Single-Molecule Mechanics of Catechol-Iron Coordination Bonds

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Single Molecule Mechanics of Catechol-Iron Coordination Bonds Yiran Li, Jing Wen, Meng Qin, Yi Cao, Haibo Ma, and Wei Wang ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 11 May 2017 Downloaded from http://pubs.acs.org on May 14, 2017

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ACS Biomaterials Science & Engineering

Single Molecule Mechanics of Catechol-Iron Coordination Bonds Yiran Li,†,# Jing Wen, ‡,# Meng Qin,† Yi Cao,*,† Haibo Ma,*, ‡ and Wei Wang*,† †

Collaborative Innovation Center of Advanced Microstructures, National Laboratory of Solid

State Microstructure, Department of Physics, ‡ Key Laboratory of Mesoscopic Chemistry of MOE, School of Chemistry and Chemical Engineering, Institute of Theoretical and Computational Chemistry, Nanjing University, Nanjing 210093, P. R. China KEYWORDS: mussel foot protein; dopa; surface adhesion; atomic force microscopy; loadbearing materials;

ABSTRACT: Metal coordination bonds are widely found in natural adhesives and load-bearing and protective materials, in which they are thought to be responsible for the high mechanical strength and toughness. However, it remains unknown how metal-ligand complexes could give rise to such superb mechanical properties. Here, we developed a single-chain nanoparticle based force spectroscopy to directly quantify the mechanical properties of individual catechol-Fe3+ complexes, the key elements accounting for the high toughness and extensibility of byssal threads of marine mussels. We found that catechol-Fe3+ complexes possess a unique combination of mechanical features, including high mechanical stability, fast reformation kinetics, and

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stoichiometry-dependent mechanics. Therefore, they can serve as sacrificial bonds to efficiently dissipate energy in the materials, quickly recover the mechanical properties when load is released, and respond to pH and Fe3+ concentrations. Especially, we revealed that the biscatechol-Fe3+ complex is mechanically ~90% stronger than the tris-catechol-Fe3+ complex. Quantum calculation study suggested that the distinction between mechanical strength and thermodynamic stability of catechol-Fe3+ complexes is due to their different mechanical rupture pathways. Our study provides the nanoscale mechanistic understanding of the coordination bondmediated mechanical properties of biogenetic materials, and could guide future rational design and regulation of the mechanical properties of synthetic materials.

INTRODUCTION It is hard for artificial materials possess both high strength and toughness properties simultaneously.

Strong materials are often brittle, whereas tough materials are usually

compliant. Natural organisms have provided a great number of means to produce materials with combined high strength and toughness for loading bearing1-9, which have also inspired many successful biomimetic efforts10-21. Mussel byssal cuticle is one of the representative examples of such biogenetic tough yet elastic materials, which possesses a peculiar combination of hardness (~0.1 GPa) and extensibility (>70% strain)3,

22

. Growing evidences have indicated that such

remarkable mechanical properties are directly related to the formation of metal coordination bonds between the catechol-containing amino acid L-3, 4-dihydroxy phenylalanine (DOPA) of the mussel cuticle protein mfp-1 and Fe3+ ions

4, 23, 24

. Mechanical measurements on the DOPA-

Fe3+ cross-linked hydrogels as well as the cohesion energy measured by surface force apparatus


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(SFA) have provided rich information about the relationship between the mechanical properties of bulk materials and the formation of catechol-Fe3+ complexes14, 24-27. However, the mechanical properties of individual catechol-Fe3+ complexes are still unknown, which greatly limit our understanding of the mechanical design of mussel byssal cuticle and current reaches of the biomimicry approaches. Metal coordination bonds between catechol and Fe3+ are thought to serve as sacrificial bonds for load dispersing and shock absorbing

4, 24

. Therefore, they should possess significant

mechanical stability to efficiently dissipate mechanical energy and prohibit the break of strong covalent bonds that hold the materials together. They should also be able to quickly reform when forces acting on the materials are released to “self-heal”. Such mechanical features are yet to be demonstrated at the single molecule level. Especially, catechol-Fe3+ complexes show pH and Fe3+ concentration dependent binding stoichiometry (mono-, bis-, and tris-complexes)14. It will be interesting to explore how different environmental conditions modulate the mechanical stability of catechol-Fe3+ complexes and eventually the materials containing such interactions. However, mechanical stability is an intrinsic property for a metal coordination complex, which cannot be predicted based on other structural and thermodynamic characterizations a priori. The mechanical stability of a metal complex is defined by both the activation barrier height along the force direction and the elongation of the complex at the transition state28. Experimentally applying the force to a metal complex is not trivial and has only been done for very limited numbers of metal complexes using atomic force microscope (AFM) based force spectroscopy28-33. Generally, the metal complexes with exact two ligands can be studied by directly flanking the ligands to the cantilever tip and the surface via covalent linkage31-33. This method is not applicable for the system with more than two ligands, such as the catechol-Fe3+


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complexes. For the metal complexes inside a metalloprotein,

28, 29

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when the protein domain is

unfolded to random coiled structure, the metal coordination bonds could still be sequentially ruptured giving rise to the force events of metal coordination bonds. However, there are many kinds of other type interactions as well as intermolecular bonds in mussel cuticle proteins. It is difficult to study the mechanics of catechol-Fe3+ complexes using these natural proteins. Here, we present a more general approach, based on synthesized single-chain polymeric nanoparticles34, to study the mechanical properties of catechol-Fe3+ complexes. In this method, the nanoparticle is analogous to the unfolded protein in the metalloprotein approach, which connects the metal complexes, except that the chemical environment is greatly simplified. Stretching a single-chain polymeric nanoparticle by AFM will lead to stepwise rupture of individual catechol-Fe3+ bonds that hold the nanoparticle, allowing the mechanical strength of these bonds being directly quantified, regardless the stoichiometry. This approach could also be used to study the reformation of the metal complexes by applying stretching-relaxation cycles. Our results indicate that the catechol-Fe3+ complex ruptures at ~100-250 pN at a pulling speed of 1000 nm s-1, stronger than typical hydrogen bonding (~150 pN)

35, 36

and hydrophobic

interactions (~30-60 pN)37, 38 but weaker than covalent bonds (~1-3 nN) 33, 39. Therefore, they can break before covalent bonds yet provide considerable cross-linking strength. Moreover, the catechol-Fe3+ bonds can be efficiently reformed when load is released, allowing the mechanical properties to be restored. The strength of catechol-Fe3+ interactions is sensitive to the ligand stoichiometry and can be modulated by both pH and Fe3+ concentrations. The bis-catechol-Fe3+ complex is mechanically ~90% stronger than the tris-catechol-Fe3+ complex. Using quantum calculation, we revealed that this is due to the distortion of tris-catechol-Fe3+ complex before the complete rupture of the catechol-Fe3+ bonds. Therefore, we propose that force may play an


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important role in the remolding of the network structures of catechol-containing materials, by changing the relative population of bis- and tris- complexes. The combination of desired mechanical stability, quick and reliable rebinding property and environmental sensitivity makes catechol-Fe3+ coordination bonds ideal building blocks for load-bearing materials.

