mixtures of enzymes that work in concert to degrade polysaccharides and sometimes, lignin (1–4). In many cases, the enzymes used by these organisms are multimodular, consisting of one or more catalytic domains of various function (2–8) linked to a carbohydrate-binding module (CBM) that targets plant cell wall polysaccharides through specific recognition mechanisms (9). To date, 67 families of CBMs have been discovered (10), and many of these families contain members important in biomass depolymerization. Nearly all known CBM-bearing lignocellulose-degrading enzymes from fungi are Family 1 CBMs (10), which are small proteins that consist of less than 40 aa. Kraulis et al. (11) solved the first Family 1 CBM structure from the well-characterized glycoside hydrolase Family 7 cellobiohydrolase from the fungus Trichoderma reesei (Hypocrea jecorina) (TrCel7A). The structure of the TrCel7A CBM revealed a β-sheet–rich structure with two disulfide bridges and a flat face decorated with aromatic and polar residues that form the putative binding face for adsorption to the hydrophobic face of crystalline cellulose microfibrils (Fig. 1) (11–15).

Fig. 1.

The NMR structure of the Family 1 CBM and the top layer of cellulose (11). The tyrosine residues are shown in purple. The O-linked mannoses are shown in cyan and blue (22, 24).

Glycosylation is an important heterogeneous posttranslational modification (PTM) in fungal enzymes that degrade biomass (16, 17). To date, few studies have been conducted to examine glycosylation in secreted fungal enzymes to determine the extent and factors that control it, including growth conditions and extracellular glycan-trimming enzymes. Catalytic domains can exhibit both N- and O-linked glycans (18, 19), whereas the linkers connecting enzymatic domains to CBMs are decorated with O-linked glycosylation, which has long been attributed to protease protection (20) and more recently implicated in substrate binding (21). For TrCel7A, Harrison et al. (22) published the original characterization of the glycosylation pattern on the TrCel7A linker. Notably, the last five residues analyzed in their study (TQSHY) form the N terminus of the CBM, and the threonine and serine residues (Thr1 and Ser3, respectively) were shown to both natively exhibit mannosylation (22). Given that these residues are highly conserved, this PTM is likely a common feature of all Family 1 CBMs (23). It is also possible that mannosylation may be natively found on the highly conserved Ser14 residue, but this PTM has not been experimentally characterized to our knowledge. We recently used free energy calculations to predict that the mannosylation will improve the CBM binding affinity to crystalline cellulose (24). Our results suggested that the glycan structure, their locations, and the number of occupied glycosylation sites will impact the affinity of CBMs for crystalline cellulose (24).

To quantitatively assess the impact of glycosylation on Family 1 CBMs, here, we present a systematic experimental study of proteolytic stability, thermostability, and cellulose binding affinity of a library of Family 1 CBM glycoforms. Because glycosylation depends on host and culture conditions, multiple glycoforms of the same protein are often observed in biological production systems, which are often difficult to separate. Thus, a method for the routine production of specific Family 1 CBM glycoforms was developed using emerging tools in chemical glycoprotein synthesis (25–27). Recent advances in this field have made it possible to generate a variety of homogeneous glycoforms for structure–function studies (28). Because chemical glycosylation is not dictated by the amino acid sequences of proteins, it allows the generation of homogeneous glycoforms, thus enabling us to assess if the effects on the stability and function of the TrCel7A CBM are general effects of glycosylation or specific to certain sites and sugar moieties.

Results

Chemical Synthesis of CBM Glycoforms.

9-Fluorenylmethoxycarbonyl (Fmoc) -based solid-phase peptide synthesis (SPPS) was used for the synthesis of glycosylated CBM variants because of its compatibility with acid-sensitive glycosidic bonds (12, 29). Our synthesis started with the optimization of the conditions of most of the steps involved in the SPPS. By using a preloaded trityl resin [Fmoc-Leu-NovaSyn tentagel 4-carboxytrityl (TGT) resin], a pseudoproline dipeptide Fmoc-Ala-Ser(psiMe,Mepro)-OH, during the SPPS process and prolonged coupling time, we could efficiently prepare the glycopeptide library (Fig. 2) (30). To make the preparation of folded CBMs less labor-intensive, we next examined the feasibility of obtaining the correctly folded CBM glycoforms through a one-pot deprotection/folding sequence. To this end, we found that all acetyl protecting groups in the crude glycopeptides can be completely removed in less than 30 min using 5% (vol/vol) hydrazine in H₂O. Importantly, we found that O-mannosylation at Thr1, Ser3, and Ser14 sites does not impair CBM folding. As anticipated, folding of the deprotected glycopeptides can be initiated by direct dilution of the deprotection mixture in a mixed glutathione-folding buffer without additional extraction or purification (31). Properly folded CBM glycoforms display much shorter retention times on HPLC than side products, allowing for facile purification. Additional details on the synthesis method can be found in SI Appendix.

