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1.  The microscopic network structure of mussel (Mytilus) adhesive plaques 
Journal of the Royal Society Interface  2015;12(113):20150827.
Marine mussels of the genus Mytilus live in the hostile intertidal zone, attached to rocks, bio-fouled surfaces and each other via collagen-rich threads ending in adhesive pads, the plaques. Plaques adhere in salty, alkaline seawater, withstanding waves and tidal currents. Each plaque requires a force of several newtons to detach. Although the molecular composition of the plaques has been well studied, a complete understanding of supra-molecular plaque architecture and its role in maintaining adhesive strength remains elusive. Here, electron microscopy and neutron scattering studies of plaques harvested from Mytilus californianus and Mytilus galloprovincialis reveal a complex network structure reminiscent of structural foams. Two characteristic length scales are observed characterizing a dense meshwork (approx. 100 nm) with large interpenetrating pores (approx. 1 µm). The network withstands chemical denaturation, indicating significant cross-linking. Plaques formed at lower temperatures have finer network struts, from which we hypothesize a kinetically controlled formation mechanism. When mussels are induced to create plaques, the resulting structure lacks a well-defined network architecture, showcasing the importance of processing over self-assembly. Together, these new data provide essential insight into plaque structure and formation and set the foundation to understand the role of plaque structure in stress distribution and toughening in natural and biomimetic materials.
doi:10.1098/rsif.2015.0827
PMCID: PMC4707855  PMID: 26631333
mussels; Mytilus; plaques; small-angle neutron scattering; electron microscopy
2.  An Underwater Surface-Drying Peptide Inspired by a Mussel Adhesive Protein 
Advanced functional materials  2016;26(20):3496-3507.
Water hampers the formation of strong and durable bonds between adhesive polymers and solid surfaces, in turn hindering the development of adhesives for biomedical and marine applications. Inspired by mussel adhesion, a mussel foot protein homologue (mfp3S-pep) is designed, whose primary sequence is designed to mimic the pI, polyampholyte, and hydrophobic characteristics of the native protein. Noticeably, native protein and synthetic peptide exhibit similar abilities to self-coacervate at given pH and ionic strength. 3,4-dihydroxy-l-phenylalanine (Dopa) proves necessary for irreversible peptide adsorption to both TiO2 (anatase) and hydroxyapatite (HAP) surfaces, as confirmed by quartz crystal microbalance measurements, with the coacervate showing superior adsorption. The adsorption of Dopa-containing peptides is investigated by attenuated total reflection infrared spectroscopy, revealing initially bidentate coordinative bonds on TiO2, followed by H-bonded and eventually long-ranged electrostatic and Van der Waals interactions. On HAP, mfp3s-pep-3Dopa adsorption occurs predominantly via H-bond and outer-sphere complexes of the catechol groups. Importantly, only the Dopa-bearing compounds are able to remove interfacial water from the target surfaces, with the coacervate achieving the highest water displacement arising from its superior wetting properties. These findings provide an impetus for developing coacervated Dopa-functionalized peptides/polymers adhesive formulations for a variety of applications on wet polar surfaces.
doi:10.1002/adfm.201600210
PMCID: PMC5102340  PMID: 27840600
3.  Rate-Dependent Stiffness and Recovery in Interpenetrating Network Hydrogels through Sacrificial Metal Coordination Bonds 
ACS macro letters  2015;4(11):1200-1204.
Four-arm poly(ethylene glycol) (PEG) star polymers modified with 3-hydroxy-4-pyridinone (HOPO) end groups were shown to form transient, coordination networks upon addition of trivalent cations In3+, Fe3+, and Al3+. These coordination-based hydrogels exhibited high activation energies of viscoelasticity (34 kT) and characteristic bond lifetimes tunable over 2 orders of magnitude and could be incorporated into poly(hydroxyethylacrylamide)-based covalent scaffolds to create interpenetrating network hydrogels. Measurements carried out in compression and tension demonstrate that the secondary coordination network imparts toughness and stiffness to the overall material, and unlike traditional interpenetrating networks (IPNs), the extent of toughening is dependent on the rate at which the materials are deformed. The dynamic character of the coordination network also allows recovery after mechanical damage following high amplitude strains.
doi:10.1021/acsmacrolett.5b00664
PMCID: PMC5096649  PMID: 27818845
4.  The staying power of adhesion-associated antioxidant activity in Mytilus californianus 
Journal of the Royal Society Interface  2015;12(111):20150614.