Figure 1. Study single molecule mechanics of catechol-Fe3+ complexes by atomic force microscopy. (a) Catechol-Fe3+ complexes are widely found in mussel byssal threads, which are responsible for their high strength and toughness. DOPA residues are highlighted in orange spheres and the other amino acids are shown as blue spheres. Smaller yellow spheres represent Fe3+ ions conjugated to the proteins. (b) Schematic illustration of measuring the rupture forces for catechol-Fe3+ complexes by unraveling the HA-catechol-Fe3+ nanoparticles. The blue ribbon represents HA molecule and orange ‘Y’ shape patterns represent catechol groups. The yellow


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sphere is Fe3+ ions. Cartoon cantilevers are shown in green schematically pulling the assembly from the titanium surface (grey panel).

(c) AFM images of HA-catechol-Fe3+ complexes

prepared at pH 7.2 in the presence of 200 µM Fe3+. The scale bar on the upper-left corner is 10 nm. 2-D figure with color scale is shown in Figure S3 of Supporting Information. (d) Dynamic light scattering (DLS) measures the hydrodynamic diameter of HA-catechol in the absence (red) and presence (blue) of 200 µM Fe3+ at pH 7.2.

RESULTS Preparation of the catechol-containing polymer for single molecule AFM study. We first synthesized a polymer containing catechol groups for the study of the mechanical properties of catechol-Fe3+ complexes. In nature, mussels secret many DOPA containing foot proteins (Figure 1a). Depending on pH and Fe3+ concentrations, DOPA can form complexes with Fe3+ of different stoichiometry (mono-, bis-, and tris-). In this study, we used a synthetic catechol-containing polymer to mimic the sequence of mussel foot proteins and the experimental scheme is sketched in Figure 1b. This approach could simplify the interpretation of single molecule AFM data and avoid complications from other types of interactions found in mussel foot proteins. Without the metal-coordination bonds, stretching the random-coiled polymer would only yield a non-linear force-extension relationship corresponding to the elasticity of the polymer backbone. When the metal-complexes are formed in the nanoparticles, some of the polymer lengths were sequestered by the metal coordination bonds and can only be lengthened when those bonds are ruptured. The stepwise rupture of metal coordination bonds in the nanoparticle will give rise to a sawtooth like force-extension curve, each peak corresponding to the rupture of an individual metal coordination bond. The structure of the synthetic polymer is


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depicted in Figure S1. It is made of a hyaluronan (HA) backbone with catechol side chains by reacting HA with dopamine and is termed as HA-catechol hereafter. The grafting ratio of catechol is ~ 10%, as determined by 1HNMR (Figure S2). Lyophilized HA-catehcol was first dissolved into deionized water at the concentration of 0.2 mg mL-1. Next desired amount of FeCl3 was added to the HA-catechol solution to prepare the stock solution. Finally, Tris buffer (containing 100 mM Tris, 50 mM NaCl, pH 7.2 or 9.7) was added to the stock solution to adjust the pH. Single-chain polymer nanoparticles were formed, as evidenced by the size of the assemblies measured by AFM imaging and dynamic light scattering (Figure 1c and 1d). Such assemblies were not found in the absence of Fe3+ or in the presence of other kinds of metal ions (Figure S3). To confirm that catechol-Fe3+ bonds were formed in these complexes, we measured Raman spectra of the complexes. As shown in Figure S4, the Raman spectrum shows characteristic absorption at 535 cm-1, 592 cm-1, and 633 cm-1, which can be assigned to the Fe3+O coordination bonds4. UV-Vis spectra of dopamine-Fe3+ were employed to verify the stoichiometric dependence of catechol-Fe3+ coordinate complex at different environmental conditions (i.e. Fe3+ to catechol ratio and pH). We used dopamine instead of HA-catechol because the content of catechol in the polymer was only ~ 10% as determined by 1HNMR, and the solubility of HA was not very high at pH 7.2, making it difficult to obtain high-quality spectroscopic data. Moreover, the absorption of HA severely overlaps with the peaks from catechol-Fe3+ complexes. The UV-Vis spectra of dopamine-Fe3+ suggested that both bis and tris complexes existed at pH 7.2, as indicated by the absorption at 575 nm and 492 nm, respectively (Figure S5)14. The population of bis and tris complexes is pH and Fe3+ concentration dependent (Figure S5). However, it is worth mentioning that HA is negatively charged at basic pH and the


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local environment may change the catechol to Fe3+ ratio in the HA-DOPA polymer, preventing quantitative comparison of the results obtained using dopamine and HA-catechol.

Figure 2. Representative force-extension curves for the unraveling of individual HAcatechol-Fe3+ nanoparticles at a pulling speed of 1000 nm s-1. The AFM cantilever was brought into contact with the titanium surface to fish individual HA-catechol-Fe3+ nanoparticles (gray traces). Stretching the single-chain nanoparticles resulted in saw-tooth like force-extension curves (black traces), corresponding to the rupture of individual catechol-Fe3+ bonds in the polymer. Red lines correspond to worm-like chain (WLC) fitting to each individual force peak. The persistence length of ~0.4 nm from WLC fitting indicates that only a single HA chain is stretched between the cantilever tip and the substrate.

Single-molecule force spectroscopy on the HA-catechol-Fe3+ complex.


