Article pubs.acs.org/Biomac
Identification and Characterization of a Cellulose Binding Heptapeptide Revealed by Phage Display Jing Guo,†,▽ Jeffrey M. Catchmark,*,‡,▽ Mohamed Naseer Ali Mohamed,§,▽ Alan James Benesi,∥ Ming Tien,†,⊥ Teh-hui Kao,†,⊥ Heath D. Watts,# and James D. Kubicki#,▽ †
Intercollege Graduate Degree Program in Plant Biology, ‡Department of Agricultural and Biological Engineering, and §School of Advanced Sciences, Crystal Growth and Crystallographic Division, VIT University, Vellore-632014, India ∥ Department of Chemistry, ⊥Department of Biochemistry and Molecular Biology, #Department of Geosciences, ▽Center for NanoCellulosics, The Pennsylvania State University, University Park, Pennsylvania 16802, United States S Supporting Information *
ABSTRACT: Cellulose nanowhiskers (CNWs) were used in conjunction with phage display technology to identify polypeptides which bind the crystalline region of cellulose. A consensus peptide WHWTYYW was identified to efficiently bind the CNWs. The binding affinities of specific phage particles were assessed using biopanning assays and enzyme-linked immunosorbent assay (ELISA). The WHWTYYW peptide was synthesized and isothermal titration calorimetry (ITC) analysis showed that the peptide exhibited a binding constant of ∼105 M−1 toward the crystalline CNWs. In order to understand how the affinity of this peptide differs for noncrystalline cellulose, binding properties were characterized using cello-oligosaccharides as substrates. Binding analysis was performed using UV spectroscopy and fluorescence quenching experiments. The specific molecular interactions of the WHWTYYW peptide with cellohexaose were examined using nuclear magnetic resonance (NMR). Interactions of this peptide with crystalline cellulose were also investigated using classical molecular modeling and quantum mechanical calculations of 13C NMR chemical shifts. The NMR experiments and calculations indicate that the WHWTYYW peptide exhibits a bent structure when bound, allowing the Y5 amino acid to form a CH/π stacking interaction and H-bond with the glucose ring of cellulose.
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INTRODUCTION Cellulose binding modules (CBMs) in cellulose-hydrolyzing enzymes are domains required for efficient enzyme binding and hydrolysis of cellulose. CBMs recognize and bind to cellulose, and thus facilitate the hydrolysis activity of the whole cellulase enzyme by concentrating the catalytic domains onto the surface of the cellulose substrate.1−5 The binding mechanisms between CBMs and cellulose have been intensively studied during the past decade. Some CBMs are featured by a planar hydrophobic ligand binding surface, which is comprised of aromatic amino acid residues and it is these aromatic amino acids, W, Y and occasionally H, F, that are often involved in the binding interaction to cellulose.5−13 Other CBMs are characterized by forming clefts to accommodate single polysaccharide chains for digestion.14 As a typical protein-carbohydrate binding process, hydrogen bonding and van der Waals force, especially the stacking interaction between aromatic amino acid side chains and glucose rings, are usually involved.15 Although the structures of some CBMs have been exploited, very little is known about the structural determinants required for CBM binding to crystalline cellulose substrates, due to the complexity of the structures of cellulose and the complex formed by CBM and cellulose. In particular, although several studies have been performed on revealing the universal critical residues (aromatic © XXXX American Chemical Society
amino acids) for binding, it is still unclear whether the specific amino acid composition is responsible for the recognition of crystalline cellulose or whether a wide array of peptides are capable of interacting with crystalline cellulose.5−13,15 In order to gain better understanding of the structural determinants of peptides important for crystalline cellulose binding, we have used phage display to screen short peptides that have specific binding to crystalline cellulose. Phage display is a bioselection method that has been used to identify new peptides/proteins with desirable binding traits. Various peptide/protein libraries can be constructed and displayed on the surface of bacteriophages, and then used as sources for selection of variants with desired binding properties. This in vitro selection method provides the direct linking of genotype and phenotype during selection procedures. Because of the ease and rapidity of DNA sequence analysis, selected molecules can be identified quickly. A previous study identified a group of phage-displayed linear heptapeptides that specifically bind to microcrystalline cellulose (Avicel).16 Binding affinities (Kapp’s) of obtained phage clones Received: February 4, 2013 Revised: April 7, 2013
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which the final eluted phages were titered. The sequence of the region of each genomic DNA encoding a putative CNW-binding peptide was determined. The detailed procedure of biopanning is provided in the Supporting Information. ELISA. ELISA procedure followed Serizawa’s method.16 ELISA plate wells were coated for each clone to be characterized with 200 μL of 100 μg/mL of CNWs in 0.1 M NaHCO3, pH 8.6. Then 100 μL of phage solution with adequate concentrations dissolved in TBS were applied into wells for 1 h at room temperature. After five rinses with 200 μL of TBST (containing 0.1% Tween), horseradish peroxidaseconjugated anti-M13 bacteriophage antibodies (GE Healthcare) was applied for 1 h at room temperature with agitation. After six rinses with 200 μL of TBST, the relative amounts of bound phages were estimated by measuring the absorbance of products from substrates (2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) at 405 nm. As a negative control to identify plastic-binding phage, CNWs were omitted from some wells. Bound amounts measured as a variable directed against phage concentrations revealed phage binding constant KP. Peptide Synthesis and Concentration Determination. The consensus heptapeptide WHWTYYW was synthesized by Biomatick Corp. and purified by reverse-phase high-performance liquid chromatography (HPLC) with 98% purity. The composition of the heptapeptide was confirmed by mass spectrometry. The concentration of heptapeptide WHWTYYW was determined by UV absorbance (280 nm) using a calculated molar extinction coefficient19 of 19 630 M−1cm−1. Binding Affinity Constant Determined by Isothermal Titration Calorimetry (ITC). ITC was performed using an iTC200 MicroCalorimeter (Microcal, Northampton, MA). All samples were dissolved in the same