Article pubs.acs.org/Langmuir

Specific Binding at the Cellulose Binding Module−Cellulose Interface Observed by Force Spectroscopy Jason R. King,†,§ Carleen M. Bowers,†,∥ and Eric J. Toone*,†,‡ †

Department of Chemistry, Duke University, Durham, North Carolina 27708, United States Department of Biochemistry, Duke University Medical School, Durham, North Carolina 27710, United States

‡

S Supporting Information *

ABSTRACT: The need for effective enzymatic depolymerization of cellulose has stimulated an interest in interactions between protein and cellulose. Techniques utilized for quantitative measurements of protein−cellulose noncovalent association include microgravimetry, calorimetry, and atomic force microscopy (AFM), none of which differentiate between specific protein− cellulose binding and nonspecific adhesion. Here, we describe an AFM approach that differentiates nonspecific from specific interactions between cellulose-binding modules (CBMs) and cellulose. We demonstrate that the “mismatched” interaction between murine galectin-3, a lectin with no known affinity for cellulose, and cellulose shows molecular recognition force microscopy profiles similar to those observed during the interaction of a “matched” clostridial CBM3a with the same substrate. We also examine differences in binding probabilities and rupture profiles during CBM−cellulose binding experiments in the presence and absence of a blocking agenta substrate specific for CBM that presumably blocks binding sites. By comparison of the behavior of the two proteins, we separate specific (i.e., blockable) and nonspecific adhesion events and show that both classes of interaction exhibit nearly identical rupture forces (45 pN at ∼0.4 nN/s). Our work provides an important caveat for the interpretation of protein−carbohydrate binding by force spectroscopy; delineation of the importance of such interactions to other classes of binding warrants further study.

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INTRODUCTION Cellulose, the β-(1→4)-linked polymer of D-(+)-glucopyranose, is a ubiquitous component of plant cell walls.1 Lignocellulosic biomass grown on marginal land with little or no value for agriculture is an intriguing source of sugars for the production of biofuels. On the other hand, lignocellulosic feedstocks remain largely recalcitrant to economically viable processing on an industrial scale, and the efficient depolymerization of cellulose remains an area of aggressive research.2 Cellulosedegrading chemotrophs overcome cellulose recalcitrance through the use of a remarkable group of modular cellulose hydrolase enzymes, or cellulases, that degrade cellulose microstructures and provide monomeric glucose for respiration.3−7 Nearly all cellulophiles incorporate cellulose-binding modules (CBMs) and one or more hydrolase domains into fully functional cellulases.8 As with lipases, cellulose activity cannot be described by classical Michaelis−Menten models of enzyme activity, since enzyme activity follows interfacial binding between the CBM and an insoluble cellulose substrate.9 Knowledge of CBM−cellulose binding is essential for the development of hydrolase kinetic models and the biophysical characterization of cellulases. Though techniques such as microgravimetry,10 surface plasmon resonance,11,12 and UV− visible spectroscopy13 are often used to probe cellulose−CBM association, none differentiate nonspecific adhesion from © 2015 American Chemical Society

specific binding. Traditional biophysical methods used to assay protein−carbohydrate interactions, including fluorescence spectroscopy, isothermal titration calorimetry, ultracentrifugation, and mass spectrometry, are of limited value in the study of lignocellulose deconstruction, due to substrate heterogeneity and the insolubility of cellulosic substrates. Force spectroscopythe forced mechanical unbinding of molecules over nanometer-scale distances using optical tweezers or an atomic force microscope (AFM)offers an attractive approach to observe binding interactions at the solid−liquid interface. We have previously demonstrated that force spectroscopy can be used to evaluate the probability of binding between a surface-immobilized receptor unbound and in the presence of free, soluble ligand and that the difference in these probabilities facilitates the quantification of immobilized receptor−ligand binding affinities.14 Here, we continue this work by exploring the binding between AFM-tip-bound CBM and cellulose immobilized at a surface. Many have demonstrated the resistance of clostridial cellulosome scaffoldin proteins to mechanical unraveling up to ∼600 pN at ∼15 nN s−1, a property that facilitates force Received: December 16, 2014 Revised: March 2, 2015 Published: March 4, 2015 3431

