Metal Chelation Dynamically Regulates the Mechanical Properties of

Oct 4, 2016 - chelation-induced shift of the conformational equilibrium of the MEP and consequently the effective cross-linking density of the hydroge...
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Metal Chelation Dynamically Regulates the Mechanical Properties of Engineered Protein Hydrogels Na Kong, Linglan Fu, Qing Peng, and Hongbin Li* Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada ABSTRACT: Engineering protein hydrogels with dynamically tunable mechanical and physical properties is of great interest due to their potential applications in biomedical engineering and mechanobiology. In our recent work, we engineered a novel dynamic protein hydrogel using a redox responsive, mutually exclusive protein (MEP)-based folding switch as the building block. By modulating the redox potential, the MEP-based folding switch can switch its conformation between two distinct states, leading to a significant change of the proteins’ effective contour length of the polypeptide chain and an effective change of the cross-linking density of the hydrogel network (Kong, N. et al. Adv. Funct. Mater. 2014, 24, 7310). Building upon this work, here we report an engineered metal-chelation based method to dynamically regulate mechanical and physical properties of MEP-based protein hydrogels. We engineered a bihistidine metal binding motif in the host domain of the MEP. The binding of bivalent ions (such as Ni2+) enhances the thermodynamic stability of the host domain and results in the shift of the conformational equilibrium between the two mutually exclusive conformations of the MEP. Thus, the bihistidine mutant can serve as a metal ion responsive-folding switch to regulate the conformational equilibrium of the MEP. Using this bihistidine mutant of MEP as building blocks, we engineered chemically cross-linked protein hydrogels. We found that the mechanical and physical properties (including Young’s modulus, resilience, and swelling degree) of this hydrogel can be regulated by metal chelation in a continuous and reversible fashion. This dynamic change is due to the metal chelation-induced shift of the conformational equilibrium of the MEP and consequently the effective cross-linking density of the hydrogel. Our results demonstrate a general strategy to engineer MEP-based dynamic protein hydrogels that may find applications in mechanobiology and tissue engineering. KEYWORDS: protein hydrogels, metal chelation, dynamic hydrogels, mutually exclusive protein, mechanical properties

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INTRODUCTION Because of the ability to precisely control the structure and function of proteins entailed by protein engineering, we have witnessed an increasing interest in engineering protein-based hydrogels with specific properties for a myriad potential applications in biomedical engineering in recent years.1−5 For tissue engineering applications, protein hydrogels provide a biomimetic three-dimensional environment, which is often static, for cells. To better mimic the dynamic nature of the cell as well as extracellular matrix, engineering dynamic protein hydrogels whose physical, chemical, as well as mechanical properties can be regulated dynamically in response to external stimuli has attracted considerable interest.6−8 Conformational changes, which are common in proteins,9 provide the feasibility to engineer such dynamic biomaterials. Through ligand binding or other physical/chemical triggers, many proteins can undergo conformational change,9−12 leading to changes in protein structure, size, stability as well as functionality. Large protein conformational changes, which lead to the change of protein size in the range of 1−2 nm, have been successfully utilized to engineer protein−polymer hybrid hydrogels,5,13−15 where protein conformational changes at the molecular level can © XXXX American Chemical Society

lead to changes in physical properties of hydrogels at the macroscopic level, such as volume change. Conformational changes in calmodulin and adenylate kinase have been used for such purposes,13−15 leading to the engineering of protein− polymer hybrid hydrogels that showed large volume change in response to Ca2+ or adenine triphosphate (ATP). However, the efforts in developing dynamic protein hydrogels have been limited. Folding/unfolding is the ultimate format of protein conformational change and results in drastic structural and functional changes in proteins. Unfolding of a folded globular domain converts the protein into a random coil whose contour length is significantly larger than the size of the folded globular domain. The size change involved in protein folding/unfolding is much larger than that involved in protein conformational changes used in constructing protein−polymer dynamic hydrogels; thus protein folding/unfolding may offer a much Special Issue: Designer Protein Biomaterials Received: June 30, 2016 Accepted: October 4, 2016