buffer containing 50 mM Tris-HCl, pH 7.5. All solutions were thoroughly degassed under vacuum prior to measurements. The system was allowed to equilibrate for 30 min at 25 °C with stirring at 1000 rpm to ensure rapid mixing and equilibrium. For a typical titration, 5 μL titrant of 2 mM peptide (WHWTYYW) was injected into a solution of 4.2 mg/mL CNWs with 180 s between each injection. A blank experiment to evaluate the heat of dilution contributed from peptide dilution was performed at 25 °C using identical injections of peptide solution into buffer, and all ITC data were corrected for the heat of dilution by subtracting the blank from test experiment. At least three independent titration experiments were performed to determine the binding constant of peptide to CNWs. A thermodynamic profile of binding interaction was determined by fitting the data to an independent binding site interaction model. Analysis of Cellohexaose Binding to Heptapeptide by UV Absorbance. Absorbance spectra were collected on a UV-2550 UV− Vis spectrophotometer with a TCC-240A temperature-controlled cell holder (Shimadzu Corp., Japan). All spectra were collected from 200 to 400 nm with a spectral bandwidth of 1 nm, an average integration time of 0.2 s, and a data interval of 0.2 nm. Experiments were conducted at room temperature in 25 mM Tris-HCl, pH 7.5. Difference spectra were obtained by first collecting a baseline spectrum on 1 mL of 0.1 mM heptapeptide. A second scan was performed by adding a saturating amount of ligands (1 mM of cellohexaose) into 1 mL of 0.1 mM heptapeptide; the sample was left to equilibrate and then scanned. Quantification of Cello-oligosaccharides Binding to Heptapeptide by Fluorescence Quenching. All fluorescence quenching analyses were performed on a Perkin-Elmer LS-50 luminescence spectrometer (Perkin-Elmer, Norwalk, CT) with a sample cuvette holder thermostatted by a recirculating heating/cooling bath. OLIS Global Works was used for data analysis. Emission scans were performed using 5.1 μM heptapeptide WHWTYYW in the presence or absence of 1 mM ligand cello-oligosaccharide in 50 mM Tris-HCl buffer, pH 7.5. Quantitative binding experiments were performed by titrating 5 μL of concentrated cello-oligosaccharides into the peptide solution sequentially until all the binding sites of the heptapeptide were saturated. The fluorescence intensity was measured using an excitation wavelength of 280 nm and emission wavelength of 353 nm. Emission and excitation slit widths were 4 nm. The emission intensities were corrected for
were quantitatively analyzed by enzyme-linked immunosorbent assay (ELISA). In this study, two groups of cellulose-binding peptides were characterized based on the binding affinities. Group 1 heptapeptides were enriched by amino acids with lateral hydroxyl groups and end amino acids with amine groups. Group 2 peptides were enriched by aliphatic amino acids. However, it is noticed that two groups of binding sequences were identified, and authors proposed that this might be due to the fact that this cellulose substrate contained significant amorphous content precluding identification of novel peptides that bind only crystalline cellulose. In addition, no consensus sequences were identified, possibly due to the variability of the substrate, and binding mechanisms between the peptide and cellulose were not completely elucidated. Here we report the screening of short peptides (heptapeptides) that are capable of tight binding to crystalline cellulose nanowhiskers (CNWs). We found that part of the identified binding heptapeptide is contained in many CBMs found in nature.17 The binding constants and mechanisms of binding of this peptide and similar peptides were characterized. In addition, soluble cello-oligosaccharides were used as simplestructured cellulose-mimics to identify the binding region on cellulose. The molecular details of the interaction of this heptapeptide with cellulose-mimics/cellulose were determined by nuclear magnetic resonance (NMR) and molecular modeling (MM) studies, respectively. These peptides can also be potentially used as linkers or binders for many applications ranging from cellulose specific protein tags to additives in cellulose composites useful for improving mechanical or chemical properties.
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EXPERIMENTAL SECTION
Materials and Methods. CNWs and Cello-oligosaccharides. CNWs were produced from Whatman CF11 cellulose by sulfuric acid hydrolysis (63.5%, w/w) according to Bondeson et al.18 CNW preparation and characterization are described in the Supporting Information. Microcrystalline cellulose (Avicel) was used as the target for bioscreening in an earlier paper.16 However, Avicel contains significant amorphous content (with ∼65% of crystalline content, measured by X-ray diffraction (XRD)), precluding identification of novel peptides that bind specifically crystalline cellulose. Therefore, CNWs, which are mainly comprised of crystalline cellulose (with ∼90% crystalline content by XRD, Figure S1, Supporting Information) were chosen as the target in this study to better understand the binding mechanisms between the peptide and crystalline cellulose. In addition, CNWs produced in this study have similar particle sizes. The homogeneous suspension (Figures S2 and S3) is more appropriate for binding studies than that of Avicel. Cello-oligosaccharides were purchased from Seikagaku (Tokyo, Japan). Phage Display Peptide Library. The Ph.D.-7 Phage Display Peptide Library was from NEB and was biopanned for screening CNWbinding phages. Biopanning. 1.5 mL of 100 μg/mL CNWs, in 0.1 M NaHCO3 (pH 8.6), was coated onto a Petri dish. A blank experiment was conducted, and it showed that no CNWs were detached and lost from the Petri dish during the biopanning process. A 100-fold representation of the library about 2 × 1011 pfu phages diluted in 1 mL of sterile TBST [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% (v/v) Tween-20] was pipet onto a coated plate for approximately 60 min at room temperature with gentle shaking. Unbound and weakly adsorbed phages were discarded. The plates with the CNW-phage conjugates were washed 10 times with TBST. The eluted phages were titered, the rest were then amplified, and the resulting phage stock was used for a second round of biopanning, which was performed exactly as described above except for a more stringent washing condition (with 0.5% Tween). A total of three rounds of biopanning were performed after B