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(Scheme 1). We previously reported that the PEG24 linker provides sufficient translational and rotational mobility to

spectroscopy evaluation of clostridial CBM−cellulose interactions.15−17 Xu and co-workers18,19 utilized AFM force spectroscopy to derive CBM−cellulose unbinding forces (∼50 pN at 100 nN/s loading rate) and binding energies of ∼15 kcal mol−1 for the Clostridium thermocellum CBM3a. Similarly, Kitayoka and co-workers20 used AFM to measure binding forces between a hexahistidine (His6)-tagged CBM from Cellulomonas fimi and a cellulose surface and obtained an unbinding force nearly 100 times larger, 6.3 nN. The biophysical significance of the reported binding strengths and energies is obscured by the ambiguous origin of the observed ruptures: the observed unbinding events could arise from nonspecific protein−cellulose adhesion and/or force-induced protein denaturation, rather than from specific binding. CBM−substrate binding has been queried with numerous cellulosic substrates that vary considerably in morphology, crystallinity, and chemical composition. Such studies depict a range of structurally distinct CBM-binding sites across multiple polysaccharide surfaces.13 A thermodynamic description of CBM ligand binding, however, is often left ambiguous due to the difficulty in distinguishing specific binding from nonspecific adsorption. The term “specific”used here in the context of ligand bindingimplies the formation of a bound complex that has defined stoichiometry and for which a true thermodynamic equilibrium can be established. Here, we use blocking (or the abolishment of binding) by the addition of saturating soluble ligand as evidence for specific binding. We hypothesized that the ability to block nonspecific adhesion events with free, soluble ligand in a force spectroscopy experiment would differentiate specific CBM−cellulose binding from nonspecific interactions.21 Here, we describe the functionalization of an AFM cantilever with clostridial CBM3a and the application of piconewton forces over nanometer distances to disrupt CBM binding to a synthetic surface of cellulose nanocrystals (NCs). We differentiate specific from nonspecific binding by first blocking the CBM− cellulose interaction with a colloidal suspension of cellulose NCs and then removing the NCs to observe the return of CBM−cellulose binding.

Scheme 1. Chemical Functionalization of AFM Cantilever and MCC Immobilizationa

Reagents and conditions: (a) NanoStrip, 75 °C, 30 s; (b) 5% HF, rt, 1 min; (c) 254 nm light at 5 cm, S1, rt, 30 min; (d) 50% TFA in CH2Cl2, rt, 30 min; (e) 15 mM NHS-dPEG24-Mal, 1% Et3N in CH2Cl2, rt, 2.5 h; (f) 10 mM S2, 5 mM TCEP, 10 mM NaH2PO4, pH 8.1, rt, 13 h; (g) 50 mM NiSO4, 20 mM NaHEPES, pH 7.0, rt, 45 min; (h) 40 μM MCC, 10 mM NaH2PO4, 140 mM NaCl, 0.05% Tween-20, pH 7.4, rt, 30 min. a

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RESULTS AND DISCUSSION Surface Immobilization of CBM3a and Cellulose. Immobilization of CBM3a to an AFM cantilever poses several challenges. To both maximize the likelihood of specific binding and to reduce nonspecific adhesion of protein to the cellulose surface, the protein must be oriented in a favorable binding geometry.22 We expressed a recombinant trimodular Clostridium cellulolyticum protein comprising CBM3a, an X-module of unknown function, and the first cohesin domain of the cellulosome integrating protein (CipC), after the approach of Pagès et al.23 This “miniCipC” (MCC) positions the CBM3a at the N-terminus and an engineered Ni(II)-binding His6 tag at the C-terminus of the protein. By separating the functional CBM from the His6 tag via the intermediate protein domains, we ensure that orientation of the CBM3a for productive binding is not precluded by Ni(II) chelation to the cantilever. In addition, Nash and others have shown that the CBM consistently denatures under tensions at which the X-module and cohesion domains remain structured (< 300 pN at ∼15 nN s−1). Thus, these domains should not impede force measurements of CBM−cellulose dissociation.15,17 The cantilever was modified with a tetracosaethylene glycol (PEG24) linker bearing an NTA moiety for Ni(II) chelation

enable productive substrate-binding geometry.14 The tip was prepared for protein immobilization using known silicon functionalization chemistry.24,25 Briefly, the cantilever was chemically etched with Nano-Strip and aqueous HF to form a H-terminated silicon nitride surface and then alkylated via ultraviolet-light-promoted hydrosilylation of the heterobifunctional tert-butyl 3,6,9,12,15-pentahexacos-25-enylcarbamate linker (S1, Supporting Information) to yield an N-Bocterminated monolayer. The N-Boc functional group was cleaved using trifluoroacetic acid, and the free amine was coupled to an N-hydroxysuccimidyl-PEG24-maleimide linker (NHS-dPEG24-Mal, Quanta BioDesign). A synthetic sulfhydryllinked NTA molecule (S2, Supporting Information) was used to incorporate the NTA Ni(II) chelator via Michael addition to the thiol-reactive maleimide monolayer. The elemental composition of the surface was monitored at each step by measuring the ratio of carbon to silicon signals using X-ray photoelectron spectroscopy (XPS; Table 1). For each sample an element sweep from 0 to 1200 eV was performed to identify the electron absorption bands of 3432