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Next, we used AFM to directly probe the mechanical stability of catechol-Fe3+ bonds based on the HA-catechol-Fe3+ single-chain polymer nanoparticles (Figure 1b). In a typical force spectroscopy experiment, the HA-catechol-Fe3+ complex was first prepared as stock solution in pure water to prevent Fe3+ hydrolysis under basic condition. Then HA-catechol-Fe3+ nanoparticles were deposited on a titanium substrate and then covered by Tris buffer (100 mM Tris, 50 mM NaCl, pH 7.2). As Tris is a weak coordinating ligand of Fe3+, it kept Fe3+ concentrations constant throughout the AFM experiments (Figure S6). It is worth mentioning that the HA-catechol-Fe3+ complex was very stable even after being placed in a large amount of Tris buffer (50 times the original volume) without additional Fe3+ ions, which is supported by the X-ray photoelectron spectroscopy (XPS) results (Figure S7). The high stability of catechol-Fe3+ complexes was consistent with the high binding equilibrium constant between DOPA and Fe3+ reported in literature (the dissociation constant Kd is ~10-37–10-40 M)

14, 40-42

. In a typical single-

molecule force spectroscopy experiment, the AFM cantilever tip was first brought into contact with the substrate surface to pick up the HA-catechol molecule and then pull back to break the catechol-Fe3+ bonds. We relied on non-specific physical adsorption to anchor the molecule between the cantilever tip and the substrate at two random positions along its contour, instead of specific interactions between catechol and the surfaces. The nonspecific interactions between HA and the cantilever tip (or the substrate) can be as high as several hundred pN (Figure S8) and may be due to the formation of multiple weak bonds between them at the anchoring sites. This method has been widely adopted to study the mechanical properties of polyproteins, polysaccharide, double stranded DNA, and synthetic polymers34, 43-45. In about 1%-2% of the total trials, we were able to successfully grasp a HA-catechol molecule and stretch it to rupture the catechol-Fe3+ bonds one at a time. Three representative force-extension traces are shown in


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Figure 2. They are both saw-tooth-like with rupture forces of ~100-250 pN. The force peaks in the force-extension curves are mainly from the rupture of the tris- and bis-catechol-Fe3+ complexes because mono-catechol-Fe3+ complexes form single linkages with the polymer chain are not subjected to forces. We used worm-like chain model (WLC) for polymer elasticity to fit each individual peak. The persistence lengths were ~0.4 nm and increased slightly from the first to the last46, 47. ( Section 9, Supporting information and Figure S9). The measured persistence length of ~0.4 nm agreed well with that for a single HA chain46, 47, which suggested only a single HA-catechol molecule was picked up each time. The force-extension traces with abnormal persistence lengths were excluded for data analysis, as they may result from simultaneously stretching multiple molecules (Figure S10). The distances between consecutive peaks varied in a wide range, because catechol groups were randomly distributed in the polymer chain. To rule out the possibility that these peaks were resulted from other types of interactions in HA molecules (e.g. hydrogen bonding or ionic interaction), we performed control experiments using unmodified HA in the presence of Fe3+ ions. No such saw-tooth like peaks with persistence length of ~0.4 nm could be observed (Figure S11). To further confirm HA cannot bind to Fe3+ in our experimental conditions, we examined the HA and HA-catechol samples mixed with Fe3+ first and then dialyzed to the same buffer as used in AFM experiments using XPS (Figure S7). The HA-catechol-Fe3+ showed clear peaks at 710 eV and 725 eV, corresponding to Fe 2p3/2 and Fe 2P1/2, respectively. However, these two peaks were absent in the HA-Fe3+ sample, supporting that HA cannot bind to Fe3+ in our experimental conditions. As catechol can potentially bind with both the silicon nitride cantilever tip and the titanium substrate, we also studied the HAcatechol polymer in the absence of Fe3+ to evaluate whether the peaks were from surface binding. Based on the AFM images, we found that in the absence of Fe3+, HA-catechol adhered


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to the titanium substrate instead of forming single-chain nanoparticles (Figure S3). The rupture forces for catechol-titanium surface were (~80 pN), consistent with those reported previously47 (Figure S11). However, in our experimental conditions, the majority of catechol in the polymer were bound with Fe3+ and only very limited catechol could bind with the surface or cantilever tip when single-chain nanoparticles were formed. Therefore, catechol-surface interactions did not contribute too much to the total events, although we could not completely preclude them.

Figure 3. Single molecule stretching-relaxation experiments of HA-catechol-Fe3+ complexes. (a) The scheme of the AFM protocol to unfold and refold a single chain HAcatechol-Fe3+ complexes. First, the complexes were stretched to break the catechol-Fe3+ bonds within the complexes while avoiding detaching the molecule from either the cantilever or the substrate. Then the molecule was relaxed to a position ~ 20 nm away from the surface and stayed there for 2 s to allow the reformation of catechol-Fe3+ complexes. In the subsequent stretching cycles, the number of catechol-Fe3+ bonds reformed during the waiting time can be estimated from the number of peaks in the force extension curves. (b) Representative force-extension


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curves obtained in the stretching-relaxation experiments. Because the molecule did not reach the surface in the stretching-relaxation experiments, all peaks in the stretching curves, except the first one, can be assigned as the break of single catechol-Fe3+ bonds with 100% confidence. (c) The rebinding probability after several stretching-relaxation cycles. The rebinding probability was calculated as ratio of the peaks in the consecutive curves. As no additional Fe3+ presented in the buffer, Fe3+ gradually detached from the HA-catechol chain, leading to gradually decrease of the number of force peaks in the force extension curves. The total number of force curves is 87 and the total rupture events in the first, second, third and fourth cycles are 404, 209, 158 and 163, respectively.

Reformation of catechol-Fe3+ complex when force is released. Next, we investigated whether the unfolded catechol-Fe3+ complex can reform when force is released. We first pulled a HA-catechol-Fe3+ complex to a distance less than its contour length to avoid its detachment from either the cantilever tip or the substrate surface (Figure 3a). The number of catechol-Fe3+ bonds that formed within the molecule and are subjected to stretching force can be counted from the number of peaks on the trace. Then, we repeatedly relaxed the molecule near the surface, waiting for 2 s, and pulled again to figure out how many catechol-Fe3+ bonds were reformed during the waiting time. As shown in Figure 3b, many of the catechol-Fe3+ bonds could reform after the molecule was relaxed to zero extension. Because the HA-catechol molecule was suspended in the solution without touching any other molecules on the surface, all force peaks shown in such repeated pull-relax traces must result from the rupture of the reformed intra-molecular catechol-Fe3+ bonds. The positions of the reformed catechol-Fe3+ bonds are stochastic, as Fe3+ ions can bind with any catechol groups in proximity within the HA-catechol