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background fluorescence caused by buffer and cello-oligosaccharides. The quenching of aromatic amino acids (3W, 2Y) fluorescence induced by sugar binding was observed, and the fraction of binding sites occupation (fa) was calculated following the method described by Yoon and Cowan in 2003.20 Plots of relative fluorescence ( fa) against total cello-oligosaccharide concentration were constructed. The association binding constants (Ka) were derived by a nonlinear leastsquares fit of the corrected data to a one-site binding model with ligand depletion using Origin v.7.0 software (Microcal, Northampton, MA). Analysis of Cellohexaose Binding to Heptapeptide by NMR. Samples for NMR spectroscopy were not isotopically 13C or 15N enriched. The heptapeptide sample contained 2.24 mM of peptide WHWTYYW dissolved in 1 mL of D2O (pH 5). The heptapeptide− cellohexaose mixture contained 2 mM cellohexaose and 2.24 mM heptapeptide (pH 5) in 1 mL of D2O. All NMR experiments were performed at 25 °C on a Bruker Avance-III-850 (20.0T) spectrometer equipped with a TCI-Cryo probe with enhanced sensitivity for 1H and 13 C. The cellohexaose, heptapeptide, and heptapeptide−cellohexaose conjugates in D2O solution were characterized using one-dimensional (1D) 1H NMR, two-dimensional (2D) 1H−13C HMQC,21 2D 1 H−13C HMBC,22 2D 1H−1H TOCSY,23 2D 1H−1H DQF-COSY,24 and 2D 1H−1H NOESY25experiments. The 1H and 13C chemical shifts were referenced using the heptapeptide threonine methyl Hγ2 and Cγ2 resonances (assigned to 1.22 and 21.6 ppm respectively) (Biological Magnetic Resonance Data Bank http://www.bmrb.wisc. edu/bmrb/). Molecular Modeling. This peptide model was generated using the xleap module of AMBER 10 (Assisted Model Building with Energy Refinement).26 An initial model was generated and then was subjected to a partial energy minimization of 15 000 steps. Atomic coordinates were then obtained after the relaxation of the initial model. This peptide model was heated from 0 to 325K in steps of 50 K up to 300 K and 25 K from 300 to 325 K. The heating steps was at least 50 ps with a 1 ps temperature coupling constant. These heating steps allow the peptide to fold slowly, in an attempt to avoid being trapped in a local minimum of the potential energy surface. Production molecular dynamics (MD) simulations of 100 ns at 325 K were performed followed by the heating stage. These 100 ns simulations were performed in 20 stages with each stage equal to 5 ns simulation. An integration time step of 2 fs was used. All MD simulations were fully unrestrained and carried out in the canonical ensemble using the SANDER module of AMBER 10. The ff99SB force field was employed27 for peptide, and GLYCAM0628 for cellulose. Solvation effects were incorporated using the Generalized Born model29 as implemented30 in AMBER. The SHAKE algorithm31 was turned on and applied to all H atoms to fix the bonds involving H atoms. Time-averaged structure of the folded peptide (Figure S4) obtained through the simulations described above was used to study the possible interactions with the cellulose model. A starting geometry of cellulose model (Figure S5) was generated from the X-ray crystal structure.32 Reducing-ends of the glucan chains were terminated with methyl groups. A peptide-cellulose complex molecular model was manually generated from the models shown in Figure S4 and S5 using the xleap module of AMBER. The peptide−cellulose complex was then solvated using a truncated octahedron shell of TIP333 H2O molecules. The complex model was heated from 0 to 325K using constant volume periodic boundary conditions, with the SHAKE algorithm turned on and applied to all H atoms. Simulation of 5 ps with a 0.5 fs time step was carried out on the complex model. Langevin dynamics were used to control the temperature using a collision frequency of 1.0 ps−1. After heating the system with this protocol, 1.225 ns equilibration MD simulations were performed by restraining the cellulose model. Restraints were considered in order to ensure the integrity of the cellulose structure upon peptide interaction. Throughout the simulations, periodic boundary conditions were applied. The particle mesh Ewald (PME) method was used to compute long-range electrostatic interactions.34 All the MD results were processed using the ptraj module of AMBER 10 for the analysis, and visualization of the MD trajectory was performed using VMD.35
The mPW1PW9136 density functionals with the 6-31G(d) basis set (37and references therein) and gauge-including atomic orbitals (38 and references therein) were used to calculate 13C NMR isotropic chemical shieldings. The multistandard method39 was used to calculate the δ13C chemical shifts. Theoretical shifts were obtained using39 δ x calc = σref − σx + δexp ,ref Here σref is the calculated shielding tensor C from the threonine γ C atom, σx is the shielding tensor for sp3 C in the model of interest, and δexp,ref is the experimental chemical shift of C (21.6 ppm) from threonine γ C. Calculated shifts for carbonyl C are referenced to N(2)acetylglycinamide (average carbonyl shift = 170.4 ppm). The computed NMR chemical shifts were compared with the experimentally observed values of the corresponding residues. δ13C NMR calculations were also performed on these model structures using the mPW1PW9136 method, the gauge-including atomic orbital method of Wolinski et al., (1990)38to obtain isotropic chemical shielding, and the multireference method of Sarotti et al., (2009)39 to obtain chemical shifts relative to tetramethylsilane (TMS).39
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RESULTS AND DISCUSSION Heptapeptide WHWTYYW Binding to CNWs Was Identified by Phage Display. The Ph.D.-7 phage display peptide library40 from NEB was chosen to identify peptides with affinity toward CNWs. The starting Ph.D.-7 library has a complexity of ∼2 × 109 independent phage clones, which can be compared with ∼1.3 × 109 different sequence combinations that are possible for a heptapeptide so that the initial library includes essentially all possible 7-mer amino acid sequences. An affinity biopanning was applied by incubating a library of phage particles with surface-coated CNWs. Unbound and weakly absorbed phages were washed out, and only the CNWspecific bound phage particles were retained, eluted, and amplified for another round of biopanning. During each round of biopanning, a small amount of input phage, biopanned elute, and amplified elute phage particles were saved for titering. The number of phages observed in these samples would provide an indicator for the enrichment of phage populations. The phages were quantified both after elution and amplification to enable the use of equal input amounts (∼2 × 1011 phages) in each round of biopanning. The enrichment of the pool in favor of binding to CNWs should result