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Langmuir Table 1. Surface Functionalization Monitored by XPS, Water Contact Angles, and Goniometric pH Titration contact angle (deg)

surfacea 3, 4, 5, 6,

R-NHBoc R-NH2 R-PEG-Mal R-Mal-NTA

7, R-NTA-Co

C-1s/Si-2p (area ratio)

Co-2p position (eV)

0.54 0.39 0.71 0.61

NDb ND ND NOc

0.64

NO 792.6 793.5

advancing

receding

pH

67 49 60 49 52 51 53 45 45 46 43 42 42 ND ND ND

58 38 46 32 35 28 27 20 20 17 16 12 10 ND ND ND

ND ND ND 2.0 3.1 4.0 5.1 6.1 7.0 8.0 9.1 10.1 11.1 5.5 6.8 7.4

Figure 1. AFM image of spun-cellulose on silicon. Surface metrics of depth and roughness were evaluated at various points (red triangles) along the white axis, depicting a uniform dispersion of nanocrystals across the silicon surface.

NCs to block specific binding interactions between the cantilever-bound CBM3a and the cellulose surface, thus differentiating specific interfacial binding events from nonspecific protein−cellulose adhesion (vide infra). Measurements of MCC−Cellulose Binding Using Force Spectroscopy. AFM is a sensitive tool, measuring cantilever tensions at piconewton loads and stage retraction over nanometer distances.32 As a result, the analysis of force spectroscopy data is subject to errors associated with surface vibrations and tip fluctuations that can alter rupture morphology and lead to inconsistent identification of ruptures. Other experimental parameters, such as contact force and dwell time, can also alter rupture morphologies and impact the probability of observing rupture events, even within a single data set.33 To minimize these effects, we rely on automated protocols developed in our laboratory previously33 for the objective analysis of rupture data. A prerequisite to the use of AFM to characterize CBM3a− cellulose binding is an assessment of the force and distance limitations of the molecular system. The CBM3a−cellulose interaction is dominated by hydrophobic contacts.34 To assess the mechanical strength of these interactions in comparison to the noncovalent coordination of the His6 tag and the NTA− Ni(II) complex, a model peptide with the primary sequence CGWGGHHHHHH was synthesized using microwave-assisted solid-phase peptide synthesis. The peptide cysteine residue was used to covalently link the peptide to a maleimide-terminated silicon surface using chemistry similar to that described above and elsewhere.22,35−38 An AFM cantilever bearing a Ni(II)loaded NTA monolayer was gently39 brought into contact with the peptide surface; upon retraction, the deflection of the cantilever yielded a force vs distance plot characteristic of forceinduced rupture of the His6−Ni(II)−NTA interaction. We repeated the cycle 250 times and plotted the frequency of observed rupture forces and lengths (Figure 2). The frequency histograms were fit to an inverse Gaussian distribution function to determine statistically significant forces and lengths. The mean Ni-dependent rupture force was 121 ± 7 pN at a retraction velocity of 200 nm s−1 (The retraction velocity corresponds to a loading rate of ∼0.4 nN s−1, as calculated for the presented molecular system by Bowers et al.33), and the mean rupture length was 15.5 ± 0.4 nm; these values agree well with a molecular system length of about 13 nm (3 nm peptide + 10 nm PEG24).

a

Surfaces from Scheme 1. bND: data was not determined. cNO: peak was not observed.

elements present on the surface. Ten scans were performed in the C-1s and Si-2p regions to quantify the C/Si ratio on the surface. (The measured C/Si ratio is normalized by multiplying by the Si/C relative sensitivity factor ratio of 1.1799, which is specific to this instrument.) Due to the poor sensitivity of nickel in XPS analysis, cobalt chelation to the NTA surface 7 was verified by the emergence of the Co-2p band using 10 scans in the Co-2p absorption region in XPS. Interestingly, metal chelation was not observed under nonbuffered soaking conditions (50 mM CoCl2 or NiSO4 in water) common to metal loading on immobilized metal-affinity chromatography resins.26 This behavior is likely due to a surface-induced increase in the pKa of the NTA moiety, an effect previously described by Whitesides and co-workers27 and also shown here using goniometric pH titration of buffered water droplets on NTA-functionalized silicon wafers. Changes in the elemental composition at the surface following each step altered the hydrophobicity of the surface, an effect that was evident in water contact angles after each reaction (Table 1). A clear decrease in contact angle with increasing pH is consistent with greater surface wetting as the NTA moiety is ionized. Although the reported28,29 pKa of NTA is 2.5 (pKaNi(II) = 1.89, 2.49, 9.73), the surface appears to perturb the pKa upward to near 5.5 (Supporting Information). As a result, buffered NiSO4 at pH >6.5 was required to ensure immobilization of Ni(II) at the surface. Cellulose nanocrystals (NCs) were spun on silicon to produce a 24.5 nm film using the methods of Edgar and Gray30 and Wågberg et al.31 AFM images confirmed that the cellulose NCs were uniformly distributed across the surface (Ra = 1.5, Rq = 1.8), allowing effective CBM3a−cellulose binding at any location on the wafer (Figure 1). Inspection of AFM images indicated that the NCs were typically 25−30 nm in width, 80− 100 nm in length, and 8 nm in depth. Unlike other cellulosic films, cellulose NCs can be prepared in a mild, aqueous buffer that is compatible with cellulose-binding proteins. This unique feature enabled the use of a colloidal suspension of cellulose 3433