A

DOI: 10.1021/acsbiomaterials.6b00374 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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a well-established molecular biology technique that was detailed previously.27 The GL5HH-I27 and polyprotein GR-(G-MEPHH-R)2 were overexpressed in E. coli strain DH5α and purified by Co2+ affinity chromatography. Purified protein was dialyzed against EDTA for 2 days to remove any residual Co2+ ion. GR-(G-MEPHH-R)2 was then lyophilized. The yield of GR-(G-MEPHH-R)2 is ∼30−40 mg per liter of bacteria culture. Fluorescence Chemical Denaturation Measurements. Chemical denaturation experiments were carried out on a Cary Eclipse fluorescence spectrophotometer. Tryptophan fluorescence of the mutually exclusive protein GL5HH-I27 was monitored at 360 nm to probe the unfolding process of the MEP in the presence of Ni2+ (from 0 to 12.8 mM Ni2+). The excitation wavelength was 280 nm. The chemical denaturation data were normalized according to standard procedures and fitted to the following equation:

larger dynamic range to tune the mechanical and physical properties of the dynamic hydrogels. Recently, we have developed a novel strategy to utilize protein folding−unfolding as a stimulus to engineer the first protein-based dynamic hydrogel whose mechanical and physical properties can be tuned dynamically by the external stimulus.16 We used the socalled mutually exclusive protein (MEP) as a folding switch to accomplish this goal.17,18 MEP is a special class of domain insertion protein which was first designed by Loh and coworkers.19 To engineer a mutually exclusive protein, a guest domain with a long end-to-end distance is inserted into one of the short surface loops of the host domain.18−21 In such an arrangement, the topological constraints of the two proteins lead to the consequence that only one of the two domains can fold at any given time. The conformational state of the MEP at equilibrium is determined by the relative thermodynamic stability of the two domains.18,20,21 The “mutual exclusiveness” of the two domains in an MEP was directly observed by our group.18 Via protein engineering, we designed an MEP, GL5CC-I27, which carries an engineered disulfide bridge that can be used to control the folding/unfolding of GL5CC-I27 reversibly via redox potential.17 The folding and unfolding of the host domain leads to drastic changes of the effective contour length of the host domain, resulting in an effective change of the chain length between two adjacent cross-linking points in the hydrogel network and consequently a change of the crosslinking density of the hydrogels. Making use of such a redox responsive folding switch, we successfully engineered protein hydrogels based on GL5CC-I27 whose physical and mechanical properties can be regulated dynamically by the redox potential.16 Building upon these prior efforts, here we endeavor to expand the range of stimuli to include metal chelation so that it can be used to regulate the conformational equilibrium of the MEP and ultimately to regulate the physical and mechanical properties of such MEP-based protein hydrogels. Protein− metal interaction is ubiquitous in nature, and the binding of metal ions to proteins can lead to changes in protein conformation, stability, and functionality.22,23 The binding of metal ions to naturally occurring proteins, such as calmodulin,14,15,24,25 as well as engineered metal binding proteins, such as elastin variants that carry metal binding motifs,26 have been used to engineer biomaterials whose physical properties are responsive to metal ions. However, the use of metal binding in engineering protein-based dynamic hydrogels has been limited. Here, we demonstrate that bihistidine-based metal chelation is a useful and robust approach to control the equilibrium between the two conformations of a designed MEP and to dynamically regulate the physical and mechanical properties of such MEP-based protein hydrogels. Our results demonstrate the feasibility to tune the mechanical properties of protein hydrogels in a continuous fashion using metal chelation.