in an increase in yield depending on the biopanning rounds, which can be estimated by the amount of eluted phages relative to input phages. The yields tended to increase with increased rounds of biopanning. Titering data are shown in Table S1. Compared to the titers in Round 1, the amount of eluted phages in Rounds 2 and 3 were 1.3 and 40.6 times higher, respectively, indicating that enrichment for CNW-binding phages occurred. A drastic enrichment was observed in the third round of biopanning with an enrichment of phages by a factor of 40 compared to the first-round titers, as shown in Figure S6. After the third round of biopanning, 43 phage plaques were chosen randomly and purified. Sequencing the appropriate regions of the genomes of phage revealed the identity of putative CNW-binding peptides. It was found that among the 43 phages examined, 9 phages displayed an identical heptapeptide WHWTYYW, which is considered the consensus sequence identified by this bioscreening process. They accounted for a considerable percentage (∼21.9%) of the total identified phages. Another peptide, WHWRAWY, showing high similarities to the consensus peptide sequence also occurred three times, suggesting that these peptides, which were highly enriched in the pool, preferably and specifically C
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particles were incubated with surface-coated CNWs in TBST. After mixing at room temperature for 60 min, unbound phages were washed and removed. Bound phages were eluted from the CNWs. A small amount of phage elute was titered, and the number of phage colonies were counted and then multiplied by the dilution factor. The same experiment was also conducted on a phage expressing WHWRAWY, a similar sequence to the consensus sequence. In addition, a control experiment on phage displaying TALVMPG (without any aromatic amino acid residues) was performed as well. It is shown in Table S3 that a much higher amount of elute phages expressed peptide WHWTYYW than the control, which confirmed the result of our phage display selection experiment that WHWTYYW was better suited for binding to CNWs. For the phages expressing WHWRAWY, compared to the consensus sequence WHWTYYW, a smaller amount of elute was detected, while still much higher than the control phage peptide. It reveals that the conserved CNW-binding motif contained within the phage is essential for binding to CNWs. In the case of WHWRAWY, small differences from the consensus sequence WHWTYYW results in a loss of elute phages. However, a stronger binding affinity relative to control phage still was observed, probably due to the presence of four aromatic amino acids. The binding affinity of obtained phage expressing consensus sequence WHWTYYW with CNWs was quantitatively analyzed by ELISA (Figure 1). Another two phages displaying similar
binded to the highly crystalline CNWs. In this consensus sequence, the aromatic amino acids (W, Y) are predominant. They compose five amino acids out of seven, and the aromatic amino acids are highly conserved among all the peptide sequences examined (a total of 31 out of 43, Table S2), which supports the hypothesis based on research involving natural CBMs that aromatic amino acids play a critical role in cellulose glucose ring stacking interactions needed for binding.4,6,8,12,17 Therefore this result confirms that the aromatic amino acids are essential for the crystalline cellulose binding. Biopanning was stopped after three rounds of phage selection. Since the Ph.D.-7 phage display library contains ∼2 × 109 different clones, theoretically, the eluted pool of phage should be fully enriched of binding sequences after only two or three rounds of biopanning. Further rounds of amplification and biopanning will not promote further consensus sequence enrichment. Instead, it might result in selection of phages that have a growth advantage over the Ph.D.-7 library phage. On the other hand, the stringency of the screening of binding experiments was enhanced by increasing the concentration of Tween-20 to prevent the nonspecific binding interactions. This should result in an increased number of conserved amino acids among the phages.41 To determine which amino acids were enriched after biopanning, the frequency of occurrence of the 20 amino acids in the selected phages was calculated by quantifying the number of times each amino acid occurred in the 43 heptapeptides (Figure S7). Aromatic amino acids W and Y are highly enriched in these displayed peptides, among which W residues are present in the most abundance. Y residues are also abundantly present, although a little less than the occurrence of W, indicating the high involvement of W and Y in the crystalline cellulose substrate recognition and binding. This result is consistent with previous reports that the binding affinity mainly arises from the aromatic ring-glucose ring polar stacking interaction.4,12,17 Furthermore, this is also in great agreement with another study that the most evolutionarily conserved amino acids in natural CBM sequences focus on W and Y.17 F residues are relatively less abundant in the displayed peptides, suggesting the less preferable selection of F for the polar stacking interaction. The second dominant amino acid residue is P, which could provide a turn/kink in peptide structure. The presence of P probably helps to reduce the structural flexibility of the heptapeptide, thus providing a constraint configuration for ligand binding. The polar basic residues H are enriched in the displayed peptides as well. It is possible that the stacking interaction between pyranose ring and H imidazole ring provide additional stacking binding force besides the aromatic amino acids. Additionally, the positive charge on H may interact with any negatively charged sulfate groups present on the CNWs from hydrolysis. The relative abundance of T, Y, and S residues might be explained by the extra hydroxyl groups of these polar residues involved in cellulose binding by forming hydrogen bonding to cellulose. Binding of Heptapeptide to CNWs Was Confirmed by Single-Phage Biopanning Assay and ELISA. To confirm that the displayed peptides interact specifically with CNWs, the binding of individual phage clones was tested by biopanning assay and ELISA. Since the phage particle harboring peptide WHWTYYW was preferentially screened and enriched during biopanning, the binding efficiency of this single phage was examined by biopanning. Approximately 1011 pfu/mL of these phage
Figure 1. The affinities of phages selected from the phage display library for binding CNWs by ELISA. The data reported are the averages and standard deviations of three independent experiments.