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With a firm understanding of the system characteristics, we next sought to characterize specific (i.e., blockable) CBM3a− cellulose interactions. Initially, the cantilever-immobilized MCC was brought into gentle contact33 with a nanocrystalline cellulose surface, and the surface was retracted to generate a force vs distance plot (Figure 3, right). Next, a blocking solution of cellulose NCs (in buffered saline, pH 7.5) was introduced into the AFM fluid cell. Initial attempts to monitor binding in the presence of a NC suspension were unsuccessful due to large background cantilever movement, presumably attributable to the presence of particulate NCs. Fortunately, the CBM3a−cellulose interaction is highly salt dependent,40 and treatment with buffered saline left intact bound MCC−NC complexes but successfully removed unbound colloidal NCs, silencing NC-induced noise. The NCs were removed from the MCC molecules with pure watera process routinely used in the purification of CBM3a via cellulose-affinity chromatography,23,40 and the MCC−cellulose binding returned in the presence of buffered saline, albeit with a large reduction in total binding events as compared to the initial data set (Figure 3). Each cycle was repeated more than 100 times to yield statistically significant frequency histograms of rupture forces and lengths. Significantly, a reduction in Pbind from 93% to 55% was observed between unblocked (NCs not present in the fluid cell) and blocked (NCs introduced to the fluid cell) experiments. The binding probability increased (73%) following removal of the NCs, indicating a significant number of specific, “blockable” interactions. The large binding probability in the presence of blocking agent is consistent with extensive nonspecific adhesion between a hydrophobic substrate and a hydrophobic protein. Gratifyingly, the highest frequency of blockable rupture events was observed at a rupture length of 32 ± 1 nm (Supporting Information), a distance consistent with a predicted molecular system of about 30 nm (20 nm protein + 10 nm PEG linker). The distribution of rupture lengths may be skewed by the presence of short, nonspecific tip−cellulose adhesion events (vide supra). The

Figure 2. His6-tag rupture force histograms depict a 121 pN rupture force (top) and a 15.5 nm rupture length (bottom).

To determine the inherent adhesive forces associated with cantilever−cellulose contact, we plotted the systematic rupture frequencies at varying distances for a “blank” NTA-terminated cantilever (i.e., a functionalized cantilever not loaded with CBM3a) in contact with a cellulose film. The probability of observing a rupture event (Pbind) was 14% over 150 pulls, indicating minimal adhesion between the tip and cellulose surface. The adhesion forces observed varied widely, though most were below 150 pN. The rupture length distribution indicated no statistically significant mean rupture length, consistent with nonspecific adhesion.

Figure 3. Binding probability (left) between tip-immobilized MCC and cellulose surfaces was assayed using AFM in the presence (blocked, blue) or absence (initial/washed, green/red) of blocking agent (here NCs). Force vs extension curves (right) depict initial contact points with 100−200 pN forces and unbinding events with dark blue circles. The approach (green) and retraction (black) cycles are overlaid in the initial, blocked, and washed experiments. 3434

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Figure 4. MCC rupture histograms depict mean forces (left) centered around 45 pN and lengths (right) centered around 20−30 nm.