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F=

exp[(m × [D] − (ΔGDH−2ON )/RT ] 1 + exp[(m × [D] − (ΔGDH−2ON )/RT ]

(1)

where F is the fraction of unfolded proteins, m is the slope of the H2O is the transition, [D] is the concentration of the denaturant, ΔGD−N thermodynamic stability of the protein in the absence of denaturant, R is the gas constant, and T is the absolute temperature in Kelvin. The 2O , of the protein can be measured using thermodynamic stability, ΔGHD−N H2O ΔGD−N = m·[D]0.5, where [D]0.5 is the concentration of denaturant at which 50% of the protein is unfolded. The difference of thermodynamic stability can be calculated using

ΔΔG = ΔGD[Ni−]N − ΔGDH−2ON

(2)

Circular Dichroism (CD) Measurement. CD spectra were recorded on a Jasco-J815 spectrometer flushed with nitrogen gas. The experiments were performed in a 1 mm path length quartz cuvette at a scan rate of 50 nm/min. The CD spectra of the GL5HH-I27 protein in buffers containing Ni2+ (from 0 to 12.8 mM) was measured in the far UV range from 260 to 195 nm. All data were corrected for buffer contributions. Hydrogel Preparation. Hydrogels were prepared using a welldeveloped Ru (II) (bpy)32+ mediated photochemical cross-linking strategy.28−31 The lyophilized protein was redissolved in Tris buffer containing 50 mM ammonium persulfate (APS) and 0.2 mM Ru(II) (bpy)32+. Protein solution was then quickly transferred to a custommade plexiglass mold containing a ring-shaped slot. Gelation was achieved by irradiating the protein solution using a 150W fiber optical white light source for 10 min. The ring-shaped hydrogels were then carefully taken out of the mold and stored in Tris buffer. Tensile Tests. The tensile tests were performed using an Instron5500R tensometer with a custom-made force gauge.30 The ring shaped hydrogel was fixed at one end and stretched by a hook from the other end. The hydrogel first stored in Tris buffer was stretched to the given length at a strain rate of 25 mm/min at a constant temperature of 25 °C. Then, the hydrogel equilibrated in the buffer containing different concentrations of Ni2+ was subsequently tested in stretching and relaxation cycles. To remove Ni2+ from the sample, the hydrogel was transferred and tested in Tris/EDTA buffer. The slope at 15% strain of the loading curve was taken as the Young’s modulus of the hydrogel. Resilience is a measure of a material’s ability to recover after deforming under an applied stress and is measured from the ratio of the area under the loading curve to the area under the relaxation curve at a given strain using custom-written software in IgorPro 6.0. Swelling Ratio Measurements. The hydrogel deswelling degree is measured by mass difference, calculated between the freshly made hydrogel and the equilibrium hydrogel in the absence and presence of Ni2+. Degree of swelling (SD) was calculated by SD = (W−W0)/W0 × 100%, where W0 is the weight of the freshly prepared hydrogel, and W is the weight of the hydrogel after reaching equilibrium.31

MATERIALS AND METHODS

Protein Engineering. The gene of the host protein GL5-I27 was constructed as described in our previous work.17,18 Residues 32 and 36 of the GL5 domain in GL5-I27 were mutated to histidine in two steps using the site-directed mutagenesis strategy to obtain GL5HH-I27,27 which was confirmed by direct DNA sequencing. The gene of GL5HH-I27 was then subcloned into the expression vector pQE80L. Polyprotein GB1-Resilin-(GB1-GL5HH-I27-Resilin) 2 (GR-(GMEPHH-R)2 for short) was constructed in an iterative manner using B

DOI: 10.1021/acsbiomaterials.6b00374 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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RESULTS AND DISCUSSION