sequences of WHWRAWY and WHWRAFF were chosen as well, and a phage with TPPPQAA (without any aromatic amino acids) was randomly chosen from the eluted phages as a negative control. The binding constant (Kp) of specific phage particle was estimated from dependence of bound phages against phage concentrations by a Langmuir fitting. There are significant trends of the amino acids with respect to affinity and selectivity depending on the peptide sequence displayed, indicating that Kp is governed by the specific peptide sequence. The phages with consensus peptide WHWTYYW shows significantly greater binding affinity than the control phage expressing TPPPQQA. This observation suggests that specific affinities exist between aromatic amino acids and crystalline cellulose. A larger amount of phage particles harboring WHWTWWY was found to bind onto CNWs compared to the phages with a similar sequence WHWRAFF, implying a preference of W/Y to F in crystalline cellulose recognition and binding. On the other hand, a slightly higher binding affinity D
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was observed for phages displaying WHWRAWY other than the ones displaying WHWTYYW. This could be explained by the electrostatic interaction between positively charged R and negatively charged CNWs. Affinity comparison of structurally similar sequences suggests variations in binding specificities, but all are based on key aromatic amino acid residues. The Kp against CNWs was relatively larger when greater amounts of aromatic amino acids and/or amino acids with hydrogen donor side chains were included in the peptides. From the single phage biopanning and ELISA analyses described above, the binding specificity of the phage-displayed peptide WHWTYYW suggests that this segment might be a major determinant for interactions with crystalline cellulose substrates. To further investigate whether this peptide is sufficient to mediate interaction with cellulose and to confirm the specific binding of isolated peptide to cellulose/cellulosemimics, a peptide corresponding to the displayed peptide WHWTYYW was independently synthesized to evaluate the binding without the presence of the phage particle. Peptide was subsequently purified by reverse-phase HPLC, and its composition was confirmed by mass spectrometry. Binding of Heptapeptide WHWTYYW to CNWs Characterized by ITC. To quantify the binding affinity and thermodynamic change of peptide WHWTYYW interacting with CNWs, ITC experiments were carried out with the synthesized peptide. By titrating aliquots of peptides into CNW suspensions, the data on heat change versus molar ratio of peptides to CNWs have been fit with a theoretical curve that models a set of identical peptide binding sites on the CNWs (Figure 2) (Alternative binding models were also explored, but this model had the best fit). The downward ITC titration peaks demonstrate that the association between peptides and CNWs is an exothermic reaction or an enthalpically driven process. The parameters derived from the ITC titrations are summarized in Table 1. The enthalpy (ΔH) and entropy (ΔS) changes of binding between the peptides and CNWs are the same within experimental uncertainty. Thus, the adsorption reaction is driven by both the π-stacking, van der Waals forces and release of H2O molecules during this hydrophobic interaction. The more hydrophobic the peptide, the larger the number of H2O molecules displaced from substrate (i.e., the more positive the ΔS of adsorption), which indicates direct contact with the substrate. Also, the number of hydrophobic residues determines the characteristics of the overall peptides and the binding mechanism with their target. Thus, although each residue interaction has a weak enthalpic contribution and a small entropic contribution, these contributions are additive, so for a number of interacting residues as suggested in this study, the overall Gibbs free energy of interaction can be significant. The binding affinity constant (Ka) was ∼105 M−1. Compared to natural CBMs, which can have Ka as high as 107 ∼109 M−1, small peptides, such as the heptapeptide studied here, do not achieve extremely high Ka values, possibly due to the absence of a tertiary structure. The binding affinity of heptapeptide freed from phage body measured by ITC was much smaller than the quantitative data obtained from ELISA. This is due to the fact that ELISA might overestimate the affinity because peptides with huge phage particles might adsorb onto CNWs nonspecifically. In addition, five copies of heptapeptide displayed on one phage particle might strengthen the affinity to CNWs through multiple interactions. Accordingly, the binding constant estimated by ITC is expected to be more realistic.
Figure 2. Representative calorimetric isotherms of binding of peptide WHWTYYW to CNWs. The titrations were performed in 50 mM Tris-HCl, pH 7.5, at 25 °C. The upper panels show the raw data of calorimetric titration of peptides into CNW suspension, and the lower panels display the integrated injection heats from the upper graphs, corrected by heat of dilution. The integrated data were fit to an independent binding site model.
However, the molarity of the CNW solution is also an estimate, which may also impact the accuracy of the ITC results. The binding energy of the peptide WHWTYYW-cellulose model was computed from the MD simulated complex using an expression Ebinding energy = Ecomplex − EWHWTYYW − Ecellulose model. For the calculation, we considered the time (0.3−0.75 ns) averaged equilibrium structure. The calculated binding energy is −9 kcal/mol, which is similar to the measured value. We conclude from this that the molecular model is at least consistent with the experimental thermodynamic measurements. This model will be compared further with NMR results below. Binding of Heptapeptide to Cello-oligosaccharides via Glucose Ring-Aromatic Acid Stacking Interaction Was Detected by UV Absorption and Fluorescence Quenching. Fluorescence quenching studies were carried out to determine binding constant and to identify minimum binding motif in cellulose for heptapeptide recognition. Here cello-oligosaccharides were used as soluble structural representatives of cellulose. Glucose and linear cello-oligosaccharides from cellobiose to cellohexaose with six sugar rings were tested since cellohexaose is the longest cello-oligosaccharide that is soluble in water. If there is binding between the heptapeptide and any cello-oligosaccharide, a complex would be expected to form and results in perturbations in both the UV absorption spectrum and intrinsic fluorescence emission spectrum of the heptapeptide. E
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Table 1. Thermodynamics of Heptapeptide Binding to CNWs at 25 °C in 50 mM Tris-HCl (pH 7.5) Ka (M−1 × 10−5)
n
ΔH (kcal/mol)
ΔG (kcal/mol)
TΔS (kcal/mol)
1.61 ± 0.9
7.5 ± 1.0
−(3.8 ± 1.2)
−(7.0 ± 0.3)
3.2 ± 1.0
Triplicate titrations were performed, and errors are the standard deviations determined from three independent experiments.