The California mussel, Mytilus californianus, adheres in the highly oxidizing intertidal zone with a fibrous holdfast called the byssus using 3, 4-dihydroxyphenyl-l-alanine (DOPA)-containing adhesive proteins. DOPA is susceptible to oxidation in seawater and, upon oxidation, loses adhesion. Successful mussel adhesion thus depends critically on controlling oxidation and reduction. To explore how mussels regulate redox during their functional adhesive lifetime, we tracked extractable protein concentration, DOPA content and antioxidant activity in byssal plaques over time. In seawater, DOPA content and antioxidant activity in the byssus persisted much longer than expected—50% of extractable DOPA and 30% of extractable antioxidant activity remained after 20 days. Antioxidant activity was located at the plaque–substrate interface, demonstrating that antioxidant activity keeps DOPA reduced for durable and dynamic adhesion. We also correlated antioxidant activity to cysteine and DOPA side chains of mussel foot proteins (mfps), suggesting that mussels use both cysteine and DOPA redox reservoirs for controlling interfacial chemistry. These data are discussed in the context of the biomaterial structure and properties of the marine mussel byssus.
doi:10.1098/rsif.2015.0614
PMCID: PMC4614499  PMID: 26468070
redox; antioxidant activity; mussels
5.  Underwater contact adhesion and microarchitecture in polyelectrolyte complexes actuated by solvent exchange 
Nature materials  2016;15(4):407-412.
Polyelectrolyte complexation is critical to the formation and properties of many biological and polymeric materials, and is typically initiated by aqueous mixing1 followed by fluid–fluid phase separation, such as coacervation2–5. Yet little to nothing is known about how coacervates evolve into intricate solid microarchitectures. Inspired by the chemical features of the cement proteins of the sandcastle worm, here we report a versatile and strong wet-contact microporous adhesive resulting from polyelectrolyte complexation triggered by solvent exchange. After premixing a catechol-functionalized weak polyanion with a polycation in dimethyl sulphoxide (DMSO), the solution was applied underwater to various substrates whereupon electrostatic complexation, phase inversion, and rapid setting were simultaneously actuated by water–DMSO solvent exchange. Spatial and temporal coordination of complexation, inversion and setting fostered rapid (~25 s) and robust underwater contact adhesion (Wad ≥ 2 J m−2) of complexed catecholic polyelectrolytes to all tested surfaces including plastics, glasses, metals and biological materials.
doi:10.1038/nmat4539
PMCID: PMC4939084  PMID: 26779881
6.  Redox Capacity of an Extracellular Matrix Protein Associated with Adhesion in Mytilus californianus 
Biochemistry  2016;55(13):2022-2030.
Adhesive mussel foot proteins (Mfps) rely in part on DOPA (3,4-dihydroxyphenyl-l-alanine) side chains to mediate attachment to mineral surfaces underwater. Oxidation of DOPA to Dopaquinone (Q) effectively abolishes the adsorption of Mfps to these surfaces. The thiol-rich mussel foot protein-6 (Mfp-6) rescues adhesion compromised by adventitious DOPA oxidation by reducing Q back to DOPA. The redox chemistry and kinetics of foot-extracted Mfp-6 were investigated by using a nonspecific chromogenic probe to equilibrate with the redox pool. Footextracted Mfp-6 has a reducing capacity of ~17 e− per protein; half of this comes from the cysteine residues, whereas the other half comes from other constituents, probably a cohort of four or five nonadhesive, redox-active DOPA residues in Mfp-6 with an anodic peak potential ~500 mV lower than that for oxidation of cysteine to cystine. At higher pH, DOPA redox reversibility is lost possibly due to Q scavenging by Cys thiolates. Analysis by one- and two-dimensional proton nuclear magnetic resonance identified a pronounced β-sheet structure with a hydrophobic core in foot-extracted Mfp-6 protein. The structure endows redox-active side chains in Mfp-6, i.e., cysteine and DOPA, with significant reducing power over a broad pH range, and this power is measurably diminished in recombinant Mfp-6.