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molecule. Interestingly, although mussel byssus fibers require several hours to regain their original mechanical strength after deformation beyond the yield point 48, we found that catecholFe3+ bonds can reform within a few seconds. This is probably because the real situation in byssus is much more complicated. In the byssus, there is a much larger diffusion length and longer diffusion time scale for replenishment of the metal in the crowded environment. Also the relaxation of the collagen protein core involves potentially other coordination interactions (e.g. Zn2+ and histidine). It would be difficult to compare our results to the byssal recovery simply. Moreover, it is worth mentioning that there were no additional Fe3+ ions in the solution. After the HA-catechol-Fe3+ was stretched, Fe3+ can only remain attached to the HA-catechol chains as mono-complex, in which the Fe3+ binding affinity became lower than the bis- or tris- complexes. Therefore, during relaxation, Fe3+ has a higher chance to dissociate from HA-catechol than the un-stretched nano-assemblies, leading to gradual loss of Fe3+ ions. Therefore, the number of peaks decreased when the HA-catechol molecule underwent more pull-relax cycles, suggesting the gradual loss of Fe3+ ions (Figure 3c), consistent with a recent report on rubredoxin49. The reformation of catechol-Fe3+ bonds is more complex if additional free Fe3+ is supplied in the buffer. Low concentrations of free Fe3+ in buffer can prevent the loss of Fe3+ from nanoparticle chain and increase the probability of reformation of catechol-Fe3+ complex. However, excessive Fe3+ may result in the increase of mono-complex, which can also cause the decrease of the number of rupture force peaks.

Distinct mechanical strength of bis- and tris-catechol-Fe3+ complexes. It is well known that the population of bis- and tris-complexes in the HA-catechol polymer can be modulated by Fe3+ concentrations and pHs14,

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. Therefore, we would expect to observe


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different rupture force distributions corresponding to bis- and tris-catechol-Fe3+ complexes, respectively, at different Fe3+ concentrations and pHs. Indeed, we found that the rupture forces for the HA-catechol-Fe3+ single-chain nanoparticles depend on the Fe3+ concentrations at which the assemblies were prepared (at pH 7.2). As shown in Figure 4a, at ~30 µM of Fe3+, the rupture forces showed only a single peak centered at ~80 pN. In this condition, because the concentration of Fe3+ was inadequate, catechol-surface interaction dominated the rupture events. With the increase of Fe3+ to ~70 µM, the force peak shifted to ~100 pN and an additional force peak at ~200 pN appeared. When the Fe3+ concentration increased from ~ 70 µM to ~100 µM or ~200 µM, although the relative populations between the low force events and high force events varied, the positions of the two peaks remained the same. Kernel density estimation, a nonparametric statistics method, was used to estimate whether the unimodal and bimodal Gaussian fittings for different rupture force histograms is appropriate. As shown in Figure S12, the calculated Kernel functions are consistent with the Gaussian fitting, indicating the fittings are suitable. We could not perform experiments using HA-catechol-Fe3+ complexes prepared with higher Fe3+ concentrations, as Fe3+ became unstable at high concentrations at pH 7.2. Based on the UV-Vis spectra of the solution of Fe3+ and catechol, the amount of the bis- complexes increases with the increase of Fe3+ concentrations. Therefore, we attributed the higher force peak at ~200 pN to the rupture of the bis-catechol-Fe3+ complex and the lower force peak at ~100 pN to the rupture of the tris-catechol-Fe3+ complex. To further confirm this, we also studied the assemblies of HA-catechol with ~200 µM of Fe3+ at an elevated pH of 9.7 (Figure 4b). At this pH, majority of the catechol Fe3+ forms tris- complexes. As expected, we only observed the lower rupture force peak at ~ 100 pN. To avoid Fe3+ hydrolysis in basic environment, we also prepared the Fe3+ solutions in Tris buffer first and then added the HA-catechol solution in situ for


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the single molecule AFM experiments. As illustrated previously, the hydrolysis of Fe3+ is inhibited due to the presence of Tris. We measured the rupture forces of the HA-catechol-Fe3+ in pH 7.2 and 9.7 using this procedure. As shown in Figure S13, the rupture forces are consistent with those measured using the previous procedure. The ratios between the low force peak and high force peak at pH 7.2 changed slightly, presumably due to the prevention of Fe3+ from hydrolyzing and polymerizing in this experimental procedure. In addition, the lower rupture force at pH 9.7 seems not from the oxidized catechol-Fe3+ interaction. The oxidization process is relative slow in freshly prepared buffer without additional oxidants. When chelated with Fe3+, oxidization process of catechol become much slower, as Fe3+ is protective for auto-oxidation of catechol in air27. In this circumstance, the rupture force distribution would not behave as a unimodal distribution if catechol was only partially oxidized. To further confirm this, we performed theoretical calculation. Our calculation found that when catechol was oxidized to quinone, it cannot chelate to Fe3+, as the binding of quinone to Fe3+ is energetically disfavored. (Figure S14) Therefore, even residual amount of catechol was oxidized in our experimental conditions, they were inert and did not contribute to the force peaks in the force-extension traces.


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Figure 4. Fe3+ concentrations and pH modulate the mechanical strength catechol-Fe3+ complexes. (a) The rupture force distribution of catechol-Fe3+ complexes with different Fe3+ concentrations at a pulling speed of 1000 nm s-1. When the concentration of Fe3+ is low (i.e. ~ 30 µM), catechol-surface interaction dominated the rupture events. The rupture force of catecholsurface interaction is ~80 pN, consistent with our previous study. With the increase of Fe3+ concentration, the rupture force histograms show bimodal distributions, with two peaks located at ~100 pN and ~200 pN, respectively. (b) The rupture force distribution of catechol-Fe3+


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nanoparticles at different pHs. At pH 7.2, there are two peaks located at ~100 pN and ~200 pN, respectively, while at pH 9.7, there is only one peak located at ~100 pN. Solid lines in the figures represent Gaussian fitting. See supporting TableS3 for statistic details. The Fe3+ concentrations were 200 µM at both pH 7.2 and pH 9.7.