As shown in Figure S8, the UV absorption spectrum of heptapeptide was perturbed upon cellohexaose binding as indicated by a reduction in the absorbance (relative to the unbound heptapeptide) at 220 and 280 nm. This result suggests that the heptapeptide selected by phage display can bind to cellulose via glucose ring-aromatic amino acid interactions. To further identify the binding motif on cellulose, the binding of several soluble cello-oligosaccharides to this heptapeptide were determined by titrating cello-oligosaccharides into peptide solution and monitoring the quenching of fluorescence emission spectrum of the heptapeptide. The binding of cellohexaose to the heptapeptide caused a reduction in the emission intensity (Figure S9A). Quantitative studies by fluorescence titrations showed an affinity of approximately 4.96 × 104 M−1 for cellohexaose and a preference for cellopentaose of 9.06 × 104 M−1 as opposed to cellohexaose (Figure S9B and Table S3), implying that the presence of one more glucose ring did not appear to confer any advantage to binding. This heptapeptide preferred cellohexaose and cellopentaose over cello-oligosaccharides with less glucose rings by factors of ∼5to 30-fold. The increased affinity suggests the specific involvement of at least five glucose rings in a linear binding configuration. Binding of Heptapeptide to Cellohexaose by NMR and MM. 1H−1H TOCSY, 1H−13C HMQC, and 1H−13C HMBC data were used to make 1H and 13C spectral assignments for the WHWTYYW heptapeptide sample and for the heptapeptide in the heptapeptide/cellohexaose mixture. The TOCSY data (Figure S10) for the heptapeptide clearly identify scalar coupled α and β hydrogens of each amino acid and separately scalar coupled aromatic hydrogens of each amino acid. The HMQC and HMBC data (Figures 3 and 4, respectively) independently provide the same 1H assignments (it was not possible to assign the W aromatic spin systems with the HMQC and HMBC data alone), and all of the 13C assignments including the quaternary carbons such as CO carbons and bridgehead aromatic carbons. The HMBC data for the heptapeptide and heptapeptide in complex with cellohexaose were used to establish connectivity between adjacent amino acids by means of the long-range (2JHC and 3JHC) couplings between the α H’s and the CO’s of their own and preceding amino acid CO’s, respectively (Figure 4a). A useful comparison was provided by the HMBC data for β H’s (not shown), which give cross peaks only for the long-range (3JHC) couplings to the CO’s of their own residue. The chemical shift values tabulated in the Biological Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu/bmrb/) are consistent with the assignments. The resulting α, β, and CO 1 H and 13C assignments are shown in Table 3. The 1H−1H NOESY data were used to characterize changes in interproton distances for the heptapeptide/cellohexaose mixture in comparison to the heptapeptide alone (Figures 5 and 6). The solution conformation of isolated heptapeptide was examined using 2D 1H−13C HMQC, 1H−13C HMBC, and 1 H−1H NOESY. Several cross-peaks indicate a close proximity of W3 with Y6/Y5 (dashed contours in Figures 3a, 4a, 5, and
Figure 3. 1H−13C HMQC spectrum of directly bonded C−H pairs of the heptapeptide (dashed contours) and of the heptapeptide/ cellohexaose mixture (solid contours). (a) α region and (b) β region. The projection shows the 1H 1D spectrum of the heptapeptide/ complex mixture.
6). Accordingly, a kink is expected to form in this heptapeptide structure between T4 and Y5 to introduce a coil/turn structure in this peptide. We hypothesize that this kink would help position W3 and Y6/Y5 to make it possible for them to interact. From the equilibrium average structure (Figure 7) of the MD simulations, we also found that the W3 Hα and Y6 Hβ distance decreases to ≈4 Å, which indicates the close proximity of these two residues. Protein and carbohydrate binding may be mediated by hydrogen bonding and van der Waals interaction.14,17,42−44 In this phage displayed-heptapeptide, van der Waals interaction possibly exists between W, Y and the glucose rings. In addition, hydrogen bonding would be expected between OH groups of glucose rings and polar groups of the heptapeptide such as OH, CON, and NH, alternatively, between the OH of T/Y side chain and the backbone of cellulose chain. The NMR results show sugar-induced conformational changes in the heptapeptide. The largest changes in chemical shift for the heptapeptide/ cellohexaose mixture were observed for W7 and Y6 (Table 3), with smaller changes for H2 and Y5. The large changes in the HMQC W7α/Cα and in the HMBC W7α/CO crosspeak shifts are shown in Figures 3a and 4a, respectively. The changes in chemical shift are also evident in Figures 3b, 4b, and 5. Also important is the total loss of Y6α/CO crosspeaks (Figure 4a) for the heptapeptide/cellohexaose mixture, and the total loss of F
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Figure 5. 1H−1H NOESY spectra of the heptapeptide (dashed contours) and of the heptapeptide/cellohexaose mixture (solid contours). (a) α to β cross peaks. (b) Aromatic to α crosspeaks. Projections show the 1D 1H spectrum of the heptapeptide/ cellohexaose mixture.
Figure 4. H− C HMBC spectra of JCH and JCH “long-range” C−H pairs of the heptapeptide (dashed contours) and of the heptapeptide/ cellohexaose mixture (solid contours). (a) Crosspeaks between α H’s and CO C’s. (b) Crosspeaks between β H’s and aromatic carbons. The projections show the 1H 1D spectrum of the heptapeptide/ complex mixture. 1
13
2
3
Table 3. NMR Assignments of α, β, and CO 13C and 1H Chemical Shifts for the Amino Acid Residues of the Heptapeptide WHWTYYW in the Absence and Presence of Cellohexaose at 25 °C residue Trp (W)1 His (H)2 Trp (W)3 Thr (T)4 Tyr (Y)5 Tyr (Y)6 Trp (W)7
peptide complex peptide complex peptide complex peptide complex peptide complex peptide complex peptide complex
α1H
β1H
β1H
α13C
β13C
4.50 4.49 4.83 4.80 4.77 4.78 4.43 4.42 4.61 4.60 4.75 4.75 4.91 4.81
3.55 3.54 3.35 3.33 3.42 3.42 4.22 4.18 2.98 2.96 3.17 3.21 3.60 3.58
3.44 3.44 3.27 3.25 3.42 3.42 4.22 4.19 2.98 2.96 3.04 2.99 3.50 3.47
56.5 56.7 55.4 55.1 57.9 58.3 62 62 58.1 58.5 57.9 58.4 57.4 59.3
30.0 30.3 30.0 30.2 30.0 30.3 70.4 70.9 38.5 39.4 38.8 39.5 30.0 30.7
13
CO
172.6 172.7 173.1 173.3 176.5 176.8 173.7 173.7 175.2 175.5 175.2 174.8 177.5 180.6
Significant chemical shift changes of amino acid residues between isolated peptide and conjugated peptide are denoted in bold. Figure 6. 1H−1H NOESY spectra of the heptapeptide (dashed contours) and of the heptapeptide/cellohexaose mixture (solid contours). (a) Aromatic to β crosspeaks. (b) α to aromatic crosspeaks. Projections show the 1D 1H spectrum of the heptapeptide/ cellohexaose mixture.