Graphical Abstract
doi:10.1021/acs.biochem.6b00044
PMCID: PMC4934423  PMID: 26998552
7.  Sugary interfaces mitigate contact damage where stiff meets soft 
Nature Communications  2016;7:11923.
The byssal threads of the fan shell Atrina pectinata are non-living functional materials intimately associated with living tissue, which provide an intriguing paradigm of bionic interface for robust load-bearing device. An interfacial load-bearing protein (A. pectinata foot protein-1, apfp-1) with L-3,4-dihydroxyphenylalanine (DOPA)-containing and mannose-binding domains has been characterized from Atrina's foot. apfp-1 was localized at the interface between stiff byssus and the soft tissue by immunochemical staining and confocal Raman imaging, implying that apfp-1 is an interfacial linker between the byssus and soft tissue, that is, the DOPA-containing domain interacts with itself and other byssal proteins via Fe3+–DOPA complexes, and the mannose-binding domain interacts with the soft tissue and cell membranes. Both DOPA- and sugar-mediated bindings are reversible and robust under wet conditions. This work shows the combination of DOPA and sugar chemistry at asymmetric interfaces is unprecedented and highly relevant to bionic interface design for tissue engineering and bionic devices.
Robust attachment between living tissues and inert materials is challenging to achieve. Here, Hwang and co-workers look at the molecular level between tissue and embedded byssal threads of Atrina pectinata and how this affects tenacity, toughness, and robustness.
doi:10.1038/ncomms11923
PMCID: PMC4912638  PMID: 27305949
8.  Mussel adhesive protein provides cohesive matrix for collagen type-1α 
Biomaterials  2015;51:51-57.
Understanding the interactions between collagen and adhesive mussel foot proteins (mfps) can lead to improved medical and dental adhesives, particularly for collagen-rich tissues. Here we investigated interactions between collagen type-1, the most abundant loadbearing animal protein, and mussel foot protein-3 (mfp-3) using a quartz crystal microbalance and surface forces apparatus (SFA). Both hydrophilic and hydrophobic variants of mfp-3 were exploited to probe the nature of the interaction between the protein and collagen. Our chief findings are: 1) mfp-3 is an effective chaperone for tropocollagen adsorption to TiO2 and mica surfaces; 2) at pH 3, collagen addition between two mfp-3 films (Wc = 5.4 ± 0.2 mJ/m2) increased their cohesion by nearly 35%; 3) oxidation of Dopa in mfp-3 by periodate did not abolish the adhesion between collagen and mfp-3 films, and 4) collagen bridging between both hydrophilic and hydrophobic mfp-3 variant films is equally robust, suggesting that hydrophobic interactions play a minor role. Extensive H-bonding, π-cation and electrostatic interactions are more plausible to explain the reversible bridging of mfp-3 films by collagen.
doi:10.1016/j.biomaterials.2015.01.033
PMCID: PMC4361793  PMID: 25770997
mussel foot proteins; collagen type-1; mfp-3
9.  α,β-Dehydro-Dopa: A Hidden Participant in Mussel Adhesion 
Biochemistry  2016;55(5):743-750.
Dopa (L-3,4-dihydroxyphenylalanine) is a key chemical signature of mussel adhesive proteins, but its susceptibility to oxidation has limited mechanistic investigations as well as practical translation to wet adhesion technology. To investigate peptidyl-Dopa oxidation, the highly diverse chemical environment of Dopa in mussel adhesive proteins was simplified to a peptidyl-Dopa analogue, N-acetyl-Dopa ethyl ester. On the basis of cyclic voltammetry and UV–vis spectroscopy, the Dopa oxidation product at neutral to alkaline pH was shown to be α,β-dehydro-Dopa (ΔD), a vinylcatecholic tautomer of Dopa-quinone. ΔD exhibited an adsorption capacity on TiO2 20-fold higher than that of the Dopa homologue in the quartz crystal microbalance. Cyclic voltammetry confirmed the spontaneity of ΔD formation in mussel foot protein 3F at neutral pH that is coupled to a change in protein secondary structure from random coil to β-sheet. A more complete characterization of ΔD reactivity adds a significant new perspective to mussel adhesive chemistry and the design of synthetic bioinspired adhesives.