Free energy landscape for the rupture of bis- and tris-catechol-Fe3+ complexes. It is intriguing to observe such distinct mechanical stability of bis- and tris-catechol-Fe3+ complexes. Especially, thermodynamically more stable tris-complex is mechanically less stable. To understand the molecular mechanism and explore the free energy landscape underlying the rupture of bis- and tris-complexes, we performed dynamic force spectroscopy experiments. As shown in Figure 5A and Figure S15, the rupture forces for both bis- and tris-complexes are pulling-speed dependent. The faster the pulling speeds, the higher the rupture forces are. Interestingly, the increase of the rupture forces for bis-complexes is more pronounced than triscomplexes, making the two peaks well separated at high pulling speeds. The relative ratio of events in the two peaks does not change with the pulling speeds and is solely determined by the population of the bis- and tris-complexes in the HA-catechol nanoparticles. The rupture forces for bis-complexes are more dependent on the loading rates than those for tris-complexes. We used the Bell-Evans model50,

51

to extract the kinetic parameters underlying the free energy

landscape for the rupture of bis- and tris-complexes from the loading-rate dependency rupture forces (Figure 5b). The elongation of the complex at the transition state along the force direction, ∆x‡, for the bis-complex is much

shorter than that for the tris-complex. Despite that the

activation energy barriers, ∆G‡, for the two are similar, the rupture forces for bis-complex are


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significantly higher than those for tris-complex, especially at high loading rates. Such different free energy landscape leads to distinct mechanical stability of bis- and tris-complexes.

Figure 5. Loading rate dependent rupture forces for the catechol-Fe3+ complexes. (a) Representative rupture force histograms for HA-catechol-Fe3+ complexes prepared at 200 µM Fe3+ and pH 7.2 at different pulling speeds. (b) The plot of rupture forces at different loading rates for bis-complexes (blue) and tris-complexes (red). The lines correspond to the fitting of Bell-Evans model to the experimental data. The R2 values (rupture force vs. logarithmic loading


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rate) are 0.901 for bis complex and 0.942 for Tris complex.

(c) Free energy landscape

underlying the rupture of bis- and tris- catechol-Fe3+ complexes based on the parameters extracted from the loading rate dependent experiments using Bell-Evans model.

Quantum chemical calculation.

Figure 6. (a-b) The optimized ground state structures of tris- and bis-catechol-Fe3+ complexes. (c) Relative energies for bis- and tris-catechol-Fe3+ complexes with different spin multiplicities under their optimized geometries by DFT calculations. (The ground state energies of sextet were taken as zero.) (d) Force-displacement relationships of the two complexes. (e) The evolution of


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four Fe-O bond lengths between Fe3+ and two pulled catechol groups of bis- (top) and triscomplex (bottom). (f) The evolution of the distance d between Fe and the C1-C2 line (top), and the change of angle∠C1FeC2 (α) in terms of increasing end-to-end displacement (bottom).

In order to provide insights into the different mechanical response of bis- and tris-catecholFe3+ complexes at the molecular level, a first-principle quantum chemical study is also incorporated in this work. Before simulating the mechanical stretching process, we firstly examine the validity of our computational scheme by comparing the calculated spin multiplicity and geometry as well as electronic spectrum with the experimental findings. Fe3+ complex has five 3d valence electrons, which may result in electronic states with different spin multiplicities (doublet, quartet, and sextet). Therefore, a thorough modeling of the mechanical response of catechol-Fe3+ complexes requires a detailed pre-examination of the possible spin multiplicities for the ground state. From Fig 6c, in which the relative energies of different spin states are shown, one may clearly find that the spin sextet states are energetically much favorable than the doublets and quartets. As a consequence, only spin sextet states will be considered in our mechanical stretching simulations of the catechol-Fe3+ complexes. Our calculation results showed that catechol-Fe3+ complexes have spin-sextet ground states and the bis-catechol-Fe3+ complex has a non-planar tetrahedral structure while the tris-one has a non-planar octahedral structure (Figure 6a-b). This is in accordance with the well-known fact that metal coordination compounds with five d electrons under weak ligand fields (e.g. catechol-Fe3+ complex) usually exist in a non-planar octahedral structure for hexa-coordination or a non-planar tetrahedral structure for tetra-coordination. The length of six Fe3+-O bonds in the equilibrium spin sextet structure of tris-complex was evaluated to be 2.04 Å, matching well with the experimental value


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of 2.04 Å 52. Our calculation also indicated that unsurprisingly the Fe3+-O bonds in tris-complex is slightly longer than those in bis-complex which have a length of 1.93 Å. Our calculated excitation energy for the first dipole allowed state by the time-dependent density functional theory (TDDFT) is 2.37 and 3.23 eV for bis- and tris-complexes respectively. The energetic order of these two peaks is also in good agreements with the experimental observation (2.16 and 2.52 eV for bis- and tris-complexes respectively)14. All these facts confirm that our simulation can give useful information which is in close agreements with the experiments and it can be expected that the further mechanical response analysis based on our simulations should be reasonable. The mechanical stretching process was simulated by moving the two terminal carbon atoms (C1 and C2 in Figure 6a-b) of two catechol groups from each other. The evolution of the average force on C1 and C2 with the C1-C2 displacement is illustrated in Figure 6d, where only one curve is shown for each complex for clarity because our simulations have verified that the different pulling directions do not change the force-displacement relationship in any important ways (Figure S17 and S18). It is clearly shown that the force peak positions are quite distinct for bisand tris-complexes, which locate at 0.8 Å and 1.8 Å respectively. Such a picture is quantitatively consistent with our experimental results that the Fe3+-O bond breaking occurs earlier in the biscomplex with a larger force comparing with the tris- complex. This confirms again that the bimodal distribution of the rupture forces of catechol-Fe3+ complexes is caused by the existence of different Fe3+ coordination stoichiometry under experimental conditions. Moreover, we also found that even the 3rd catechol is constrained in the quantum chemical calculation, the rupture forces were not noticeably changed compared with those without constraining the 3rd catechol, and remained significantly lower than the rupture forces for bis-complexes (Figure S19 and S20).


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Therefore, the two distinct force peaks in our AFM experiments for Fe3+ complex should be mainly caused by the different coordination patterns (bis- vs. tris-) and the restraints on the 3rd catechol group in tris-complex have only a marginal effect on that. It is necessary to note that our theoretically predicted atomic forces for the breaking of Fe3+-O coordination bond are in the same magnitude order with a recent computational work of metal-catecholate complexes by Xu47, but one order of magnitude higher than our experimental values. The possible reason for such a discrepancy is that the thermal fluctuation and environmental effects (pH, ionic strength, salt, pulling speed, etc.) were not well considered in the current quantum chemical calculations due to the limitation of huge computational costs47. A recent single molecule study has also highlighted the importance of environmental effects on the mechanical strength of metal coordination bond48. But such kind of quantitative discrepancy will not change our qualitative conclusion from theoretical simulations. To further understand the different rupture behaviors of Fe3+-O coordination bonds in bisand tris- complexes, we analyzed the evolution of the distances between Fe3+ and four oxygen atoms at the two pulled catechol groups in Figure 6C. The bis- and tris- complexes show distinct Fe3+-O bond breaking sequences. Fe3+-O bonds are ruptured much earlier in the bis- complex than in the tris- one and the bonds are ruptured sequentially and one at a time. The two Fe3+-O bonds at the same side of the complexes remain attached after pulling for both bis- and triscomplexes, indicating that Fe3+ can remain chelated with single catechol groups in the polymer chain after rupturing the bis- and tris-complexes. We then analyzed the structural characteristics of optimized geometries at different C1-C2 displacements for both complexes and the main results were illustrated in Figure 6f. For a biscomplex of a tetrahedron structure, Fe3+ ion is in the middle of two catechol groups and