Design Principle of the Mutually Exclusive Protein with a Metal Chelation Site. The conformation of a MEP depends on the relative thermodynamic stability of the host and guest domains.18,21 For example, in the MEP GL5-I27,18 the host domain GL5 is thermodynamically less stable than the guest domain, thus the dominant equilibrium conformation for GL5-I27 is GL5(U)-I27(F), where the host domain GL5 is unfolded, and the guest domain I27 is folded. If one can tune the relative thermodynamic stability of the host and guest domains, one should be able to control the equilibrium between the two conformations of the MEP. Here, we employ metal chelation as a means to tune the relative thermodynamic stability of the host and guest domains. It has been demonstrated that bihistidine-based metal chelation is an efficient means to enhance protein’s thermodynamic stability.32−37 By engineering a bihistidine (bi-His) motif, which consists of two histidines in a configuration allowing for the binding of a bivalent transition metal ion (such as Ni2+, Cu2+, and Zn2+), onto the surface of a protein domain, the engineered metal chelation site can be easily introduced into a folded protein. In the unfolded state, the engineered bi-His metal binding site will be disrupted. Thus, divalent metal ions bind to the native state preferentially over the unfolded state, resulting in a significant enhancement of the protein’s thermodynamic stability upon metal binding.37 In our previous work,27 a series of bi-His mutants of GB1 were constructed, and their thermodynamic stability showed significant enhancement upon the binding of divalent transition metal ions (such as Ni2+, Cu2+, Co2+, and Fe2+). On the basis of these prior results, we reason that if a bi-His metal binding site can be engineered into the host domain GL5 to obtain GL5HH, the binding of bivalent metal ions, such as Ni2+, should enhance the thermodynamic stability of the host GL5HH and thus shifting the conformational equilibria from GL5(U)-I27(F) toward GL5(F)-I27(U) (Figure 1). Since the effective contour length of the MEP protein GL5-I27 is significantly different for the two conformations (∼2.6 nm for GL5(F)-I27(U) and ∼26 nm for GL5(U)-I27(F), shifting the conformational equilibrium between GL5(U)-I27(F) and GL5(F)-I27(U) in this MEP-based hydrogel would lead to a significant change of the effective chain length between two adjacent cross-linking points and thus effective change of the cross-linking density of the hydrogel. On the basis of this rationale, here we endeavored to develop a metal chelationbased method to dynamically tune the mechanical properties of protein hydrogels. Engineered Bi-His Motif in GL5HH-I27 Can Efficiently Bind Ni2+. Figure 1 shows our design of the mutually exclusive protein with the bi-His binding motif GL5HH-I27. In our previous work on GB1, we showed that the bihistidine motif at residues 32 and 36 can readily form a bi-His motif that binds bivalent metal ions (such as Ni2+) and enhances the thermodynamic stability of GB1.27 Here, we mutated residues 32 and 36 in GL5 to engineer the bi-His metal chelation motif. To confirm that the engineered bi-His motif in GL5HH-I27 can efficiently bind to bivalent transition metal ions and shift the conformation equilibrium between GL5HH(U)-I27(F) and GL5HH(F)-I27(U), we carried out circular dichroism spectroscopy experiments to characterize the stabilization effect of the metal chelation on GL5HH-I27. As a proof-of-principle, we used Ni2+ in this work, as most of our prior studies27,38 on bi-

Figure 1. Schematic of the engineered mutually exclusive protein with the bi-His metal chelation site in the host domain GL5. (A) Construction of the designed MEP GL5HH-I27. Guest domain I27 (gray) is inserted into the second loop of the host protein GL5HH (green). The mutated His 32 and His36 are labeled in orange. (B) The conformational equilibrium between the two conformations of GL5HH-I27 can be regulated by metal chelation. In the absence of Ni2+, GL5HH(U)-I27(F) is the dominant conformation, where the effective contour length (Lec, denoted by the dotted line) between the two termini of GL5HH(U)-I27(F) is ∼26 nm (61 aa*0.36 nm/aa+4.0 nm = 26.0 nm, where 61 aa is the number of residues in GL5, 0.36 nm/aa is the length of an amino acid residue, and 4 nm is the distance between the N- and C-termini of the I27 domain); upon binding of Ni2+, metal chelation stabilizes the GL5HH(F)-I27(U) conformation, and shifts the equilibrium toward GL5HH(F)-I27(U), where the folded host domain GL5HH has a significantly smaller Lec of 2.6 nm between its N- and C-termini. EDTA can chelate Ni2+ and shift the equilibrium toward GL5HH(U)-I27(F).