Graphical abstract
doi:10.1021/acs.biochem.5b01177
PMCID: PMC4760353  PMID: 26745013
10.  Bridging Adhesion of Mussel-Inspired Peptides: Role of Charge, Chain Length, and Surface Type 
Langmuir  2014;31(3):1105-1112.
The 3,4-dihydroxyphenylalanine (Dopa)-containing proteins of marine mussels provide attractive design paradigms for engineering synthetic polymers that can serve as high performance wet adhesives and coatings. Although the role of Dopa in promoting adhesion between mussels and various substrates has been carefully studied, the context by which Dopa mediates a bridging or nonbridging macromolecular adhesion to surfaces is not understood. The distinction is an important one both for a mechanistic appreciation of bioadhesion and for an intelligent translation of bioadhesive concepts to engineered systems. On the basis of mussel foot protein-5 (Mfp-5; length 75 res), we designed three short, simplified peptides (15–17 res) and one relatively long peptide (30 res) into which Dopa was enzymatically incorporated. Peptide adhesion was tested using a surface forces apparatus. Our results show that the short peptides are capable of weak bridging adhesion between two mica surfaces, but this adhesion contrasts with that of full length Mfp-5, in that (1) while still dependent on Dopa, electrostatic contributions are much more prominent, and (2) whereas Dopa surface density remains similar in both, peptide adhesion is an order of magnitude weaker (adhesion energy Ead ∼ −0.5 mJ/m2) than full length Mfp-5 adhesion. Between two mica surfaces, the magnitude of bridging adhesion was approximately doubled (Ead ∼ −1 mJ/m2) upon doubling the peptide length. Notably, the short peptides mediate much stronger adhesion (Ead ∼ −3.0 mJ/m2) between mica and gold surfaces, indicating that a long chain length is less important when different interactions are involved on each of the two surfaces.
doi:10.1021/la504316q
PMCID: PMC4310636  PMID: 25540823
11.  Layer-by-layer polyelectrolyte deposition: a mechanism for forming biocomposite materials 
Biomacromolecules  2013;14(6):1715-1726.
Complex coacervates prepared from poly-Aspartic acid (polyAsp) and poly-L-Histidine (polyHis) were investigated as models of the metastable protein phases used in the formation of biological structures such as squid beak. When mixed, polyHis and polyAsp form coacervates whereas poly-L-Glutamic acid (polyGlu) forms precipitates with polyHis. Layer-by-layer (LbL) structures of polyHis-polyAsp on gold substrates were compared with those of precipitate-forming polyHis-polyGlu by monitoring with iSPR and QCM-D. PolyHis-polyAsp LbL was found to be stiffer than polyHis-polyGlu LbL with most water evicted from the structure but with sufficient interfacial water remaining for molecular rearrangement to occur. This thin layer is believed to be fluid and like preformed coacervate films, capable of spreading over both hydrophilic ethylene glycol as well as hydrophobic monolayers. These results suggest that coacervate-forming polyelectrolytes deserve consideration for potential LbL applications and point to LbL as an important process by which biological materials form.
doi:10.1021/bm400448w
PMCID: PMC4104756  PMID: 23600626
Complex coacervates; biomaterials; iSPR; QCM; polyelectrolytes; Layer-by-layer
12.  High-performance mussel-inspired adhesives of reduced complexity 
Nature Communications  2015;6:8663.
Despite the recent progress in and demand for wet adhesives, practical underwater adhesion remains limited or non-existent for diverse applications. Translation of mussel-inspired wet adhesion typically entails catechol functionalization of polymers and/or polyelectrolytes, and solution processing of many complex components and steps that require optimization and stabilization. Here we reduced the complexity of a wet adhesive primer to synthetic low-molecular-weight catecholic zwitterionic surfactants that show very strong adhesion (∼50 mJ m−2) and retain the ability to coacervate. This catecholic zwitterion adheres to diverse surfaces and self-assembles into a molecularly smooth, thin (<4 nm) and strong glue layer. The catecholic zwitterion holds particular promise as an adhesive for nanofabrication. This study significantly simplifies bio-inspired themes for wet adhesion by combining catechol with hydrophobic and electrostatic functional groups in a small molecule.