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accordingly the increase of C1-C2 displacement will not change d (the distance between Fe and the C1-C2 line) and ∠C1FeC2 (α) much (Figure 6f). On the contrary, the tris-complex has a nonplanar octahedron structure. Therefore, when gradually increasing the C1-C2 distance, the Fe3+ ion together with the third catechol group will descend slowly towards the C1-C2 line to stabilize the weakened Fe3+-O bonds. As a consequence, d and α will change remarkably for the triscomplex, as illustrated in Figure 6f. Interestingly, the positions of the smallest d or the largest α (0.8 Å and 1.8 Å for bis- and tris-complexes respectively) are very close to those predicted by atomic force analysis in Figure 6d or bond distance analysis in Figure 6e. Such a good agreement verifies again that the break of Fe3+-O coordination bond occurs earlier in bis-complex than that in the tris-complex and indicates that such distinct mechanical response of bis- and triscomplexes can be ascribed to the significant difference between a tetrahedral structure for the bis- one and an octahedral structure for the tris- one.

DISCUSSION Catechol-Fe3+ coordination bonds are widely exploited as load-bearing cross-links in mussel byssus. They are proposed as sacrificial bonds to dissipate energy and are responsible for the extreme toughness of mussel byssus. However, direct experimental verification of such hypothesis is yet to be provided. In this work, we quantified the bond strength of catechol-Fe3+ complexes using single molecule force spectroscopy, for the first time, which could yield tremendous insights into the molecular mechanism underlying the high mechanical stability of mussel byssus. We discovered that the rupture forces for catechol-Fe3+ bonds are ~100-250 pN at a pulling speed of 1000 nm s-1, which are comparable with those for stable mechanical proteins in load-bearing tissues, such as titin

6, 7

and tenascin8. Therefore, catechol-Fe3+ bonds could


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efficiently serve as sacrificial bonds to increase the total energy needed to permanently crack the materials. Comparing with protein-based sacrificial bonds, catechol-Fe3+ bonds can be implemented in synthetic materials much more easily to enhance the toughness. A deeper understanding of the mechanical properties of such bonds will further stimulate the engineering and tailoring of the mechanical performance of mussel-inspired materials. It is worth noting that other types of metal coordination bonds, such as zinc/copper-histidine and magnesium/calciumphosphorylated serine bonds, are also ubiquitous in load-bearing biomaterials10. The experimental approaches presented in this study could serve as a general tool to explore the complicated mechanical stability of different types of coordination bonds and provide quantitative understanding of the mechanical design of load-bearing biomaterials. Moreover, the mechanical stability of catechol-Fe3+ bonds is also similar to other coordination bonds reported in literature, but are significantly lower than that of covalent bonds53-59. The mechanical stability of a bond is not just determined by its bond strength. Instead, it is determined by the free energy barrier as well as the position of the transition state. Other types of weak interactions, such as hydrogen bonding and hydrophobic interactions can only be used as sacrificial bonds when cooperatively implemented in a folded protein structures because they are transient individually due to low free energy barrier of the transition states. However, catecholFe3+ interaction is quite stable with low spontaneous dissociation rates, which is also a critical factor for its function as load-bearing sacrificial bonds. Even when the bis- and tris- complexes are unfolded, Fe3+ can still retain with catechol groups as mono-complexes, which serve as the intermediates for the fast and efficient reformation of bis- and tris-complexes. Previous studies on main-chain metallopolymers have highlighted the importance of slow dissociation kinetics on the mechanical properties of the materials60-62. Here, we demonstrated, at the single molecule


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level, that slow dissociation rates of catechol-Fe3+ complexes could be linked to both the high mechanical strength and the fast association kinetics of the bonds. The slow dissociation kinetics may also be a common feature of all metallo-complexes found in load-bearing biomaterials. Furthermore, we discovered that the bis- and tris-catechol-Fe3+ complexes exhibit distinct mechanical strength. This is probably due to their different ligand binding geometry (tetrahedral vs. octahedral). The pulling directions in bis- and tris-complexes are defined by the relative positions of the two catechols being stretched. Due to their different geometry, the force-induced rupture of bis- and tris- complexes undergoes distinct pathways. Combining loading rate dependent experiments and quantum calculation, we showed that the tris- complexes are mechanically less stable than the bis-ones because of the relatively larger deformation of the complexes at the transition state. Therefore the bis-complexes are mechanically more stable than the tris-ones. Moreover, we revealed that the coordination bonds in bis-and tris-complexes are ruptured in different orders. Similar pulling direction dependent mechanics of metal coordination complexes has been found in rubredoxin28. This highlights the importance of optimizing the force bearing geometry of metallo-complexes for the construction of hard and tough materials. Moreover, the toughness of a material depends on both the number of and the mechanical strength of the sacrificial bonds inside. A tradeoff between the two factors is found in materials containing catechol-Fe3+ bonds, which may make the mechanical properties of the materials robust under different environmental conditions and after stretching-relaxation cycles. It is also worth mentioning that the pH and iron concentration dependent mechanical properties of the catechol-Fe3+ bonds may make them ideal candidates for the construction of stimuliresponsive materials. We could envision that it is possible to tailor the mechanical properties of catechol-Fe3+ bond containing materials by adjusting the environmental pH and Fe3+


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concentrations. This has been verified by a few recent studies on natural mussel proteins24 and synthetic catechol-containing hydrogels14, 27. Water can have significant effects on many weak interactions such as hydrogen bonding, hydrophobic interactions and ionic interactions. Therefore, it can dramatically affect the overall mechanical performance of polymer materials. However, whether water molecules will affect the mechanical properties of catechol-Fe3+ bonds at the molecular level remains an open question and is certainly worth further investigation.