His mutants of GB1 were carried out using Ni2+. Figure 2A shows the CD spectra of GL5HH-I27 as a function of Ni2+ concentration. In the absence of Ni2+, GL5HH(U)-I27(F) is the dominant conformation, and the CD spectrum is characterized by one minimum at 206 nm, which results from a convolution of unfolded GL5HH and folded I27, which has a typical β-barrel structure. With increasing Ni2+ concentration, the CD spectrum of GL5HH-I27 showed significant changes. In the presence of 12.8 mM Ni2+, the CD spectrum is characterized by two minima at 208 and 220 nm, largely resembling that of the folded GL5 domain,18 which contains a β-sheet packing against an α-helix. These results suggest that the addition of Ni2+ ion leads to the folding of GL5HH and thus shifting the conformational equilibrium of the MEP from GL5HH(U)-I27(F) toward GL5HH(F)-I27(U). In addition, equilibrium chemical denaturation studies, which use tryptophan fluorescence as a probe, were carried out to directly demonstrate that the binding of Ni2+ can stabilize host domain GL5HH. Since I27 used in the MEP does not contain any tryptophan residue,18 the tryptophan fluorescence measured from GL5HH-I27 solely reflects the chemical unfolding process of the host protein GL5HH, thus reflecting the unfolding behaviors of the conformation GL5HH(F)-I27(U) in equilibrium. As shown in Figure 2B, GL5HH-I27 exhibits an increased [D]1/2, at which 50% of the protein is unfolded, with the increasing of Ni2+ concentration, indicating that the thermodynamic stability of GL5HH increases in the presence of Ni2+ and confirms the metal chelation capability of GL5HHC

DOI: 10.1021/acsbiomaterials.6b00374 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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site can significantly enhance the thermodynamic stability of the host protein GL5HH in the MEP GL5HH-I27. Designing Dynamic Protein Hydrogels with GL5HHI27. Having confirmed that Ni2+ binding can modulate the conformational equilibrium between the two conformations of the MEP GL5HH-I27, we used GL5HH-I27 as a building block to engineer protein-based dynamic hydrogels. We constructed an artificial elastomeric protein GB1-R-(GB1-GL5HH-I27-R)2 (hereinafter GR-(G-MEPHH-R)2) that incorporates the mutually exclusive protein GL5HH-I27 as a protein folding switch to shift the conformation equilibrium of the host domain GL5HH (Figure 3A). GB1-Resilin (G-R) based elastomeric

Figure 3. (A) Schematic of the protein hydrogel network constructed from GR-(G-MEPHH-R)2. In the absence of Ni2+, the effective chain length between two adjacent cross-linking points is large since GL5HH(U)-I27(F) is the dominant conformation in the hydrogel; in the presence of Ni2+, metal chelation induced shifts in the conformational equilibrium toward GL5HH(F)-I27(U) and reduces the effective chain length between two neighboring cross-linking points, leading to the deswelling of the hydrogel. (B) Photographs of a ring shaped transparent hydrogel sample constructed from GR-(GMEPHH-R)2 (120 mg/mL) in Tris buffer (left) and in 10 mM Ni2+ (right). The unit of the scale in the photographs is cm.

Figure 2. Far-UV CD and tryptophan fluorescence spectroscopy indicate that Ni2+ binding to GL5HH-I27 leads to the shift of the conformational equilibrium of GL5HH-I27. (A) Far-UV CD spectra of GL5HH-I27 in the presence of different concentrations of Ni2+. (B) GdmCl denaturation curves of GL5HH-I27 in the presence of different concentrations of Ni2+. The solid lines are fits to the experimental data using eq 1. (C) Changes in the thermodynamic stability of GL5HH-I27 upon addition of Ni2+. Fitting the data to eq 3 yields a dissociation constant Kd of 870 μM for the binding of Ni2+ to GL5HH(F)-I27(U).

proteins have been used to engineer biomaterials to mimic the passive elastic properties of muscles.30 The incorporation of GB1 and resilin allows us to use the well-developed Ru2+mediated photo-cross-linking strategy to construct chemically cross-linked protein hydrogels, which cross-links two tyrosine residues in proximity into a dityrosine adduct.28−30 Tuning GL5HH-I27’s conformation with the addition of Ni2+ will allow us to effectively change the length of GL5HH in the protein network, thus allowing us to change the effective chain length between two cross-linking points and change the cross-linking density of the protein hydrogel. In doing so, the mechanical properties of resultant protein hydrogels can be regulated using Ni2+ as a stimulus. We found that an aqueous solution of GR(G-MEPHH-R)2 can be readily cross-linked into a solid, transparent hydrogel upon illumination with white light when the protein concentration is higher than 50 mg/mL (Figure 3B). Metal Chelation Regulates the Mechanical Properties of Protein Hydrogels. To investigate the influence of metal binding triggered folding switch behavior on the mechanical properties of the GR(G-MEPHH-R)2 hydrogel, we carried out tensile tests on the GR-(G-MEPHH-R)2 hydrogel in the absence and presence of Ni2+(Figure 4A). In Tris buffer (Ni2+-free), the