JHC coupling crosspeaks between the α H’s and preceding residue CO’s of the heptapeptide/cellohexaose mixture. These findings suggest that there is faster relaxation (and therefore faster loss of crosspeak intensity) during the HMBC mixing time (50 ms) for the heptapeptide/cellohexaose mixture than for the heptapeptide alone, as would be expected if the heptapeptide spends part of its time bound to the cellohexaose.
3
If it is assumed that the association reaction rate between heptapeptide and cellohexaose is diffusion-limited, the relatively G
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Figure 7. Time-averaged equilibrium structure of isolated WHWTYYW peptide. In (a), 4.01 Å indicates the distance between Hα of W3 and Hβ of Y6 and 2.6 Å represents the distance between Hα of W3 and backbone carbonyl oxygen of Y6. (b) Shows the Hα-Hα distance (4.5 Å) of the residues W3 and W7.
low Ka value indicates that the complex is short-lived (ca. ∼1 μs). Thus, it was anticipated that the heptapeptide-cellohexaose sample would yield NMR spectra that show chemical shifts that are averaged by fast exchange between the bound and unbound values. This expectation was verified by the absence of discernible NOESY cross peaks between the heptapeptide and the cellohexaose in the mixture. The most notable changes in the NOESY spectra are apparent in the α to aromatic expansion, Figure 6b. A crosspeak between T4α and Y5ε (and possibly Y6ε) is evident in the mixture, but not in the heptapeptide alone. Similarly, there is also a new crosspeak between T4α and Y5δ (or possibly Y6δ) in the mixture that is not present in the heptapeptide alone. Interproton distances reported in Table 4 also indicating a possible crosspeak between T4α and Y5δ (5.53 Å). It is also noted from Table 4 and Figure S11 that T4α shows possible interaction with W3α and W3ζ. To test the hypothesis of amino acid residues binding that are involved in carbohydrate recognition, the WHWTYYWcellulose model complex attained through the MD simulations was analyzed. From the structural analyses, we noted that an aromatic ring of Y5 aligns with the α face of the glucose residue of the cellulose model (Figure S12a). This type of stacking has been called CH/π stacking. The interplane spacing in the present complex ranges from 2.6 to 3.1 Å (Figure S12a), which agrees well with other similar systems.45,46 In addition to inter peptide-cellulose Y CH/π stacking, we also noted an intrapeptide−cellulose weak CH···O H-bond. The H-bond donor in this case comes from the cellulose glucose residue and the acceptor atom is the carbonyl O of Y5. From the analysis of the MD trajectory of the complex, the peptide slides along the cellulose surface by exchanging the CH/π contacts among the aromatic groups. Another model structure shown in Figure S12b arises due to different environments on the cellulose surface (hydrophilic and hydrophobic nature of the glucan chains). The hydrophilic region is predominant in the region between the glucan chains, i.e., two glucan chains are separated by a hydrophilic region formed by sugar hydroxyls. Destabilization of CH/π interactions in the structure in Figure S12b support this hypothesis. Therefore, it is interesting to note here that the WHWTYYW is not locked into a single binding region of the cellulose surface; instead it hops on the cellulose surface from one glucan chain to the other.
To study the close proximity of aromatic groups with respect to the α face (H1, H3 and H5) of the sugar rings radial distribution of Y−Cγ···H1−Cellulose, Y−Cγ···H3−Cellulose, Y−Cγ···H5-Cellulose and the Y−Cδ2−H (1, 3, and 5) pairs were analyzed using the pair distribution analysis plug-in of VMD. This analysis is shown in Figure 8. From Figure 8 it is clear that the probability of stacking for Y5 with sugar residues of cellulose is appreciably larger than other aromatic groups. As expected for fast exchange between bound and free status, the interaction between the nuclei of the heptapeptide and cellohexaose was not directly detectable in the 2D NOESY experiments. However, changes in the structure of the heptapeptide were observable. Figures 5 and 6 show changes of NOSEY spectra of heptapeptide complex with cellohexaose compared to native peptide. The binding of cellohexaose to peptide induced some structural changes relative to the native peptide structure. Changes in the NOESY crosspeaks for Y5, Y6, W7, T4 and H2 were detected (Figures 5 and 6). Multistandard 13C NMR chemical shifts using quantum mechanical method at mPW1PW91/6-31G(d) basis set were calculated and were compared (Figure 9) with the data obtained through experiment (Table 3). The changes between the 13C chemical shifts of complex and peptide obtained both from theory and experiment are listed in Table 5. This comparison will be useful to test the reproducibility of experimental 13C shifts by the quantum mechanical method for the model obtained from MD simulations. The multistandard NMR calculations with mPW1PW91/6-31G(d) method fairly reproduced the experimental data as evident from the correlation R2 value (0.998) and nearly a “unit” slope. From the data reported in Table 5 it is noted that the change in shifts of α13C, β13C carbons of Y5 residue followed the same trend in both experiment and calculation even though the calculated α13C shift is relatively smaller than that observed experimentally. Furthermore, the calculated β13C shift was almost the same as the experimentally observed one. Calculated shifts for W7, however, showed significant difference in shifts obtained from experiment. This discrepancy is probably due to the noninteracting configuration of W7 of peptide with the cellulose in the MD simulation and suggests that W7 should be interacting more closely with the cellulose (Figure S12a). H
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8.49 6.74 5.62 6.17 10.98 8.42 7.42 5.27 T4/Hα H2/Hα
a Values in parentheses correspond to isolated peptide. †Average structure is the structure obtained by minimizing the averaged structures between the simulations period mentioned in the table. See Figure S12a,b for interacting residues of cellulose and peptide complex.