Mussels use strong filaments to adhere to rocks, preventing them from being swept away in strong currents. Here, the authors borrow and simplify chemistries from the mussel foot to create a one component adhesive system which holds potential for employment in nanofabrication protocols.
doi:10.1038/ncomms9663
PMCID: PMC4667698  PMID: 26478273
13.  Halogenated DOPA in a Marine Adhesive Protein 
The Journal of adhesion  2009;85(2-3):126.
The sandcastle worm Phragmatopoma californica, a marine polychaete, constructs a tube-like shelter by cementing together sand grains using a glue secreted from the building organ in its thorax. The glue is a mixture of post-translationally modified proteins, notably the cement proteins Pc-1 and Pc-2 with the amino acid, 3,4-dihydroxyphenyl-L-alanine (DOPA). Significant amounts of a halogenated derivative of DOPA were isolated from the worm cement following partial acid hydrolysis and capture of catecholic amino acids by phenylboronate affinity chromatography. Analysis by tandem mass spectrometry and 1H NMR indicates the DOPA derivative to be 2-chloro-4, 5-dihydroxyphenyl-L-alanine. The potential roles of 2-chloro-DOPA in chemical defense and underwater adhesion are considered.
doi:10.1080/00218460902782188
PMCID: PMC2757296  PMID: 20126508
2-chloro-4,5-dihydroxyphenylalanine; Adhesive protein; Cement; Phragmatopoma californica; Sandcastle worm
14.  Mussel Coating Protein-Derived Complex Coacervates Mitigate Frictional Surface Damage 
The role of friction in the functional performance of biomaterial interfaces is widely reckoned to be critical and complicated but poorly understood. To better understand friction forces, we investigated the natural adaptation of the holdfast or byssus of mussels that live in high-energy surf habitats. As the outermost covering of the byssus, the cuticle deserves particular attention for its adaptations to frictional wear under shear. In this study, we coacervated one of three variants of a key cuticular component, mussel foot protein 1, mfp-1 [(1) Mytilus californianus mcfp-1, (2) rmfp-1, and (3) rmfp-1-Dopa], with hyaluronic acid (HA) and investigated the wear protection capabilities of these coacervates to surfaces (mica) during shear. Native mcfp-1/HA coacervates had an intermediate coefficient of friction (μ ∼0.3) but conferred excellent wear protection to mica with no damage from applied loads, F⊥, as high as 300 mN (pressure, P, > 2 MPa). Recombinant rmfp-1/HA coacervates exhibited a comparable coefficient of friction (μ ∼0.3); however, wear protection was significantly inferior (damage at F⊥ > 60 mN) compared with that of native protein coacervates. Wear protection of rmfp-1/HA coacervates increased 5-fold upon addition of the surface adhesive group 3,4-dihydroxyphenylalanine, (Dopa). We propose a Dopa-dependent wear protection mechanism to explain the differences in wear protection between coacervates. Our results reveal a significant untapped potential for coacervates in applications that require adhesion, lubrication, and wear protection. These applications include artificial joints, contact lenses, dental sealants, and hair and skin conditioners.
doi:10.1021/acsbiomaterials.5b00252
PMCID: PMC4642218  PMID: 26618194
biomimetic; adhesion; wear protection; interface; Mytilus californianus foot protein 1; mcfp-1; hyaluronic acid; HA
16.  Tough Coating Proteins: Subtle Sequence Variation Modulates Cohesion 
Biomacromolecules  2015;16(3):1002-1008.
Mussel foot protein-1 (mfp-1) is an essential constituent of the protective cuticle covering all exposed portions of the byssus (plaque and the thread) that marine mussels use to attach to intertidal rocks. The reversible complexation of Fe3+ by the 3,4-dihydroxyphenylalanine (Dopa) side chains in mfp-1 in Mytilus californianus cuticle is responsible for its high extensibility (120%) as well as its stiffness (2 GPa) due to the formation of sacrificial bonds that help to dissipate energy and avoid accumulation of stresses in the material. We have investigated the interactions between Fe3+ and mfp-1 from two mussel species, M. californianus (Mc) and M. edulis (Me), using both surface sensitive and solution phase techniques. Our results show that although mfp-1 homologues from both species bind Fe3+, mfp-1 (Mc) contains Dopa with two distinct Fe3+-binding tendencies and prefers to form intramolecular complexes with Fe3+. In contrast, mfp-1 (Me) is better adapted to intermolecular Fe3+ binding by Dopa. Addition of Fe3+ did not significantly increase the cohesion energy between the mfp-1 (Mc) films at pH 5.5. However, iron appears to stabilize the cohesive bridging of mfp-1 (Mc) films at the physiologically relevant pH of 7.5, where most other mfps lose their ability to adhere reversibly. Understanding the molecular mechanisms underpinning the capacity of M. californianus cuticle to withstand twice the strain of M. edulis cuticle is important for engineering of tunable strain tolerant composite coatings for biomedical applications.