mean rupture force of 50 ± 1 pN is slightly larger than the 33.3 pN rupture force reported by Xu and co-workers19 at 16 nN/s and is well below the binding threshold for the His6−Ni(II)− NTA interaction (120 pN here and 40−500 pN elsewhere21,41,42 using different loading rates43). A significant reduction in the frequency of rupture events was also apparent after the initial stage of the experiment. This drop in binding frequency upon blocking and washing is also consistent with substantial levels of nonspecific adhesion in the initial stage of the experiment. Because we cannot rule out elevation of the mean rupture force and diminution of rupture lengths as a result of background adhesion between MCC and the cellulose surface, we sought to remove nonspecific contacts between the AFM tip and cellulose surface through the use of buffer additives. Pulling experiments were repeated in the presence of Tween-20, a nonionic detergent that should disrupt weak hydrophobic contacts between the PEG24 linker and protein with the cellulose surface. As shown in Figure 3, the addition of up to 0.05% Tween-20 resulted in a slightly lower initial Pbind of 89% but yielded a satisfactory blocking efficiency (Pbind = 34%) and a substantial return of binding upon removal of the blocking agent (Pbind = 76%). MCC−cellulose rupture force and length profiles are presented in Figure 4. We observed an initial mean rupture force of 52 ± 1 pN, a value that agrees well with the initial force in the absence of Tween-20. However, blocking with a NC suspension followed by removal of the blocking agent with pure water shifted the mean to a smaller rupture force of 45 ± 2 pN, closer to the values of Xu and co-workers.19 The initial mean rupture length of 17.5 ± 0.3 nm shifted to 28 ± 1 nm after removal of the blocking agent. (Deviations in the rupture lengths toward smaller distances is expected when considering the cone shape of the cantilever tip. Few molecules are located near the edge of the tip, where the rupture distance is likely to demonstrate the molecular length of the system.) The shift to lower rupture forces and longer rupture lengths upon addition

and removal of the blocking agent suggests that a different set of binding sites exists in the initial and washed states. Such an evolution could arise if the cellulose blocking agent reversibly inhibits specific CBM3a binding sites but irreversibly (on the time scale of the AFM experiment) disrupts nonspecific protein−cellulose interactions. Force Spectroscopy Using Galectin-3 as the Binding Protein: Elucidating Specific Ruptures. To probe this hypothesis, we exchanged MCC with the similarly sized lectin murine galectin-3 (G3, 35 kDa), a protein with no known affinity for cellulosic substrates. We have previously detailed the specific affinity (Ka ∼ 6400 M−1) of G3 for small galactosebased carbohydrates and demonstrated its suitability for force spectroscopy experiments.14,25,33,44 If the cellulose NC blocking agent does irreversibly inhibit nonspecific protein−cellulose interactions, we should initially observe G3−cellulose interactions blockable by the NC suspension, but binding should not reappear after removal of the NCs from the fluid cell. G3 was immobilized to the AFM cantilever via a NTA− Ni(II)-mediated His6 tag ligation of the protein.14,22,25,33 The tip was brought into gentle contact with cellulose for 1 s and the surface was removed at a retraction velocity of 200 nm/s. The process was repeated for 250 pulls over three cycles in the absence (initial/washed) or presence (blocked) of the cellulose NC suspension. The observed rupture morphologies for G3− cellulose interactions were nearly identical to those recorded in our MCC−cellulose experiments and the G3−lactose force curves reported previously;14,25,33 a representative rupture curve and Pbind values are shown in Figure 5. In this instance, Pbind diminished from 75% to 45% under blocked conditions, in qualitative agreement with blockable interactions. P bind increased to 60% in the washed experiment, an unexpected result given the lack of known affinity of G3 for cellulose. On the other hand, the differences in Pbind are small and may simply reflect larger than expected errors. 3435

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unexpected, as qualitative observations of a highly salt dependent binding between two very flat cognate CBM and cellulose binding interfaces clearly illustrate the possibility of a mechanically weak (i.e., low force barrier to unbinding) yet thermally stable (i.e., large thermal barrier to unbinding) binding driven predominantly by hydrophobic desolvation.34,40,45 Unable to differentiate specific from nonspecific binding using force distributions, we analyzed rupture length distributions to identify qualitative differences between MCC− cellulose and G3−cellulose ruptures (Figure 6, right). In the initial G3−cellulose unbinding experiment, two classes of rupture lengths characterized by short (17 nm) or long (50 nm) lengths were apparent. As with MCC, the two length distributions were attributed to short interactions between the linker or tip and cellulose and longer protein−cellulose interactions. Under the conditions of the blocked experiment, each class of ruptures is ablated, and gratifyingly, neither the short nor long class of ruptures appears to regenerate after removal of the blocking agent. Comparison between the G3− cellulose and MCC−cellulose rupture lengths clearly differentiates reversible and irreversible binding. The initially observed G3−cellulose binding in the expected protein− cellulose contact range of 40−60 nm was irreversibly blocked by NCs, yet the MCC−cellulose binding in this range (25−40 nm) was indeed regenerated in the washed experiment.

Figure 5. G3−cellulose force vs extension curves (left) exhibit similar morphology to MCC−cellulose curves. Binding probabilities for G3− cellulose (right) seem to be mildly reduced in the blocked experiments and moderately regenerated as the blocking agent is washed away.