Fig. 2.

One-pot synthesis of the TrCel7A CBM. Reagents and conditions: (a) peptide synthesis: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), N,N-diisopropylethylamine, DMSO; piperidine, 1,8-diazabicyclo[5.4.0]undec-7-ene, DMSO; (b) peptide deprotection: TFA:H₂O:triisopropylsilane (95:2.5:2.5); (c) glycan deprotection: NH₂NH₂, H₂O (for glycosylated CBMs); (d) CBM folding: Tris⋅acetate, l-glutathione reduced, l-glutathione oxidized, H₂O (pH 8.2).

Using our one-pot synthesis/deprotection/folding/purification method, we first synthesized a library of CBM glycoforms containing a mono-, di-, or trisaccharide at each of three separate glycosylation sites for a total of nine CBM glycoforms (2–10) (32). Subsequently, we synthesized two additional series of CBMs that had specific glycans at more than one of three glycosylation sites (11–20) (Fig. 2). Depending on the glycosylation patterns, the yields for the synthesis of these glycovariants ranged from 20% for 12 to 3% for 20. Glycoform identities and homogeneity were experimentally verified by liquid chromatography–MS (SI Appendix, Figs. S1–S20). Their conformations were examined by CD. The CD spectra of 2–20 were similar to that of 1, indicating that the glycosylation at positions Thr1, Ser3, and Ser14 did not significantly alter the secondary structure of the TrCel7A CBM (SI Appendix, Figs. S21 and S22 and Table S1).

Site-Specific Impacts of O-Mannosylation on CBM Properties.

We investigated how mannose at each of the three O-glycosylation sites affects proteolytic stability, thermostability, and cellulose binding affinity (Fig. 3A and SI Appendix, Table S2). To elucidate the potential site-specific effects of CBM glycans on the proteolytic stability, nine monoglycosylated variants, 2–10, were compared with the nonglycosylated CBM 1 by thermolysin digestion. Thermolysin is capable of digesting the CBM into several small fragments. One cleavage site is close to the N terminus of CBM, and the truncation causes a detectable change in molecular mass (SI Appendix, Fig. S23). Therefore, by monitoring the first-order exponential decay of the full-length CBM using quantitative MALDI-TOF MS, the CBM glycoform half-life to thermolysin degradation was calculated (33–35). As shown in Fig. 3A, Top, O-mannosylation can substantially impact and improve the proteolytic stability of the CBM in a site-specific and glycan size-dependent manner. The nonglycosylated CBM 1 has a half-life of thermolysin degradation of about 0.2 h, whereas the Ser3 glycosylated CBM variants, 6 and 7, have half-lives of more than 2 h, an increase of over 10-fold. In contrast, the glycoforms bearing large O-linked mannoses at Thr1 and Ser14 sites show much less or no increase in half-lives compared with the unglycosylated CBM 1.

Fig. 3.

The effects of O-mannose glycans on the proteolytic stability (half-life to thermolysin degradation), thermostability (melting temperatures measured by variable temperature CD), and binding affinity (K_ads values on bacterial microcrystalline cellulose) of the TrCel7A CBM. (A) The site-specific contribution of mono-, di-, and trimannoses at each of three O-glycosylation sites. (B) The combined effects of multiple O-linked glycans on CBMs. (C) The effects of glycosylation density on the properties of CBMs. Bold numbers represent the identity of CBM glycoforms as per Fig. 2. In Top, numbers in parentheses represent the glycoform pattern [i.e., (100) representing a single mannose at Thr1, (010) representing a single mannose at Ser3, and (001) representing a single mannose at Ser14]. All error bars reported are SDs of data achieved from three (thermolysin half-life and melting temperature) and two (K_ads values on BMCC) separate trials. The hatched pattern indicates the glycoforms with multiple enhanced properties. *Observable binding noted; nonlinear least squares curving fitting failed to converge.