doi:10.1021/bm501893y
PMCID: PMC4514026  PMID: 25692318
17.  Interfacial pH during mussel adhesive plaque formation 
Biofouling  2015;31(2):221-227.
Mussel (Mytilus californianus) adhesion to marine surfaces involves an intricate and adaptive synergy of molecules and spatio-temporal processes. Although the molecules, such as mussel foot proteins (mfps), are well characterized, deposition details remain vague and speculative. Developing methods for the precise surveillance of conditions that apply during mfp deposition would aid both in understanding mussel adhesion and translating this adhesion into useful technologies. To probe the interfacial pH at which mussels buffer the local environment during mfp deposition, a lipid bilayer with tethered pH-sensitive fluorochromes was assembled on mica. The interfacial pH during foot contact with modified mica ranged from 2.2−3.3, which is well below the seawater pH of ~8. The acidic pH serves multiple functions: it limits mfp-Dopa oxidation, thereby enabling the catecholic functionalities to adsorb to surface oxides by H-bonding and metal ion coordination, and provides a solubility switch for mfps, most of which aggregate at pH ≥ 7-8.
doi:10.1080/08927014.2015.1026337
PMCID: PMC4420479  PMID: 25875963
Dopa; mussel interfacial pH; pH sensitive surface; Oregon Green 488 DHPE
18.  A Mussel-Derived One-Component Adhesive Coacervate 
Acta biomaterialia  2013;10(4):1663-1670.
Marine organisms process and deliver many of their underwater coatings and adhesives as complex fluids. In marine mussels, one such fluid, secreted during the formation of adhesive plaques, consists of a concentrated colloidal suspension of a mussel foot protein (mfp) known as Mfp-3S. Results of this study suggest that Mfp-3S becomes a complex fluid by a liquid-liquid phase separation from equilibrium solution at a pH and ionic strength reminiscent of conditions created by the mussel foot during plaque formation. The pH dependence of phase separation and its sensitivity indicate that inter/intra-molecular electrostatic interactions are partially responsible for driving the phase separation. Hydrophobic interactions between the nonpolar Mfp-3S proteins provide another important driving force for coacervation. As complex coacervation typically results from charge-charge interactions between polyanions and polycations, Mfp-3S is thus unique in being the only known protein that coacervates with itself. The Mfp-3S coacervate was shown to have an effective interfacial energy of ≤ 1 mJ/m2 which explains its tendency to spread over or engulf most surfaces. Of particular interest to biomedical applications is the extremely high adsorption capacity of coacervated Mfp-3S on hydroxyapatite.
doi:10.1016/j.actbio.2013.09.007
PMCID: PMC3960351  PMID: 24060881
19.  Structural Proteins from Whelk Egg Capsule with Long Range Elasticity Associated with a Solid-state Phase Transition 
Biomacromolecules  2014;15(1):30-42.
The robust, proteinaceous egg capsules of marine prosobranch gastropods (genus Busycotypus) exhibit unique biomechanical properties such as high elastic strain recovery and elastic energy dissipation capability. Capsule material possesses long-range extensibility that is fully recoverable and is the result of a secondary structure phase transition from α-helix to extended β-sheet rather than of entropic (rubber) elasticity. We report here the characterization of the precursor proteins that make up this material. Three different proteins have been purified and analyzed, and complete protein sequences deduced from messenger ribonucleic acid (mRNA) transcripts. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectra indicate that the proteins are strongly α-helical in solution and primary sequence analysis suggests that these proteins have a propensity to form coiled-coils. This is in agreement with previous wide-angle x-ray scattering (WAXS) and solid-state Raman spectroscopic analysis of mature egg capsules.
doi:10.1021/bm401598z
PMCID: PMC3956310  PMID: 24350603
Egg capsule; elasticity; coiled-coil; α-β transition; shape memory; self-healing
20.  Optimized DPPH assay in a detergent-based buffer system for measuring antioxidant activity of proteins 
MethodsX  2014;1:233-238.