CONCLUSION In summary, by using atomic force microscope based single molecule force spectroscopy, we measured the mechanical properties of catechol-Fe3+ complexes for the first time. Our results indicate that catechol-Fe3+ complexes possess unique combination of mechanical features including high mechanical stability, fast reformation kinetics, and environmental sensitive mechanics. These make them ideal to act as sacrificial bonds in load-bearing materials of high strength and toughness. Our study also reveals the molecular origin of such mechanical features, which could further deepen the understanding of the mechanical design of metal coordination complexes found ubiquitously in biogenetic load-bearing materials. Our findings could also inspire and serve as the bases for the rational design of catechol-Fe3+ bond based materials for a broad range of biomedical applications. Besides catechol-Fe3+ interactions, other metal-ligand coordination (such as histidine with divalent transition metal ions and carboxyl with calcium) were also exploited in artificial materials to create tough yet strong hydrogels or mimic the mechanical properties of natural tissues. The single molecule force spectroscopy method used in this study can be easily extended to investigate those interesting systems. We anticipate that the


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molecular mechanism underlying the toughness of biomaterials with metal-ligand coordination can be understood in more details in the near future.

EXPERIMENTAL SECTION Synthesis of HA-catechol. We used the EDC/NHS coupling reaction to conjugate dopamine and hyaluronan (HA), as reported in our previous work47. First, 60.0 mg of HA (MW: 150 kDa; polydispersity: 1.4, Freda Biopharm, Shandong, China) was dissolved in deionized water. Next, 46.5 mg of EDC (Sigma-Aldrich) was added to HA solution and stirred for 0.5 h. 27.3 mg of dopamine and 27.9 mg of NHS (Sigma-Aldrich) were pre-dissolved in DMSO and then transferred into the HA/EDC solution. The reaction was conducted overnight at room temperature with mild magnetic stirring. The unreacted reactants were removed by dialysis against excess deionized water using the dialysis tubing (Zhuyan Biotech, Nanjing, China) of a cutoff molecular weight of 5 kDa. The final product was lyophilized and stored at -20 °C. As determined by 1HNMR analysis, the graft ratio of catechol was ~10% (Figure S2). Preparation of HA-catechol-Fe3+ assemblies. HA-catechol was first dissolved in deionized water at a concentration of 0.2 mg mL-1. In such low HA-catechol concentrations, the inter-chain distance was estimated to be ~110 nm, much longer than the radius of gyration of HA-catechol estimated using freely-jointed chain model of ~30 nm. Then, desired amount of FeCl3 stock solution (10 mM) was added into the HA-catechol solution to prepare HA-catechol-Fe3+ assemblies with different Fe3+ concentrations. Tris buffers (containing 100 mM Tris, 50 mM NaCl, pH7.2 or 9.7) were used to adjust the solution pH. Additional of Fe3+ caused the decrease instead of increase of the nanoparticle size based on DLS measurement suggested that singlechain nanoparticles were formed.


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Raman and UV-Vis spectra. Raman spectrum was used to confirm the interaction between HA-catechol and Fe3+. 100 µM of freshly prepared HA-catechol-Fe3+ solution (pH 7.2) was first lyophilized and then analyzed by a reflective Raman spectrometer (LabRAM Aramis, Horiba, Japan). UV-Vis spectra were used to analyze the bind stoichiometry of catechol-Fe3+ complexes at different Fe3+ concentrations and pH. The samples containing different amount of dopamine and Fe3+ were measured by a UV-Vis Spectrometer (JASCO, Japan).In our experiments, the UVVis spectra at 3 different pHs (5.5, 7.2, 9.7 in 100 mM Tris, 50 mM NaCl) and 8 different Fe: dopamine ratios (FeCl3 :dopamine =5:1, 1:1, 1:2, 1:3, 1:5, 1:8, 1:10, 1:15 at constant pH of 7.2 in buffer containing 100 mM Tris, 50 mM NaCl.) were measured and the absorption from the buffer was subtracted. Single molecule force spectroscopy experiments.

Single molecule force spectroscopy

experiments were carried out on a commercial AFM (JPK Nanowizard II). The force-distance curves were recorded by commercial software from JPK and analyzed by custom-written procedures based on Igor pro 6.12 (Wavemetrics, Inc.). Before each force spectroscopy experiment, titanium surface was cleaned ultrasonically in deioned water for 10 min and then further cleaned by immersing into chromic acid for 30 min to remove the possible impurities. After cleaning, several drops of HA-Catechol-Fe3+ solution were deposited on the titanium surface and incubated for 20 min to make the complexes stick to the surface by nonspecific adhesion. The loosely adhered compounds were removed by rinsing the surface extensively. The fluid chamber was filled in with 1.5 mL of Tris buffer (100 mM of Tris, 50 mM of NaCl and pH 7.2 or 9.7). AFM experiments were conducted after allowing the system to equilibrate for 30 min. The cantilever was first brought to the surface with a constant speed of 1000 nm s-1, and hold for ~2 s at a constant force of ~1-2 nN to make the HA-catechol-Fe3+ compounds adsorb to


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the tip surface non-specifically. Then the cantilever was retracted with the same speed. Silicon nitride cantilevers (D type of MLCT from Bruker) were used in all experiments. The spring constants of the cantilevers were calibrated in buffer by the thermal fluctuation method, which were in the range of 0.03~0.05 N m-1. All AFM force measurements were carried out at 25 ± 1 °C. Kernel density estimation analysis. Kernel density estimation (KDE) was used to estimate the probability function of the rupture force distributions for HA-catechol-Fe3+ complexes. The kernel density estimate was calculated by a custom-written procedure based on Igor pro. We used Gaussian kernel in our calculation and thumb method to calculate bandwidth (h). Quantum chemical calculation. The geometries of the equilibrium ground state and the structures during the coordination bond dissociation of the coordination complexes were optimized by the density functional theory (DFT) with long-range M06-2X exchange-correlation functional63 and TZVP64 basis set. The excitation energy is calculated by the time-dependent density functional theory (TDDFT) combined with the same functional and basis set. During the mechanical tensile process, two terminal carbon atoms (C1 and C2 in Figure 6A) of two catechol groups were displaced from each other step by step (0.2 Å for each step). Constrained geometry optimizations were performed after each movement and the forces were evaluated by using the same method and basis set. All calculations were implemented with the consideration of water solvation by polarizable continuum model (PCM) model and carried out by using the Gaussian 09 program65.