O-Mannosylation can also affect the CBM thermostability in a site-specific manner. The thermostability of each variant was assessed by its melting temperature, which can be directly measured by variable temperature CD (28). As shown in Fig. 3A, Middle, O-mannosylation at Ser3 leads to the most substantial stabilization, with the increase in melting temperature of 11 °C compared with nonglycosylated CBM 1, which has a melting point of 62 °C. Mannosylation at Ser14 also leads to noticeable but less pronounced stabilization than Ser3 mannosylation. Thr1 mannosylation displayed the least stabilizing ability. The glycan size does not seem to be directly related to the magnitude of the stabilizing effect, similar to observations for protein N-glycosylation (36, 37).

The binding affinity of the TrCel7A CBM glycoforms to crystalline cellulose was also measured using a similar method to our previous studies (21). The binding affinity of each CBM glycoform for bacterial microcrystalline cellulose (BMCC) was fitted to a Langmuir isotherm and is reported as K_ads, which correlates with the strength of CBM adsorption to BMCC. As shown in Fig. 3A, Bottom, all three sites are potentially involved in CBM–substrate interactions. The addition of a single monomannose motif to the Ser3 or Ser14 position provided a substantial increase in affinity, and a dimannose motif to Thr1 caused a similarly large increase in binding affinity. Conversely, increased affinity to BMCC is diminished with attachment of larger glycans beyond a monomannose at Ser3 or Ser14 or a dimannose at Thr1. Overall, by systematically comparing the properties of the monoglycosylated variants, the importance and site-specific and size-dependent effects of O-mannosylation were shown. Two glycoforms, 5 and 6, were identified with multiple enhanced properties (Fig. 3A).

Effects of O-Mannosylation at Multiple Glycosylation Sites.

To understand the impact of O-glycosylation on the TrCel7A CBM in the physiological context (22), it is necessary to examine glycoforms with O-mannose glycans at multiple glycosylation sites. Thus, we conducted two additional series of comparative studies (Fig. 3B and SI Appendix, Table S3). Because glycoforms with O-mannose residues at Ser3 (5 and 6) have multiple enhanced properties, we focused our studies on changes of the properties of these two variants. In each series of studies, the effects of the addition of O-linked glycans at Thr1 and/or Ser14 on the proteolytic and thermostability and binding affinity of the CBM were examined.

As shown in Fig. 3B, Top, the attachment of an additional monomannose to Thr1 or Ser14 can lead to additional increase in the half-life of CBM to thermolysin degradation (compare 5, 11, and 12 with 6, 15, and 16), although the half-life of monomannosylated 2 is essentially the same as that of 1. The greatest half-life enhancement was achieved with higher glycan density, such as additional attachment of glycans at more positions and greater length of glycans (compare 5, 13, and 14 with 6, 17, and 18). The correlation between thermostability and glycan density is much less obvious than that of the proteolytic stability (Fig. 3B, Middle). The mannosylation of Ser3 with a dimannose leads to the most significant increases in the melting temperature (compare 1 with 6). Additional glycosylation site occupancy and the greater glycan length past the dimannose structure have much less impact (compare 6 with 15, 16, 17, and 18). An interesting affinity trend was identified from the BMCC adsorption studies of multimannosylated CBM glycoforms. As shown in Fig. 3B, Bottom, it seems that the three O-mannosylation sites synergistically modulate the binding affinity enhancements. The CBM glycoforms with fully occupied glycosylation sites show better binding than those with partially occupied sites (compare partially occupied 11, 12, 15, and 16 with fully occupied 13 and 17). Intriguingly, increases in glycan sizes lead to decreased binding affinities.

Lastly, to further confirm the influence of the size of O-linked mannoses on the properties of CBM, we compared the stability and binding affinity of 1, 13, 19, and 20 (Fig. 3C and SI Appendix, Table S4). As expected, glycoform 20, which contains a trimannose at each glycosylation site, has a higher proteolytic stability, a similar thermostability, and a much lower binding affinity compared with 13. The overall properties of 19 are better than those of 20 but less favored than those of 13.