To better understand the differential Pbind values between the blocked and washed G3−cellulose binding experiments, the change in rupture force and length distributions were examined under initial, blocked, and washed conditions (Figure 6). Little variation in the force distributions was observed between different pulling cycles. Strikingly, the forces observed for G3− cellulose unbinding (∼46 pN) were nearly identical to the MCC−cellulose rupture forces observed in the regenerated binding (washed) experiments (45 pN). Again, given the lack of specific affinity of G3 for cellulose, the similarity in rupture forces presumably demonstrates that nonspecific protein− cellulose contacts are mechanically similar to specific CBM3a− cellulose interactions. This observation is not entirely

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EXPERIMENTAL SECTION

General. Nano-Strip was purchased from Cyantek (Fremont, CA). TCEP and IPTG were purchased from GoldBio Technology (St. Louis, MO). Syntheses of carbamate S1 and NTA-thiol S2 are presented in the Supporting Information. All other chemicals were purchased and used without modification from Sigma-Aldrich (St. Louis, MO). XPS spectra were collected on a Kratos Analytical Axis Ultra spectrometer and analyzed using the CasaXPS platform. Cellulose film thickness was measured using a Nanometrics 210

Figure 6. G3−cellulose rupture force (left) and length (right) histograms depict rupture forces near 45 pN and rupture lengths from 10 to 100 nm. The failure to regenerate rupture length distributions in the washed experiments is likely symptomatic of the lack of specificity for G3−cellulose binding. 3436

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locations on the surface and the data collected using an automated program.39,47 The sample surface was brought into contact with the tip, held for a dwell time of 1 s, and then retracted at a constant velocity of 200 nm s−1. The force loading rate (383 ± 183 pN s−1) was determined from the cantilever spring constant (120 pN nm−1) provided by the manufacturer and the slope on the force curve before an unbinding event; see the Supporting Information of Bowers et al.33 As per our prior findings, pulls with contact forces in excess of 350 pN or below 30 pN were removed for analysis. Unblocked protein experiments (initial and washed) and His6 unbinding experiments were carried out in the presence of PBS-T (30 μL, see AFM cantilever functionalization procedure). For the blocked experiments, the flow cell was initially flooded with 30 μL of cellulose NC suspension (1.6 wt % in PBS-T, filtered through 0.2 μm PES membrane). After 10 min, the NCs were removed with PBS-T (10 × 30 μL), and the data collection was continued. Then, the NCs were removed with water (10 × 30 μL) and PBS-T (3 × 30 μL), and data collection was continued for the washed experiments. The photodetector was calibrated prior to data collection so as not to foul the tip with excessive force. The data was analyzed using a custom Matlab (MathWorks, Natick, MA) script,47 and the resulting rupture force and length histograms were fit to inverse Gaussian distributions using the Matlab curve fitting toolkit (Supporting Information). (Though the inverse Gaussian function more qualitatively matched the generated histograms, the means obtained with normal distribution functions were nearly identical in each case to the means from the Gaussian function.)