ASSOCIATED CONTENT


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Supporting Information. Identification of HA-catechol by 1HNMR and the combination of catechol-Fe by Raman as well as UV-Vis spectrum, Kernel density estimation and quantum chemical calculation details and other experimental details. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * [email protected] * [email protected] * [email protected] Author Contributions #

These authors contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work is funded by the National Natural Science Foundation of China (Nos. 21522402, 21373109, 11374148, and 11334004), and the 973 Program of China (No. 2012CB921801 and 2013CB834100). ABBREVIATIONS


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Single Molecule Mechanics of Catechol-Iron Coordination Bonds Yiran Li,†,# Jing Wen, ‡,# Meng Qin,† Yi Cao,*,† Haibo Ma,*, ‡ and Wei Wang*,†

For Table of Contents use only


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Figure 1. Study single molecule mechanics of catechol-Fe3+ complexes by atomic force microscopy. (a) Catechol-Fe3+ complexes are widely found in mussel byssal threads, which are responsible for their high strength and toughness. DOPA residues are highlighted in orange spheres and the other amino acids are shown as blue spheres. Smaller yellow spheres represent Fe3+ ions conjugated to the proteins. (b) Schematic illustration of measuring the rupture forces for catechol-Fe3+ complexes by unraveling the HAcatechol-Fe3+ nanoparticles. The blue ribbon represents HA molecule and orange ‘Y’ shape patterns represent catechol groups. The yellow sphere is Fe3+ ions. Cartoon cantilevers are shown in green schematically pulling the assembly from the titanium surface (grey panel). (c) AFM images of HA-catecholFe3+ complexes prepared at pH 7.2 in the presence of 200 µM Fe3+. The scale bar on the upper-left corner is 10 nm. 2-D figure with color scale is shown in Figure S3 of Supporting Information. (d) Dynamic light scattering (DLS) measures the hydrodynamic diameter of HA-catechol in the absence (red) and presence (blue) of 200 µM Fe3+ at pH 7.2. 225x159mm (300 x 300 DPI)



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Figure 2. Representative force-extension curves for the unraveling of individual HA-catechol-Fe3+ nanoparticles at a pulling speed of 1000 nm s-1. The AFM cantilever was brought into contact with the titanium surface to fish individual HA-catechol-Fe3+ nanoparticles (gray traces). Stretching the single-chain nanoparticles resulted in saw-tooth like force-extension curves (black traces), corresponding to the rupture of individual catechol-Fe3+ bonds in the polymer. Red lines correspond to worm-like chain (WLC) fitting to each individual force peak. The persistence length of ~0.4 nm from WLC fitting indicates that only a single HA chain is stretched between the cantilever tip and the substrate. 64x46mm (300 x 300 DPI)


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Figure 3. Single molecule stretching-relaxation experiments of HA-catechol-Fe3+ complexes. (a) The scheme of the AFM protocol to unfold and refold a single chain HA-catechol-Fe3+ complexes. First, the complexes were stretched to break the catechol-Fe3+ bonds within the complexes while avoiding detaching the molecule from either the cantilever or the substrate. Then the molecule was relaxed to a position ~ 20 nm away from the surface and stayed there for 2 s to allow the reformation of catechol-Fe3+ complexes. In the subsequent stretching cycles, the number of catechol-Fe3+ bonds reformed during the waiting time can be estimated from the number of peaks in the force extension curves. (b) Representative force-extension curves obtained in the stretching-relaxation experiments. Because the molecule did not reach the surface in the stretching-relaxation experiments, all peaks in the stretching curves, except the first one, can be assigned as the break of single catechol-Fe3+ bonds with 100% confidence. (c) The rebinding probability after several stretching-relaxation cycles. The rebinding probability was calculated as ratio of the peaks in the consecutive curves. As no additional Fe3+ presented in the buffer, Fe3+ gradually detached from the HA-catechol chain, leading to gradually decrease of the number of force peaks in the force extension curves. The total number of force curves is 87 and the total rupture events in the first, second, third and fourth cycles are 404, 209, 158 and 163, respectively. 108x75mm (300 x 300 DPI)



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Figure 4. Fe3+ concentrations and pH modulate the mechanical strength catechol-Fe3+ complexes. (a) The rupture force distribution of catechol-Fe3+ complexes with different Fe3+ concentrations at a pulling speed of 1000 nm s-1. When the concentration of Fe3+ is low (i.e. ~ 30 µM), catechol-surface interaction dominated the rupture events. The rupture force of catechol-surface interaction is ~80 pN, consistent with our previous study. With the increase of Fe3+ concentration, the rupture force histograms show bimodal distributions, with two peaks located at ~100 pN and ~200 pN, respectively. (b) The rupture force distribution of catechol-Fe3+ nanoparticles at different pHs. At pH 7.2, there are two peaks located at ~100 pN and ~200 pN, respectively, while at pH 9.7, there is only one peak located at ~100 pN. Solid lines in the figures represent Gaussian fitting. See supporting TableS3 for statistic details. The Fe3+ concentrations were 200 µM at both pH 7.2 and pH 9.7. 136x152mm (300 x 300 DPI)


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Figure 5. Loading rate dependent rupture forces for the catechol-Fe3+ complexes. (a) Representative rupture force histograms for HA-catechol-Fe3+ complexes prepared at 200 µM Fe3+ and pH 7.2 at different pulling speeds. (b) The plot of rupture forces at different loading rates for bis-complexes (blue) and triscomplexes (red). The lines correspond to the fitting of Bell-Evans model to the experimental data. The R2 values (rupture force vs. logarithmic loading rate) are 0.901 for bis complex and 0.942 for Tris complex. (c) Free energy landscape underlying the rupture of bis- and tris- catechol-Fe3+ complexes based on the parameters extracted from the loading rate dependent experiments using Bell-Evans model. 145x156mm (300 x 300 DPI)



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Figure 6. (a-b) The optimized ground state structures of tris- and bis-catechol-Fe3+ complexes. (c) Relative energies for bis- and tris-catechol-Fe3+ complexes with different spin multiplicities under their optimized geometries by DFT calculations. (The ground state energies of sextet were taken as zero.) (d) Forcedisplacement relationships of the two complexes. (e) The evolution of four Fe-O bond lengths between Fe3+ and two pulled catechol groups of bis- (top) and tris-complex (bottom). (f) The evolution of the distance d between Fe and the C1-C2 line (top), and the change of angle∠C1FeC2 (α) in terms of increasing end-to-end displacement (bottom). 201x207mm (300 x 300 DPI)


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Single-Molecule Mechanics of Catechol-Iron Coordination Bonds

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