Discussion

Protein glycosylation is one of the most prevalent posttranslational modifications, with more than 50% of proteins in eukaryotes containing glycans (38). Glycosylation can modulate both the physical and biological properties of proteins (39, 40) and aid in protein folding and secretion (41). Indeed, O-linked glycans on cellulase linkers confer proteolytic resistance (20, 42) and have been shown to impart affinity to crystalline cellulose (21). Furthermore, it has been shown that small O-linked glycans exist on the TrCel7A CBM near the binding face (Fig. 1) (22), which were predicted through computational studies to improve CBM affinity to crystalline cellulose (24). Alternatively, O-linked glycans distant from the binding face of a Family 2a CBM do not affect cellulose affinity (43), and large high mannose-type N-linked glycans near the Family 2a CBM binding face detrimentally affect cellulose affinity (44). Family 1 CBM experimental studies to date have examined the functional role of many structural features, but no work has systematically considered the effects of the natural O-mannosylation (9, 12, 13, 15, 43, 45). To address the various potential effects of O-mannosylation on the TrCel7A CBM, we performed the comparative study using synthetic homogenous glycoforms.

Using synthetic glycoforms, we systematically showed that O-glycosylation enhances the stability and cellulose binding affinity of a model Family 1 CBM. This study also shows the feasibility and reliability of chemical synthesis in exploring the effects of glycosylation and allows for the identification of the O-glycosylation site that has the largest impact on the functional properties of CBM (Ser3) and the identification of the glycoforms with better overall properties (13 and 17) (Fig. 3, hatching). In addition, this study provides unique insights into the varied roles of different O-linked mannoses in modulating the properties directly related to the performance of the CBM, which would not be possible using heterogeneous natural mixtures of glycoforms.

During biomass depolymerization in nature, organisms secrete proteolytic enzymes capable of cellulase degradation. The secretion of proteases aids in the competition for resources and also is a means for pathogen defense mechanisms (46). It is clear from previous studies that glycans can protect against proteolysis (20, 42). Our results also show such protection, showing that glycans can protect the peptide backbone from proteolytic attack, likely through a steric hindrance mechanism. It is also clear that mannosylation at Ser3 leads to larger increases in proteolytic stability, possibly because glycosylation hinders thermolysin access to the N-terminal cleavage site at Tyr5. Although the protease protection conferred by mannosylation shows site-specific differences, the density of glycans on the surface of CBM causes far more dramatic increases in the thermolysin resistance. The more heavily glycosylated CBMs all have much longer half-lives to proteolytic degradation by thermolysin, indicating that the backbone of the CBM can be more effectively shielded by increased glycan length and density (40).

Thermostability is a highly preferred trait of industrial enzymes. As such, many studies have engineered cellulases for improved thermostability through amino acid substitutions or domain and sequence shuffling (47, 48). We observed that marked increases in CBM thermostability are conferred by glycosylation and that mannosylation at Ser3, specifically, plays a more substantial role in increasing the melting temperature (T_m) of CBM than glycans at Ser14 and Thr1. The results show that even a monomannose glycan at Ser3 substantially increases the CBM thermostability. Dimannose at Ser3 leads to a T_m enhancement of 11 °C compared with the nonglycosylated molecule, which is the largest increase observed by the addition of a glycan to a single site. Additional attachment of mannose to the CBM only induces minor changes in T_m. This finding is similar to the observations for studies of N-glycosylation, indicating that the large enhancements caused by O-mannosylation at Ser3 might be at least partially caused by interactions between the first two mannose units attached to Ser3 and its local amino acid residues (36, 49).