spectrometer. Contact angles were collected on an NRA C. A. model 100 Rame-Hart contact angle goniometer. AFM images were collected on a Digital Instruments Dimension 3100 scanning probe microscope and rendered using Nanoscope SPM software. Cellulose Surface Preparation. Cellulose nanocrystal suspensions were prepared and spin-cast onto a polymer-precoated silicon wafer as reported by Wågberg et al.31 AFM images were collected in tapping mode using bare Ultrasharp NSC 15/AIBS tips (Mikromasch, Lady’s Island, SC). Expression and Purification of His6-Tagged MCC. MCC was expressed and purified in a similar manner to pETscaf3, as reported by Fierobe et al.46 and described in detail in the Supporting Information. Expression and Purification of His6-Tagged Murine Galectin3. Murine galectin-3 was expressed and purified as described by Bowers et al.33 with minor modifications, as described in the Supporting Information. AFM Cantilever Functionalization. The AFM cantilevers were functionalized as described via a combined approach of Bowers et al.25 and Shestopalov et al.24 In a clean environment, AFM tips [NP, nominal spring constant of 0.120 N/nm; tip radius 20 nm (nom) to 60 nm (max); Bruker, Billerica, MA] were soaked in Nano-Strip at 75 °C for 30 s, rinsed with ultrapure water, and dipped into 5% aqueous HF for 1 min. The acid was blown away with N2, and the tip immediately coated with 10 μL of alkene S1. The tip(s) were transferred to a nitrogen-filled glovebox and irradiated with UV light for 30 min at room temperature (UVP 11sc lamp, 4400 μC cm−2 at 5 cm distance above tips). The tips were then removed from the clean room and rinsed with filtered EtOH, Milli-Q H2O, EtOH, and CH2Cl2. The tips were submerged in 50% trifluoroacetic acid in CH2Cl2 for 30 min and then rinsed with EtOH, Milli-Q H2O, EtOH, and CH2Cl2. The tips were submerged in a solution of NHS-dPEG24-Mal (15 mM, 1% Et3N in CH2Cl2; Quanta BioDesign, Powell, OH) for 2.5 h and rinsed with CH2Cl2 and EtOH. The tips were transferred to a freshly prepared solution of NTA-thiol S2 (10 mM with respect to reduced sulfhydryl, 5 mM TCEP, 10 mM aqueous sodium phosphate, pH 7.5, filtered at 0.2 μm) for 13−16 h. The tips were rinsed with Milli-Q H2O, soaked in nickel solution (50 mM NiSO4, 20 mM NaHEPES, pH 7.0) for 30 min, and rinsed with water. The tips were soaked in bind buffer (5 mM imidazole, 10 mM sodium phosphate, 0.15 M NaCl, 0.05% Tween-20, pH 7.5) for 15−30 min and then transferred to the protein solution [∼250 μL per tip; ∼40 μM protein in PBS-T (10 mM sodium phosphate, 0.14 M NaCl, 0.05% Tween-20, pH 7.4)] using a mass spectrometry vial cap placed in a covered glass weighing dish as the reaction vessel. The protein soaking step was omitted in the blank tip and His6 unbinding experiments. The tips were rinsed with phosphatebuffered saline immediately before the unbinding experiments. Functionalization of Silicon Surfaces with His6 Peptide. In a clean room, 1 cm2 silicon wafers were soaked in NanoStrip for 10 min at 75 °C, rinsed with water, and soaked in 5% aqueous HF for 5 min at room temperature. The acid was blown away under a N2 stream and the surface immediately covered with alkene S1. The surface was transferred to a N2-filled glovebox and irradiated with UV light for 2 h at room temperature (UVP 11sc lamp, 4400 μC cm−2 at 2 cm distance above surface). The surface was removed from the glovebox and clean room and rinsed with CH2Cl2, EtOH, water, EtOH, and CH2Cl2. The surface was dried under Ar and soaked in 50% TFA:CH2Cl2 for 30 min. The acid was rinsed away with water, EtOH, and CH2Cl2, and the surface was soaked in the NHS-dPEG24-Mal solution as described above for 2.5 h. The surface was rinsed with CH2Cl2, EtOH, and water and placed in a 0.2-μm-filtered solution of the CGWGGHHHHHH peptide (4 mM in 20% DMSO:water, 10 mM TCEP, pH 7.5; Supporting Information) for 16 h. The surface was loaded with NaHEPES-buffered NiSO4 as described above prior to use in His6 unbinding experiments. Unbinding Experiments. The unbinding experiments were performed as outlined by Bowers et al.33 Automated pulling experiments were carried out on a custom-built three-axis AFM composed of a MultiMode head (Digital Instruments, Santa Barbara, CA) mounted on xy- and z-positioning stages (Physik Instrumente, Auburn, MA).39,47 At least 250 pulls were generated over four random

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CONCLUSIONS The work described here offers a general approach for the study of proteins that bind immobilized surfaces not well modeled by soluble substrates. A generally useful procedure is described for the stepwise chemical modification of silicon nitride AFM cantilevers with hydrosilylation chemistry and orthogonal chemical ligation protocols to instill NTA moieties for the Ni(II)-mediated immobilization of His6-tagged proteins. Frequency analysis of the forced unbinding profile of an immobilized clostridial CBM3a containing MCC protein in complex with a nanocrystalline cellulose surface clearly demonstrated the mechanical rupture of specific MCC− cellulose interactions under a force load of 45 pN at 0.4 nN/ s over a distance of 28 nm. Specific binding could be disrupted with a suspension of cellulose nanocrystals and regenerated by the removal of the NCs with pure water. Upon repeating the experiment with murine galectin-3, which does not bind cellulose, nearly identical force−failure profiles were observed as compared to MCC. Our work offers a cautionary tale regarding the interpretation of AFM data in terms of specific binding events. Rather, several types of data must be considered in concert to develop a complete and accurate picture of molecular events at surfaces. Evaluation of rupture frequencies as a function of rupture length shows that distributions of G3-specific ruptures were not regenerated by washing, indicating that no specific interactions existed between G3 and cellulose. On the other hand, MCC− cellulose rupture length distributions could clearly be regenerated in the washed stage of the MCC−cellulose binding experiment. These data illustrate how rupture length profiles and blocking agents must be used together to differentiate specific (i.e., reversible and blockable) from nonspecific adhesion. Furthermore, our results offer the first clear indication of specific binding at the CBM3a−cellulose interface by way of accounting for nonspecific protein−cellulose contacts in the use of a galectin-3-based negative control. 3437

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functionalized surfaces; goniometric titration of NTA-terminated surfaces; table of binding probabilities from unbinding experiments; inverse Gaussian fitting results from unbinding force and extension histograms; and proposed CMB3a-cellulose binding model. This material is available free of charge via the Internet at http://pubs.acs.org.