I27. Changes in the thermodynamic stability of GL5HH upon binding of Ni2+ was plotted against the Ni2+ concentration (Figure 2C). Fitting the data to eq 3,39 we measured a dissociation constant of Kd of 870 μM, which is similar to that determined previously for the GB1 bi-His mutant G6−53 (260 μM):38 ⎛ K + [M2 +] ⎞ ⎛ K + [M2 +] ⎞ ⎟ ⎟ − RT ln⎜ MD ΔΔG = RT ln⎜ MN KMN KMD ⎠ ⎝ ⎝ ⎠ (3)

where ΔΔG is the difference in thermodynamic stability of the protein in the presence and absence of metal ions, R is the gas constant, and T is the absolute temperature in Kelvin. KMN and KMD denote the dissociation constants for the binding of metal ions to proteins in the native and denatured states, respectively. These results clearly indicate that the engineered bi-His motif in the mutually exclusive protein GL5HH-I27 can effectively bind to the Ni2+. The binding of the Ni2+ to the metal chelation D

DOI: 10.1021/acsbiomaterials.6b00374 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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an increase in effective cross-linking density of the hydrogel. Under such conditions, the GR-(G-MEPHH-R)2-based hydrogel is expected to become stiffer. Indeed, after reaching equilibrium in 10 mM Ni2+, the GR-(G-MEPHH-R)2 hydrogel’s Young’s modulus increased to 12.9 ± 0.8 kPa. In addition, the hysteresis between the stretching and relaxation curves also increased significantly in the presence of Ni2+, indicating a significant increase in energy dissipation during the stretching of the GR(G-MEPHH-R)2 hydrogel in the presence of Ni2+ and a significant decrease in the resilience of the hydrogel. The breaking strain of the hydrogel in the presence of Ni2+ was 110%, which did not show significant change compared with that in the absence of Ni2+. Moreover, in the presence of Ni2+, the hydrogel deswelled to ∼60% of its original size (Figure 3B), resulting in a SD of around −40%. In comparison, protein hydrogels made of (G-R)4 do not show any change in their tensile tests in buffers containing Ni2+ (Figure 4B), suggesting that the behaviors we observed on GR-(G-MEP HH -R) 2 hydrogels are indeed due to the changes caused by the binding of Ni2+. It is of note that the GR-(G-MEPHH-R)2 hydrogels showed significant change in energy dissipation in the presence and absence of Ni2+. In the absence of Ni2+, GL5HH(U)-I27(F) is the dominant conformation. Since I27 is mechanically very stable,40 the hysteresis of the hydrogel in the absence of Ni2+ is likely originating from the stretching of unfolded GL5HH polypeptides chain. In the presence of Ni2+, GL5HH(F)I27(U) is the dominant conformation and is mechanically less stable than the I27 domain. Similar to the GB1-resilin-based hydrogels,30 it is likely that the hysteresis observed in the GR(G-MEPHH-R)2 hydrogel in the presence of Ni2+ is largely due to the force-induced unfolding of some GL5HH(F) domains, which is a nonequilibrium process on the time scale of the tensile experiments, as well as the stretching of the unfolded I27 polypeptide chain, which is much longer than GL5HH. These results further demonstrate the feasibility of tuning the mechanical stability of folded globular domains of elastomeric proteins to regulate the resilience of protein hydrogels. Depending on the intended applications of the hydrogels, one can use mechanically stable domains to entail the hydrogels with higher resilience or use mechanically weaker domains to generate larger hysteresis. Metal chelation is fully reversible, and hence, we anticipate that metal chelation regulated changes of the mechanical properties of the protein hydrogel are also reversible. Indeed, after the hydrogel gel is immersed in EDTA-containing buffer to reach equilibrium, the hydrogel exhibited mechanical and physical properties that are similar to those of the hydrogel in the absence of Ni2+, that is, a low Young’s modulus, high resilience, and low swelling degree (Figure 4A). The mechanical and physical properties of GR-(G-MEPHH-R)2 hydrogels can be cycled reversibly using Ni2+ and EDTA for many cycles. Figure 5 shows experimental results from such an experiment where the GR-(G-MEPHH-R)2 hydrogel was cycled three times between Ni2+-free and Ni2+-bound states using Ni2+ and EDTA. These results strongly indicate that hydrogels based on GR-(G-MEPHH-R)2 exhibit dynamically tunable mechanical and physical properties that can be regulated reversibly via metal chelation, thus presenting a novel approach to engineer dynamic protein hydrogels using metal chelation. Having confirmed that metal chelation can result in dynamically tuned mechanical and physical properties of GR(G-MEPHH-R)2-based hydrogels, we carried out measurements