An improvement of CBM-mediated adsorption onto insoluble crystalline cellulose has been shown experimentally to be beneficial for activity of the Humicola grisea glycoside hydrolase Family 7 cellobiohydrolase on crystalline cellulose (50). Although previous Type A CBM glycoengineering efforts were met with limited success (43, 44), we have shown that small mannosyl residues at each of three glycosylation sites are able to increase the binding affinity of CBM to BMCC (Fig. 3A, Bottom). Greater affinity enhancement could be achieved with a dimannose moiety at Thr1 or a monomannose at Ser3 or Ser14. Intuitively, this enhancement may be because of the greater distance of Thr1 from the binding face than the other glycosylation sites (Fig. 1). Therefore, to gain any increase in hydrogen bonding potential with the cellulose surface, a longer glycan would be required on Thr1. Moreover, the addition of extra mannosyl units actually decreased affinity for crystalline cellulose, similar to work by Boraston et al. (44), which reported large N-linked glycans as detrimental to a Family 2 CBM adsorption to cellulose. This result could be explained by a steric hindrance effect, such that longer glycans can interfere with the interactions between the hydrophobic surface of BMCC and highly conserved Tyr5, Tyr31, and Tyr32 residues, which has been hypothesized previously (Fig. 1) (43, 44).

O-Linked mannoses at the three sites studied here act synergistically to enhance the binding affinity of CBM to BMCC. From the data presented in Fig. 3B, Bottom, it was also confirmed that the chief binding enhancement could be achieved through addition of monosaccharides on all three glycosylation sites. This observation further supports the theory that large glycans inhibit additional affinity of the Cel7A CBM to BMCC. Taken together, these data show that binding affinity of Family 1 CBMs is intimately tied to O-mannosylation patterns and that the affinity is best enhanced with smaller glycan structures. Notably, the trend from the experimental binding data is quite similar to that reported by Taylor et al. (24), signifying that the combination of computational predictions and experimental verifications could be a useful tool in understanding cellulase glycosylation.

The affinity values achieved for the nonglycosylated CBM 1 are in agreement with other studies (21, 45, 51). We report affinity enhancements of similar magnitudes as the enhancements reported by Takashima et al. (50) and Linder et al. (12), with affinity of CBMs 13 and 17 approaching or within range of K_ads values obtained for both the whole TrCel7A or the multiglycosylated TrCel7A linker CBM domain (21, 52–55), suggesting that glycoengineering of Family 1 CBMs is a viable strategy for activity enhancement of cellulases. Moreover, the O-linked glycosylation sites studied here are highly conserved across Family 1 CBMs (23), suggesting that the glycosylation-enhancing properties observed here likely occur throughout this ubiquitous CBM family. It has also been proposed that other cellulolytic enzymes contain similarly beneficial glycosylation for substrate binding, such as the glycans near the binding surfaces of lytic polysaccharide monooxygenases (56, 57). Collectively, these studies suggest that glycans impart multiple beneficial properties to cellulases and that glycosylation may be a strategy commonly used by biomass-degrading organisms for cellulase enhancement.

Conclusions

In summary, we used chemical synthesis to develop a practical one-pot method for quickly and conveniently obtaining a collection of representative glycoforms of a Family 1 CBM. These homogeneous glycoforms are valuable tools for developing a quantitative understanding of protein glycosylation. Using these structurally well-defined glycoforms, we have shown that O-linked mannose residues increase proteolytic stability of CBM in a glycan size-dependent manner, thermostability in a glycosylation site-specific manner, and binding affinity in a glycosylation pattern-dependent manner. Our data also support the theory that large glycans decrease the ability of CBMs to bind to crystalline cellulose. Taken together, our study shows the importance of O-mannosylation in regulating the properties of the Family 1 CBM. This regulation may allow biological systems to fine tune how the CBM binds to crystalline cellulose during degradation. We anticipate that the concepts put forth here will find broad applicability in the study of other protein posttranslational modifications and the glycoengineering of industrially and therapeutically important proteins.

Methods

Chemical Synthesis and Folding.

Fmoc-based SPPS was used to synthesize the glycosylated CBM analogs (21). The O-mannosylated amino acid building blocks were synthesized following the method published previously (32). After SPPS, the glycopeptides were cleaved from the resin, and side chain-protecting groups were removed by stirring in TFA:triisopropylsilane:H₂O (95:2.5:2.5). The acetyl-protecting groups on attached mannose residues were removed by addition of a 0.94 M solution of hydrazine in water for 30 min at room temperature under helium atmosphere. The crude unprotected glycopeptides were folded by dilution in refolding buffer (0.2 M Tris⋅acetate, 0.33 mM oxidized glutathione, 2.6 mM reduced glutathione, pH 8.2) and stirred overnight under helium atmosphere. The products were purified with HPLC on a C18 column and lyophilized. Liquid chromatography–MS was used to confirm the identity and purity of the products.

Thermolysin Digests.

The digestions were performed at 37 °C in 100 μL solution (50 mM Tris⋅HCl buffer, 0.5 mM CaCl₂, pH 8.0) with an initial CBM variant concentration of 270 μM. The CBM variant and thermolysin were initially present in a 20:1 molar ratio; 10-μL aliquots were taken at specific time intervals and quenched with an equal volume of 5% AcOH. Each sample was analyzed by quantitative MALDI-TOF MS (described below) to calculate the change in CBM concentration with time. The digestion rate was determined by monitoring and fitting data to the first-order exponential decay of the full-length CBM glycoform over time (33–35).

Quantitative MALDI-TOF MS.

For absolute CBM quantitation, internal reference standard solutions of each CBM glycoform were prepared per experiment by serial dilution (10 μL per concentration) (58). To all sample aliquots, 150 pmol CBM internal standard peptide (Δm/z ≥ 162 Da) in H₂O:MeCN:AcOH (1:1:3.3% 3 μL) was added; 0.5 μL each sample was spotted directly on a 100-well MALDI target plate with 1.126 μL α-cyano-4-hydroxycinnamic acid matrix (6.2 mg/mL) in MeOH:MeCN:H₂O (36:56:8) and allowed to air dry (∼5 min). Spectra were acquired on a Voyager-DE STR MALDI-TOF mass spectrometer (Applied Biosystems) in linear positive ion mode with 50 shots per spectra. The laser intensity was set to 1,950, the accelerating voltage was set to 20,000 V, the extraction delay time was 100 ns, and the grid voltage was set to 94%. The low mass gate was set to 500 Da, and data were collected from 3,200 to 5,000 Da (5,500 Da for glycoform 20). An in-house MATLAB program was written to determine the ratio of analyte ion intensities between the CBM and the CBM internal standard. From these data, a standard linear calibration curve was generated for each experiment to calculate the absolute CBM concentration from CBM to CBM internal standard ion intensity ratios.

Thermostability Assay.

All CD spectra were acquired using an Applied Photophysics Chirascan-Plus CD spectrometer in a 0.5-mm quartz cuvette under nitrogen at a flow rate of 1 L/min. Lyophilized CBM glycoforms were suspended in 10 mM sodium acetate (pH 5.2) at a concentration of 0.2 mg/mL. The melts were performed by ramping the temperature of the sample from 20 °C to 94 °C at a rate of 1 °C/min while monitoring the CD signal at 217 nm. The melts resulted in roughly sigmoidal melting curves, and the point of inflection of the curve was interpreted to be the melting point of the analog (59).

BMCC Adsorption Assay.

Adsorption isotherms were performed as described elsewhere (12, 45). Briefly, lyophilized CBM glycoforms were suspended and serially diluted in 50 mM sodium acetate and 50 mM sodium chloride buffer (pH 5.0). CBM suspensions were added 1:1 with 2.4 mg/mL bacterial microcrystalline cellulose from Acetobacter xylinus ssp Sucrofermentans in 50 mM sodium acetate and 50 mM sodium chloride (pH 5.0; total volume = 100 μL) in microcentrifuge tubes containing magnetic stir bars. The samples were stirred to equilibrium at 1,100 rpm at 4 °C for 2 h before centrifugation at 14,000 × g for 10 min. Two 10-μL aliquots were taken from the supernatant and analyzed by quantitative MALDI-TOF MS (see above) to calculate unbound CBM concentration. Data were fitted to a single-site Langmuir adsorption model (Eq. 1) using OriginPro 9 software,

$$\left[B\right]\hspace{0.17em}=\hspace{0.17em}\frac{B_{max}\times K_{ads}\times \left[F\right]}{1+K_{ads}\times \left[F\right]},$$ [1]

where B_max represents the total binding capacity of the CBM glycoform, K_ads represents the binding affinity, and [B] and [F] represent bound and free concentrations, respectively (SI Appendix, Figs. S24–S43), which has been done elsewhere (51, 54, 60).