Given the large discrepancy in reported unbinding forces for CBM−cellulose association (51 pN at 100 nN/s19 vs 6.3 nN20) and that force-derived CBM−cellulose binding energies (∼14 kcal mol−1)19 exceed protein-folding energies (∼7 kcal mol−1),48 our results demonstrate the limitations of determination of binding energies via the interpretation of binding forces alone. Rather, we suggest that changes in binding probabilities during a competitive binding titration14 offer a more robust method for determining immobilized protein− substrate binding energies via AFM. With respect to our larger objective to probe the nature of CBM3a−cellulose association and enzyme targeting in cellulosome action, our results offer important insight into the cellulose-binding mode of C. cellulolyticum CBM3a and support the binding mode put forth by Tormo, Shimon, and others.45,49 Our results clearly demonstrate that CBM3a specifically binds crystalline cellulose in a reversibly blockable and salt-dependent manner. Alternatively, our previous ITC results show that the CMB3a does not bind soluble linear oligosaccharides, at least through the pentaose, while CBM3a− avicel binding is entropically driven at 298 K, suggesting a role of hydrophobic desolvation in binding.50 Although a CBM− cellulose cocrystal structure has not been reported, an examination of the CBM3a crystal structure (PDB ID: 1g43) offers clues as to how such binding might occur.45 The lower βsheet of the protein, containing the canonical sugar-binding residues (S14, S19, Y23, W58, H61, Y70, W123, and Y140; Supporting Information), is ideally structured to interact with a perimeter ring of saccharide residues on adjacent chains and encompassing the large, flat face of a cellulose sheet; such a binding would presumably be driven largely by hydrophobic desolvation. Such a binding mode would also rationalize the lack of binding of even large linear oligosaccharides, since critical binding contacts require adjacent polymer chains. Rather, the mode put forth by Tormo and co-workers would exploit the unique crystalline structure of cellulose and offer a powerful mechanism of enforcing specificity in a milieu contaminated with myriad soluble substrates of identical linear (or primary) structure. This proposed binding mode is also consistent with the Oring theory of Bogan and Thorn,51 in which a ring of residues at the binding interface of the protein occludes water from a “hot spot” residue at the center of the protein binding site. Hot spots, first described by Clackson and Wells,52 are protein residues that account for the majority of the binding free energy in protein−protein interactions. Application of the O-ring theory to CBM3a−crystalline cellulose binding provides a molecular rationale for the apparent specificity of CBM3a for very flat carbohydrate surfaces and the lack of specificity to soluble cellodextrins. The implications of this rationale provide insight into the enzyme targeting effects proposed by Fierobe and co-workers.53−55 Thus, we suggest that future work to evaluate targeting effects in the cellulosome should focus on identifying potential hot spots for affinity on the CBM3a surface and also to evaluate the role of cellulose crystallinity for CBM3a−cellulose association.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +1-919-681-3484. Fax: +1-919-660-1591. Present Addresses §

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02142. ∥ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge generally helpful experimental input from D. A. Carlson and B. E. Watts. We acknowledge the lab of D. G. McCafferty for help with peptide synthesis. We are grateful to S. L. Craig for invaluable time on the AFM. Force spectroscopy data were collected and analyzed using Matlab code provided by M. Rivera and R. L. Clark. The Duke University Shared Materials and Instrument Facility (SMiF) provided and maintained the necessary clean environment and surface characterization tools for surface chemistry. C.M.B. was supported under the NSF award CMMI-10000724.

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ABBREVIATIONS USED Boc, tert-butyloxycarbonyl; HEPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; His6, hexahistidine; MCC, miniCipC; NHS, N-hydroxysuccinimide; NTA, nitrilotriacetate; PBS-T, phosphate-buffered saline with Tween-20; TCEP, tris(carboxyethyl)phosphine; TFA, trifluoroacetic acid; XPS, Xray photoelectron spectroscopy

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

Syntheses of S1, S2, and CGWGGHHHHHH peptide; expression and purification of recombinant proteins; MCCencoding DNA and protein sequences; UV/vis and Ellman’s test of S2-derived NTA-thiol reactivity; XPS characterization of 3438

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