Figure 4. Mechanical properties of GR-(G-MEPHH-R)2-based hydrogels can be regulated via metal chelation. (A) Typical stress−strain curves of the GR-(G-MEPHH-R)2 hydrogel (120 mg/mL) in Tris (Ni2+-free state), Tris +10 mM Ni2+, and Tris +20 mM EDTA. The GR-(G-MEPHH-R)2 hydrogel is resilient and soft, with a Young’s modulus of 5 kPa in the absence of Ni2+ (in red), but is stiff (with a Young’s modulus of 13 kPa) in the presence of 10 mM Ni2+ and shows significant hysteresis in their stress−strain curves (in green). Removing Ni2+ using EDTA restores the mechanical properties of the hydrogel back to the Ni2+-free state (in yellow). The mechanical properties of the hydrogels are reversible in response to metal chelation. (B) The stress−strain curves of the (G-R)4 hydrogel in Tris (Ni2+-free state), 10 mM Ni2+ (in the presence of Ni2+), and 20 mM EDTA. The mechanical properties of the (G-R)4 hydrogel do not change in response to metal presence. (C) The GR-(G-MEPHH-R)2 hydrogel can be stretched to 120% of its original length before breaking.

GL5HH-I27 mainly exists as GL5HH(U)-I27(F), and the GR(G-MEPHH-R)2 hydrogel shows a slight swelling (a degree of swelling (SD) of ∼10%). The hydrogel is elastic and can be stretched to 120% of its original length before breaking (Figure 4C) and shows a Young’s modulus of 5 ± 0.3 kPa. There is a small hysteresis between the stretching−relaxation curves, indicative of high resilience of the hydrogel, which is a measure of a material’s ability to recover after deforming under an applied stress. The GR-(G-MEPHH-R)2 hydrogel was subsequently immersed in 10 mM Ni2+ and allowed to equilibrate. In the presence of high concentrations of Ni2+, more GL5HH-I27 will be converted into the GL5HH(F)-I27(U) conformation, leading to a reduced effective contour length of GL5HH and E

DOI: 10.1021/acsbiomaterials.6b00374 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 6. Titrating the mechanical properties of GR-(G-MEPHH-R)2 hydrogels using Ni2+. (A) Stress−strain curves of GR-(G-MEPHH-R)2 hydrogel as a function of Ni2+ concentration. Upon increasing of Ni2+ concentration, the hydrogel shows a gradual increase in Young’s modulus and gradual decrease in resilience. (B) Young’s modulus and resilience as a function of Ni2+ concentration.