5.64 10.28 3.77 9.13 8.60 9.92
7.47 4.13
6.83 (3.61) 10.43 (8.97) 3.67 (4.02) 8.15 (7.97) 7.47 (8.08) 4.11 (3.91) 8.13 (8.08) 10.09 (7.94) 8.49 (8.89) 6.86 (5.70) 5.53 (5.20) 6.29 (6.09) 7.27 (7.04) 5.32 (6.05) T4/Hα H2/Hα
10.97 (11.35) 8.44 (7.70)
W3 W1 W7 W3 W1 Y6/Y5Hα Y6/Hδ Y5/Hδ Y6/Hε Y5/Hε residues
Table 4. 1H−1H Distances of W1, H2, W3, T4, Y5, Y6 and W7 in the Mixturea
Article
†Average Minimized Structure (0.3−0.75 ns) 7.46(7.50)/ 4.54(4.53) 9.79 (9.42) 4.85 (4.75) 7.39(7.35)/ 8.12(7.93) 4.54 (4.57) 4.24 (4.32) †Average Minimized Structure (1.050−1.225 ns) 7.45/4.55 9.77 4.83 7.35/8.04 4.54 4.24
Hα
Hζ3
W7
Biomacromolecules
Figure 8. Radial distribution functions of atoms Caromatic···Hsugar pairs. Maximum distribution for Y5 indicates the stacking with the sugar α face compared to other aromatic groups.
Figure 9. Correlation of experimental and calculated (using mPW1PW91/6-31G(d)) 13C chemical shifts of α, β, and carbonyl (C′) carbons of Y5, Y6, and W7 residues. Here R2 is the correlation coefficient between the experimental and calculated shifts.
Table 5. Comparison of Relative Changes in Calculated 13C Chemical Shifts of α, β, and Carbonyl (C′) Carbons of Y5, Y6, and W7 of WHWTYYW Peptide and Its Complex with That of the Experimentally Observed Data Shown (Red Font) in Table 3 Relative change in 13C chemical shift (δcomplex−δWHWTYYW) α13C
β13C
13
CO
residue
exptl.
cal.
exptl.
cal.
exptl.
cal.
Tyr (Y)5 Tyr (Y)6 Trp (W)7
0.4 0.5 1.9
0.02 0.8 0.02
0.9 0.7 0.7
0.8 0.1 0.2
0.3 0.4 3.1
0.1 0.2 0.05
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CONCLUSION By screening a Ph.D.-7 phage display library, we have successfully identified a heptapeptide that binds crystalline CNWs with significant binding preference. This peptide WHWTYYW appears to be essential for the affinity and selectivity for CNWs. In particular, the five aromatic amino acids in the consensus sequence indicate preferential involvement of aromatic amino acids in the binding to crystalline cellulose. The observations suggest that the screened heptapeptides may utilize well-known van der Waals interaction and hydrogen bonding to potentially recognize crystalline cellulose, as are commonly found in natural CBM-cellulose I
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Notes
binding. Single phage binding assay confirmed that this heptapeptide and similar peptides binded to CNWs better than the peptides without any aromatic amino acids. ELISA revealed that specific affinities for CNWs were obtained for the phage pool containing this peptide and similar sequences. On the other hand, even though only one consensus sequence was screened, other similar sequenced peptides also exhibit comparable, although with lower binding affinity in terms of the single phage binding assay and ELISA analysis. This observation supports the hypothesis that the library has been selected for common regular structures against crystalline cellulose, rather than one specific structure. This identified peptide had been synthesized, and its binding activity to crystalline cellulose was examined by ITC with a binding constant of ∼105 M−1. Compared to natural CBMs, which can have Ka as high as 107−109 M−1, short peptides, like the heptapeptide studied here, seem to be unable to achieve extremely high Ka possibly due to the absence of a tertiary structure. The folded peptide spans approximately five glucose rings in length. Therefore, the minimum binding motif on cellulose for the heptapeptide binding was demonstrated to be at least five glucose rings with the affinity up to 105 M−1. The binding process was revealed to some extent by the comparison of NMR of native peptide and bound peptide in mixture with cellohexaose, as well as MM studies, which suggested that in aqueous solution, the heptapeptide adopted a folded structure to allow Y5 aromatic ring to form CH/π stacking interaction with the glucose ring of cellulose and other aromatic residues of the heptapeptide. In addition, hydrogen bonds were likely to be formed between side chain of Y5 and cellulose. A search of the CBM database revealed that the entire consensus sequence obtained was not conserved among the natural CBMs, implying that the phage displayed peptides were novel peptides. However, the YYW motif has been shown to be present in a wide variety of natural CBMs, indicating that two or more adjacent aromatic amino acids are important for binding to crystalline cellulose. The consensus peptide appears to be a promising architecture for the creation of small peptide linkers with cellulose-binding activities, with the potential for improvement of binding affinities by using peptides with a larger number of amino acids. This is the first report on the binding of CNWs with short peptides by phage display in combination with MM techniques. Such cellulose-binding peptides could be further used in many applications such as materials synthesis, purification, and even possibly enzyme engineering. Future work on understanding the roles of individual amino acids or specific peptide sequences or structures may enable peptides to be engineered with desired binding characteristics.
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors express their thanks to Dr. Wenbin Luo for her valuable time during NMR data analysis. Computational support was provided by the Research Computing and Cyberinfrastructure (RCC) group at The Pennsylvania State University. This work was funded by United State Department of Agriculture National Research Initiative (USDA-NRI) Competitive Grants Program. Grant #: [2007-35504-18339]. Funding from the United States Department of Agriculture (USDA) through the Grant “Improved Sustainable Cellulosic Materials Assembled Using Engineered Molecular Linkers” is gratefully acknowledged.
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ASSOCIATED CONTENT
S Supporting Information *
Ligand (CNW) preparation, characterization and biopanning procedures are described in detail in the Supporting Information. Some supplementary results are also included. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: 001-814-863-0414. J
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K
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