The 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) assay is well established for the in vitro determination of antioxidant activity in food and biological extracts. The standard DPPH assay uses methanol or ethanol as solvents, or buffered alcoholic solutions in a ratio of 40%/60% (buffer/alcohol, v/v) to keep the hydrophobic hydrazyl radical and phenolic test compounds soluble while offering sufficient buffering capacity at different pHs tested. Following this protocol, we were unable to keep proteinaceous antioxidants soluble at different pHs to test for their antioxidant activity. Thus, the assay protocol was modified as follows to improve its utility: Non-ionic detergents were added to keep the DPPH radical soluble and to provide a mild and non-denaturing environment for the antioxidant protein.Maximal concentration of DPPH was limited to 100 μM to stay within the sensitivity range of the detector at the given wavelength (515 nm) and to increase the dynamic range of the assay.0.1 M citrate phosphate buffer was introduced to prevent experimental artifacts due to changing buffer compositions at different pHs
doi:10.1016/j.mex.2014.10.004
PMCID: PMC4268772  PMID: 25530949
DPPH radical; antioxidant proteins; non-ionic detergent; pH range
21.  Biomimetic Control of Calcite Morphology with Homopolyanions 
Crystal growth & design  2009;9(10):4335-4343.
Biomineralization is an intricate process that relies on precise physiological control of solution and interface properties. Despite much research of the process, mechanistic details of biomineralization are only beginning to be understood, and studies of additives seldom investigate a wide space of chemical conditions in mineralizing solutions. We present a ternary diagram-based method that globally identifies the changing roles and effects of polymer additives in mineralization. Simple polyanions were demonstrated to induce a great variety of morphologies, each of which can be selectively and reproducibly fabricated. This chemical and physical analysis also aided in identifying conditions that selectively promote heterogeneous nucleation and controlled cooperative assembly, manifested here in the form of highly organized cones. Similar complex shapes of CaCO3 have previously been synthesized using double hydrophilic block copolymers. We have found the biomimetic mineralization process to occur interfacially and by the assembly of precursor modules, which generate large mesocrystals with high dependence on pH and substrate surface.
doi:10.1021/cg900166u
PMCID: PMC2782844  PMID: 20161392
22.  Versatile tuning of supramolecular hydrogels through metal complexation of oxidation-resistant catechol-inspired ligands 
Soft matter  2013;9(43):10.1039/C3SM51824H.
The mussel byssal cuticle employs DOPA-Fe3+ complexation to provide strong, yet reversible crosslinking. Synthetic constructs employing this design motif based on catechol units are plagued by oxidation-driven degradation of the catechol units and the requirement for highly alkaline pH conditions leading to decreased performance and loss of supramolecular properties. Herein, a platform based on a 4-arm poly(ethylene glycol) hydrogel system is used to explore the utility of DOPA analogues such as the parent catechol and derivatives, 4-nitrocatechol (nCat) and 3-hydroxy-4-pyridinonone (HOPO), as structural crosslinking agents upon complexation with metal ions. HOPO moieties are found to hold particular promise, as robust gelation with Fe3+ occurs at physiological pH and is found to be largely resistant to oxidative degradation. Gelation is also shown to be triggered by other biorelevant metal ions such as Al3+, Ga3+ and Cu2+ which allows for tuning of the release and dissolution profiles with potential application as injectable delivery systems.
doi:10.1039/C3SM51824H
PMCID: PMC3838863  PMID: 24285981
23.  Optimized DPPH assay in a detergent-based buffer system for measuring antioxidant activity of proteins 
MethodsX  2014;1:233-238.
Graphical abstract
The free radical method using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) is a well established assay for the in vitro determination of antioxidant activity in food and biological extracts. The standard DPPH assay uses methanol or ethanol as solvents, or buffered alcoholic solutions in a ratio of 40%/60% (buffer/alcohol, v/v) to keep the hydrophobic hydrazyl radical and phenolic test compounds soluble while offering sufficient buffering capacity at different pHs tested. Following this protocol, we were unable to keep proteinaceous antioxidants soluble at different pHs to test for their antioxidant activity. Thus, the assay protocol was modified as follows to improve its utility:•Non-ionic detergents were added to keep the DPPH radical soluble and to provide a mild and non-denaturing environment for the antioxidant protein.•Maximal concentration of DPPH was limited to 100 μM to stay within the sensitivity range of the detector at the given wavelength (515 nm) and to increase the dynamic range of the assay.•0.1 M citrate phosphate buffer was introduced to prevent experimental artifacts due to changing buffer compositions at different pHs.
doi:10.1016/j.mex.2014.10.004
PMCID: PMC4268772  PMID: 25530949
DPPH radical; Antioxidant proteins; Non-ionic detergent; pH range
24.  Boronate Complex Formation with Dopa Containing Mussel Adhesive Protein Retards pH-Induced Oxidation and Enables Adhesion to Mica 
PLoS ONE  2014;9(10):e108869.
The biochemistry of mussel adhesion has inspired the design of surface primers, adhesives, coatings and gels for technological applications. These mussel-inspired systems often focus on incorporating the amino acid 3,4-dihydroxyphenyl-L-alanine (Dopa) or a catecholic analog into a polymer. Unfortunately, effective use of Dopa is compromised by its susceptibility to auto-oxidation at neutral pH. Oxidation can lead to loss of adhesive function and undesired covalent cross-linking. Mussel foot protein 5 (Mfp-5), which contains ∼30 mole % Dopa, is a superb adhesive under reducing conditions but becomes nonadhesive after pH-induced oxidation. Here we report that the bidentate complexation of borate by Dopa to form a catecholato-boronate can be exploited to retard oxidation. Although exposure of Mfp-5 to neutral pH typically oxidizes Dopa, resulting in a>95% decrease in adhesion, inclusion of borate retards oxidation at the same pH. Remarkably, this Dopa-boronate complex dissociates upon contact with mica to allow for a reversible Dopa-mediated adhesion. The borate protection strategy allows for Dopa redox stability and maintained adhesive function in an otherwise oxidizing environment.
doi:10.1371/journal.pone.0108869
PMCID: PMC4193769  PMID: 25303409
25.  Asymmetric collapse in biomimetic complex coacervates revealed by local polymer and water dynamics 
Biomacromolecules  2013;14(5):1395-1402.
Complex coacervation is a phenomenon characterized by the association of oppositely charged polyelectrolytes into micron-scale liquid condensates. This process is the purported first step in the formation of underwater adhesives by sessile marine organisms, as well as the process harnessed for the formation of new synthetic and protein-based contemporary materials. Efforts to understand the physical nature of complex coacervates are important for developing robust adhesives, injectable materials, or novel drug delivery vehicles for biomedical applications, however their internal fluidity necessitates the use of in situ characterization strategies of their local dynamic properties—capabilities not offered by conventional techniques such as x-ray scattering, microscopy, or bulk rheological measurements. Herein, we employ the novel magnetic resonance technique Overhauser dynamic nuclear polarization enhanced nuclear magnetic resonance (DNP), together with electron paramagnetic resonance (EPR) lineshape analysis, to concurrently quantify local molecular and hydration dynamics, with species- and site-specificity. We observe striking differences in the structure and dynamics of the protein-based biomimetic complex coacervates from their synthetic analogues, which is an asymmetric collapse of the polyelectrolyte constituents. From this study we suggest charge heterogeneity within a given polyelectrolyte chain to be an important parameter by which the internal structure of complex coacervates may be tuned. Acquiring molecular-level insight to the internal structure and dynamics of dynamic polymer complexes in water through the in situ characterization of site- and species-specific local polymer and hydration dynamics should be a promising general approach that has not been widely employed for materials characterization.
doi:10.1021/bm4000579
PMCID: PMC4090114  PMID: 23540713
Complex coacervates; DNP; EPR; Mytilus Foot Protein; Mussel adhesive protein

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