Figure 5. Physical and mechanical properties of the GR-(G-MEPHHR)2 hydrogel can be cycled reversibly in the Ni2+ free and Ni2+-bound states. (A) Young’s modulus, (B) swelling ratio, and (C) resilience. Within each cycle, the sample was repeatedly tested in Tris buffer (Ni2+ free), 10 mM Ni2+ (reduced state), and in Tris buffer after incubating in 20 mM EDTA. The sample was incubated in the specific buffer for 3 h to reach equilibrium between each measurement.

of hydrogel mechanical properties in response to increasing concentrations of Ni2+ (Figure 6A,B). Since thermodynamic stability of GL5HH in the MEP is dependent upon Ni2+ concentration, the equilibrium between the two conformations GL5HH(U)-I27(F) and GL5HH(F)-I27(U) is also dependent upon Ni2+ concentration. Thus, we anticipate that the mechanical properties of GR-(G-MEPHH-R)2-based hydrogels should be also dependent on Ni2+ concentration. As expected, when Ni2+ concentration increased from 0 to 10 mM, the Young’s modulus of the hydrogel increases monotonically from ∼5 to ∼13 kPa, and the resilience decreases from 85% to 60%. These results demonstrate the feasibility of “titrating” the mechanical properties of protein hydrogels in a continuous fashion. Moreover, since the engineered bi-His site can chelate different divalent transition metal ions, we anticipate that a range of metal ions can be utilized to tune the mechanical properties of GR-(G-MEPHH-R)2-based hydrogels. As an example, we tested the use of Cu2+ as a stimulus for GR-(GMEPHH-R)2-based hydrogels (Figure 7). It is evident that upon binding of Cu2+, the Young’s modulus of the hydrogel increased from ∼5 kPa to ∼10 kPa. Since different metal ions bind the biHis site with different affinity, we anticipate that systematic work on different bivalent metal ions may lead to even larger dynamic ranges in which one can tune the mechanical and physical properties of such G-R-(G-MEPHH-R)2-based hydro-

Figure 7. Stress−strain curves of GR-(G-MEPHH-R)2 hydrogel in the absence and presence of 10 mM Cu2+. The binding of Cu2+ to GL5HH results in the increased Young’s modulus and reduced resilience of the hydrogel.

gels, thus opening up the possibility of tailoring dynamic protein hydrogels with defined mechanical properties to meet the requirements of specific applications.

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CONCLUSIONS Our results demonstrate that metal chelation can serve as an effective means to control the conformational state and equilibrium of the mutually exclusive protein. On the basis of this unique property, the effective chain length between two cross-linking points in the hydrogel can be effectively modulated using metal ions (such as Ni2+) and metal chelator F

DOI: 10.1021/acsbiomaterials.6b00374 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

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(such as EDTA) as stimuli, thus enabling the dynamic modulation of the hydrogel’s mechanical properties, including Young’s modulus, resilience, and swelling degree. Compared with chemical methods that introduce additional cross-links or remove some of the existing cross-links in order to tune the mechanical properties of polymer hydrogels,7 the metalchelation-based method does not involve chemical reactions and instead relies on metal chelation. This method offers an effective means to tune the mechanical and physical properties of protein hydrogels in a fully reversible fashion. Another unique feature of this protein hydrogel system is that the mechanical/physical properties of the hydrogels can be tuned in a continuous fashion. By titrating metal ion concentrations in the buffer, it is possible to shift the conformational equilibrium of the mutually exclusive protein continuously, thus allowing “titrating” the mechanical and physical properties of the hydrogels in a broad range continuously. This unique feature is in contrast with the simple “on−off” control mode used in many smart hydrogels and offers a convenient way to achieve desirable mechanical and physical properties of hydrogels. One limitation of the current hydrogel system is its relatively narrow dynamic range of the mechanical and physical properties of the MEP-based hydrogels one can achieve. This is likely due to the limited extent of the conformational equilibrium shift of the MEP upon binding of metal ions. Improved design of the MEP and bi-His metal chelation site will allow one to further expand the dynamic range of the mechanical properties. Furthermore, engineered metal chelation is a common and robust method to stabilize protein. Thus, our design should provide a general strategy to engineer MEPbased dynamic protein hydrogels for applications in mechanobiology and tissue engineering.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Sciences and Engineering Research Council of Canada, Canada Foundation for Innovation, and Canada Research Chairs Program.

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DOI: 10.1021/acsbiomaterials.6b00374 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsbiomaterials.6b00374 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX