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Protamines are natural polyguanidiniums, arginine(R)-rich proteins involved in the compaction of chromatin during vertebrate spermatogenesis. Salmine, a protamine isolated from salmon sperm, contains 65 mol% R residues, with positively charged guanidino (Gdm+) sidechains, and no other amino acids with ionizable or aromatic sidechains. Salmine sulfate solutions undergo liquid-liquid phase separation (LLPS) with a concentration-dependent upper critical solution temperature (UCST). The condensed liquid phase comprises 50 wt % water and &gt, 600 mg&middot, ml&minus, 1 salmine with a constant 1:2 ratio of sulfate (SO42&minus, ) to Gdm+. Isothermal titration calorimetry, titrating Na2SO4 into salmine chloride above and below the UCST, allowed isolation of exothermic sulfate binding to salmine chloride from subsequent endothermic condensation and exothermic phase separation events. Synthetic random polyacrylate analogs of salmine, with 3-guanidinopropyl sidechains, displayed similar counterion dependent phase behavior, demonstrating that the LLPS of polyguanidiniums does not depend upon subunit sequence or polymer backbone chirality, and was due entirely to Gdm+ sidechain interactions. The results provide experimental evidence for like-charge pairing of Gdm+ sidechains, and an experimental approach for further characterizing these interactions.

Nuclear magnetic resonance (NMR) and X-ray diffraction (XRD) experiments reveal the structural importance of divalent cation-phosphate complexes in the formation of β-sheet nanocrystals from phosphorylated serine-rich regions within aquatic silk from caddisfly larvae of the species Hesperophyla consimilis. Wide angle XRD data on native caddisfly silk show that the silk contains a significant crystalline component with a repetitive orthorhombic unit cell aligned along the fiber axis with dimensions of 5.9 Å × 23.2 Å × 17.3 Å. These nanocrystalline domains depend on multivalent cations, which can be removed through chelation with ethylenediaminetetraacetic acid (EDTA). A comparison of wide angle X-ray diffraction data before and after EDTA treatment reveals that the integrated peak area of reflections corresponding to the nanocrystalline regions decreases by 15-25% while that of the amorphous background reflections increases by 20%, indicating a partial loss of crystallinity. (31)P solid-state NMR data on native caddisfly silk also show that the phosphorylated serine-rich motifs transform from a rigid environment to one that is highly mobile and water-solvated after treatment with EDTA. The removal of divalent cations through exchange and chelation has therefore caused a collapse of the β-sheet structure. However, NMR results show that the rigid phosphorus environment is mostly recovered after the silk is re-treated with calcium. The (31)P spin-lattice (T1) relaxation times were measured at 7.6 ± 3.1 and 1 ± 0.5 s for this calcium-recovered sample and the native silk sample, respectively. The shorter (31)P T1 relaxation times measured for the native silk sample are attributed to the presence of paramagnetic iron that is stripped away during EDTA chelation treatment and replaced with diamagnetic calcium.

SUMMARY The marine sandcastle worm bonds mineral particles together into underwater composite dwellings with a proteinaceous glue. The products of at least four distinct secretory cell types are co-secreted from the building organ to form the glue. Prominent hetereogeneous granules contain dense sub-granules of Mg and the (polyphospho)proteins Pc3A and B, as well as at least two polybasic proteins, Pc1 and Pc4, as revealed by immunolabeling with specific antibodies against synthetic peptides. Equally prominent homogeneous granules comprise at least two polybasic proteins, Pc2 and Pc5, localized by immunolabeling with anti-synthetic peptide antibodies. The components of the sub-micrometer granule types are unknown, though positive staining with a redox-sensitive dye suggests the contents include o-dihydroxy-phenylalanine (dopa). Quantitative PCR and in situ hybridization demonstrated that a tyrosinase-like enzyme with a signal peptide was highly expressed in both the heterogeneous and homogeneous granules. The contents of the granules are poorly mixed in the secreted mixture that forms the glue. Subsequent covalent cross-linking of the glue may be catalyzed by the co-secreted tyrosinase. The first three parapodia of the sandcastle worm also contain at least two distinct secretory tissues. The Pc4 protein was immunolocalized to the anterior secretory cells and the tryosinase-like gene was expressed in the posterior secretory cells, which suggests these proteins may have multiple roles.

This paper describes the design, construction and characterization of the first anti-HIV drug delivery system that is triggered to release its contents in the presence of human semen. Microgel particles were synthesized with a crosslinker containing a peptide substrate for the seminal serine protease prostate specific antigen (PSA) and were loaded with the HIV-1 entry inhibitor sodium poly(styrene-4-sulfonate) (pSS). The particles were composed of N-2-hydroxyproplymethacrylamide and bis-methacrylamide functionalized peptides based on the PSA substrates GISSFYSSK and GISSQYSSK. Exposure to human seminal plasma (HSP) degraded the microgel network and triggered the release of the entrapped antiviral polymer. Particles with the crosslinker composed of the substrate GISSFYSSK showed 17 times faster degradation in seminal plasma than that of the crosslinker composed of GISSQYSSK. The microgel particles containing 1 mol% GISSFYSSK peptide crosslinker showed complete degradation in 30 hours in the presence of HSP at 37 °C and pSS released from the microgels within 30 minutes reached a concentration of 10 µg/mL, equivalent to the published IC90 for pSS. The released pSS inactivated HIV-1 in the presence of HSP. The solid phase synthesis of the crosslinkers, preparation of the particles by inverse microemulsion polymerization, HSP-triggered release of pSS and inactivation of HIV-1 studies are described.

Random clones were sequenced from a cDNA library constructed from the adhesive gland of Phragmatopoma californica, a marine polycheate that builds protective shells by gluing together sand grains and biogenic mineral fragments. As many as 14 new proteins and two phenoloxidase enzymes were found that may be structural components of or involved in processing the bioadhesive. Glue protein classification was based on the following criteria: (i) the presence of predicted secretion signal peptides, (ii) low complexity sequences, (iii) strongly skewed amino acid compositions enriched with G, Y, K, H, A, or S, (iv) repeating peptide motifs, and (v) homology to known glue proteins, other structural proteins, or enzymes. The new genes provide probes for further characterization of the adhesive gland as well as potential biotechnological resources and insight.

Polyacrylate glue protein analogs of the glue secreted by Phragmatopoma californica, a marine polycheate, were synthesized with phosphate, primary amine, and catechol sidechains with molar ratios similar to the natural glue proteins. Aqueous mixtures of the mimetic polyelectrolytes condensed into liquid complex coacervates around neutral pH. Wet cortical bone specimens bonded with the coacervates, oxidatively crosslinked through catechol sidechains, had bond strengths nearly 40% of the strength of a commercial cyanoacrylate. The unique material properties of complex coacervates may be ideal for development of clinically useful adhesives and other biomaterials.

A new method was developed for modifying gold electrode surfaces through electrochemically triggered adsorption of acrylamide copolymers containing a low percentage of tyrosineamide side chains. The amount of copolymer adsorbed, as monitored by surface plasmon resonance (SPR), was proportional to the percent of tyrosineamide side chains in the polymer over the range 0−3 mol %. The modified gold surfaces were hydrophilic and resisted nonspecific adsorption of green fluorescent protein (GFP). Incorporation of nickel-binding nitrilotriacetic acid (NTA) side chains into the tyrosineamide copolymers allowed specific immobilization of His6-tagged GFP. The Ni(II)-dependent GFP binding was measured by SPR and verified by fluorescence microscopy. The method may find utility as a means to electrically address the immobilization of unique ligands in biosensors or other diagnostic devices based on arrayed ligands.

Dityrosine cross-links are widely observed in nature in structural proteins such as elastin and silk. Natural oxidative cross-linking between tyrosine residues is catalyzed by a diverse group of metalloenzymes. Dityrosine formation is also catalyzed in vitro by metal-peptide complexes such as Gly-Gly-His-Ni(II). On the basis of these observations, a system was developed to specifically and covalently surface immobilize proteins through dityrosine cross-links. Methacrylate monomers of the catalytic peptide Gly-Gly-His-Tyr-OH (GGHY) and the Ni(II)-chelating group nitrilotriacetic acid (NTA) were copolymerized with acrylamide into microbeads. Green fluorescent protein (GFP), as a model protein, was genetically tagged with a tyrosine-modified His6 peptide on its carboxy terminus. GFP-YGH6, specifically associated with the NTA-Ni(II) groups, was covalently coupled to the bead surface through dityrosine bond formation catalyzed by the colocalized GGHY-Ni(II) complex. After extensive washing with EDTA to disrupt metal coordination bonds, we observed that up to 75% of the initially bound GFP-YGH6 remained covalently bound to the bead while retaining its structure and activity. Dityrosine cross-linking was confirmed by quenching the reaction with free tyrosine. The method may find particular utility in the construction and optimization of protein microarrays.

The mineralized tube of the sandcastle worm Phragmatopoma californica is made from exogenous mineral particles (sand, shell, etc.) glued together with a cement secreted from the "building organ" on the thorax of the worm. The glue is a cross-linked mixture of three highly polar proteins. The complete sequences of Pc-1 (18 kDa) and Pc-2 (21 kDa) were deduced from cDNAs derived from previously reported peptide sequences (Waite, J. H., Jensen, R., and Morse, D. E. (1992) Biochemistry 31, 5733-5738). Both proteins are basic (pI approximately 10) and exhibit Gly-rich peptide repeats. The consensus repeats in Pc-1 and -2 are VGGYGYGGKK (15 times), and HPAVXHKALGGYG (eight times), respectively, in which X denotes an intervening nonrepeated sequence and Y is modified to 3,4-dihydroxyphenyl-l-alanine (Dopa). The third protein, Pc-3, was deduced from the cement to be about 80 mol % phosphoserine/serine, and the cDNA was obtained by exploiting the presence of poly-serine repeats. Pc-3 consists of a family of at least seven variants with 60-90 mol % serine most of which is phosphorylated in the cement. Pc-1, -2, and -3 contain cysteine some of which reacts to form 5-S-cysteinyl-Dopa cross-links during the setting process.

SUMMARY Phragmatopoma californica is a marine polychaete that builds protective tubes by joining bits of shell and sand grains with a secreted proteinaceous cement. The cement forms a solid foam (closed cells) via covalent crosslinking, as revealed by electron and laser scanning confocal microscopy. The cement contains extractable calcium and magnesium,and non-extractable phosphorus. Amino acid analysis demonstrated that the phosphorus is in the form of phosphoserine and that >90% of serine in the cement (i.e. 28 mol% of residues) is phosphorylated. In addition to previously identified basic proteins, the cement contains a highly acidic polyphosphoserine protein as a major component. We propose a model for the structure and bonding mechanism of the cement that has the following major features: (1) within the secretory pathway of cement gland cells, the electrostatic association of the oppositely charged proteins and divalent cations (Ca2+ and Mg2+) condense the cement proteins into dehydrated secretory granules; (2) the condensation of the cement leads to the separation of the solution into two aqueous phases (complex coacervation) that creates the closed cell foam structure of the cement; (3)rehydration of the condensed cement granules after deposition onto tube particles contributes to the displacement of water from the mineral substrate to facilitate underwater adhesion; and (4) after secretion, covalent cross-linking through oxidative coupling of DOPA gradually solidifies the continuous phase of the cement to set the porous structure.

A model describing swelling changes of covalently crosslinked gels caused by unfolding of crosslinks of folded chains built-in in the gel matrix was proposed. By unfolding, a new interacting surface is exposed which may differ from the interacting sites of the gel polymer. Non-ionic as well as ionic interactions are considered. A certain force generated by the swelling pressure acts on the ends of the folded crosslinks and unfolding starts when this force exceeds a certain limiting value. The continuation or arrest of unfolding depends on whether the exposure of the new interacting surface decreases or increases this force. The unfolding process is considered to be irreversible but possible reversibility can be considered. This model relates to biohybrid gels in which the folded domain of the crosslink is composed of elements of the muscle protein titin. The model is based on the statistical-mechanical theory of swelling of crosslinked polymers. A change in external variables such as temperature or degree of ionization can cause an abrupt change in volume of the swollen gel (transition) within a certain range of parameters characterizing the gel and polymer-solvent interactions.

Hybrid hydrogels of hydrophilic synthetic polymers cross-linked by protein modules undergo externally triggered volume transitions as a result of protein conformational changes. To investigate the influence of coiled-coil protein structure and stability on hydrogel volume transition, a series of block proteins containing interspersed naturally derived recombinant coiled-coils was synthesized. Proteins were characterized using circular dichroism, size exclusion chromatography, gel electrophoresis, and analytical ultracentrifugation. The block proteins formed self-associating oligomers and displayed thermal unfolding profiles indicative of a hierarchic higher-order structure. Hybrid hydrogels were assembled from an N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer and His-tagged block proteins through metal complexation. A temperature-induced decrease in hydrogel swelling was observed, and the onset temperature of the volume transition corresponded to the onset temperature of protein unfolding. We conclude that stimuli-responsive properties of hybrid hydrogels can be tailored by engineering the structure and properties of protein cross-links.

Bacterial bioluminescence, catalyzed by FMN:NAD(P)H oxidoreductase and luciferase, has been used as an analytical tool for quantitating the substrates of NAD(P)H-dependent enzymes. The development of inexpensive and sensitive biosensors based on bacterial bioluminescence would benefit from a method to immobilize the oxidoreductase and luciferase with high specific activity. Toward this end, oxidoreductase and luciferase were fused with a segment of biotin carboxy carrier protein and produced in Escherichia coli. The in vivo biotinylated luciferase and oxidoreductase were immobilized on avidin-conjugated agarose beads with little loss of activity. Coimmobilized enzymes had eight times higher bioluminescence activity than the free enzymes at low enzyme concentration and high NADH concentration. In addition, the immobilized enzymes were more stable than the free enzymes. This immobilization method is also useful to control enzyme orientation, which could increase the efficiency of sequentially operating enzymes like the oxidoreductase-luciferase system.

Tube-building sabellariid polychaetes have major impacts on the geology and ecology of shorelines worldwide. Sandcastle worms, Phragmatopoma californica (Fewkes), live along the western coast of North America. Individual sabellariid worms build tubular shells by gluing together mineral particles with a multipart polyelectrolytic adhesive. Distinct sets of oppositely charged components are packaged and stored in concentrated granules in separate cell types. Homogeneous granules contain sulfated macromolecules as counter-polyanion to polycationic Pc2 and Pc5 proteins, which become major components of the fully cured glue. Heterogeneous granules contain polyphosphoproteins, Pc3A/B, paired with divalent cations and polycationic Pc1 and Pc4 proteins. Both types of granules contain catechol oxidase that catalyzes oxidative cross-linking of L-DOPA. Co-secretion of catechol oxidase guarantees rapid and spatially homogeneous curing with limited mixing of the preassembled adhesive packets. Catechol oxidase remains active long after the glue is fully cured, perhaps providing an active cue for conspecific larval settlement.

Two different types of proteins were imaged in buffer solution with tapping mode atomic force microscopy (AFM) in liquids : the globular proteins lysozyme and monomeric actin (G-actin) ; the filamentous proteins actin (F-actin) and microtubules. To calibrate the AFM in contact and tapping modes in liquids, a sample was prepared with features that are comparable to the height of a single protein molecule: steps in mica with a height of 1 nm. Single globular molecules of lysozyme and G-actin could be readily imaged in physiological buffer at pH 7 and lower, whereas F-actin could only be imaged stably without any visible damage at around pH 6. The helical pitch of the actin filaments was measured to be 37 nm, which is in good agreement with data from X-ray diffraction and transmission electron microscopy (TEM). The negatively charged microtubules could not be imaged on freshly cleaved mica ; instead, a method was established to adsorb them to silanized glass. Both protein types could be imaged stably with loading forces of about 200 pN. The height of the proteins was larger than the expected height measured by X-ray diffraction on protein crystals. Mechanical properties and/or electrostatic interactions may contribute to the image formation. Further work is needed to understand the height measured by tapping mode in liquids. We show here that single, globular protein molecules and protein filaments can be imaged easily and stably in buffer solution.

The general topic of this review is protein-based underwater adhesives produced by aquatic organisms. The focus is on mechanisms of interfacial adhesion to native surfaces and controlled underwater solidification of natural water-borne adhesives. Four genera that exemplify the broad range of function, general mechanistic features, and unique adaptations are discussed in detail: blue mussels, acorn barnacles, sandcastle worms, and freshwater caddisfly larva. Aquatic surfaces in nature are charged and in equilibrium with their environment, populated by an electrical double layer of ions as well as adsorbed natural polyelectrolytes and microbial biofilms. Surface adsorption of underwater bioadhesives likely occurs by exchange of surface bound ligands by amino acid sidechains, driven primarily by relative affinities and effective concentrations of polymeric functional groups. Most aquatic organisms exploit modified amino acid sidechains, in particular phosphorylated serines and hydroxylated tyrosines (dopa), with high-surface affinity that form coordinative surface complexes. After delivery to the surfaces as a fluid, permanent natural adhesives solidify to bear sustained loads. Mussel plaques are assembled in a manner superficially reminiscent of in vitro layer-by-layer strategies, with sequentially delivered layers associated through Fe(dopa)(3) coordination bonds. The adhesives of sandcastle worms, caddisfly larva, and barnacles may be delivered in a form somewhat similar to in vitro complex coacervation. Marine adhesives are secreted, or excreted, into seawater that has a significantly higher pH and ionic strength than the internal environment. Empirical evidence suggests these environment triggers could provide minimalistic, fail-safe timing mechanisms to prevent premature solidification (insolubilization) of the glue within the secretory system, yet allow rapid solidification after secretion. Underwater bioadhesives are further strengthened by secondary covalent curing.

Aquatic caddisflies diverged from a silk-spinning ancestor shared with terrestrial moths and butterflies. Caddisfly larva spin adhesive silk underwater to construct protective shelters with adventitiously gathered materials. A repeating (SX)(n) motif conserved in the H-fibroin of several caddisfly species is densely phosphorylated. In total, more than half of the serines in caddisfly silk may be phosphorylated. Major molecular adaptations allowing underwater spinning of an ancestral dry silk appear to have been phosphorylation of serines and the accumulation of basic residues in the silk proteins. The amphoteric nature of the silk proteins could contribute to silk fiber assembly through electrostatic association of phosphorylated blocks with arginine-rich blocks. The presence of Ca(2+) in the caddisfly larval silk proteins suggest phosphorylated serines could contribute to silk fiber periodic substructure through Ca(2+) crossbridging.

The challenges of developing medical adhesives for the wet environment of open surgery are analogous to the adhesion problems solved by marine organisms living at the watery interface of land and ocean. These organisms routinely bond dissimilar materials together under seawater with little if any surface preparation. One such organism is the sandcastle worm (Phragmatopoma californica). Our goal is to copy this marine worm’s mechanisms of underwater bonding to create synthetic water-borne underwater medical adhesives, and in turn, to use the synthetic adhesives to test mechanistic hypotheses about the natural adhesive. Biomimetic underwater adhesives were formulated with polyelectrolytic analogues of the natural glue proteins. The copolymers condensed into complex coacervates—dense partially water-immiscible cohesive fluids poised between soluble polymers and insoluble polymeric salts. The boundary between fluid coacervate phases and solid or gelled states was dependent on divalent cation species as well as the pH and temperature, which demonstrated that these environmental factors can trigger the adhesive setting reaction (Fig. 1). The results provide, respectively, empirical support for the natural pH-triggered set hypothesis and practical triggers for controlled setting of mimetic medical adhesives.

The I28 immunoglobulin (Ig)-like module of human cardiac titin, an elastic muscle protein, was used to cross-link acrylamide (AAm) copolymers into hybrid hydrogels. Cross-linking was accomplished through metal coordination bonding between terminal histidine tags (His tags) of the I28 module and metal-chelating nitrilotriacetic acid (NTA)-containing side chains on the copolymer. In solution, the beta-sheet structure of the I28 module unfolded with a transition midpoint of about 58 degrees C as the temperature was elevated. Hydrogels cross-linked with the I28 module demonstrated positive temperature responsiveness; they swelled to 3 times their initial volume at temperatures above the melting temperature of the cross-links. Positive temperature responsiveness is unusual for synthetic hydrogels. The I28 hybrid hydrogels demonstrate that cross-linking synthetic polymers with natural, well-characterized protein modules is a practical strategy for creating new materials with unique environmental responsiveness predictably determined by the mechanical properties of the protein cross-links. These new materials may be useful for controlled chemical delivery.

A bifunctional N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer containing nitrilotriacetic acid (NTA) and benzophenone (BP) groups was synthesized by free-radical copolymerization of HPMA, 2-methacrylamidobutyl nitrilotriacetic acid (MABNTA), and 4-methacrylamido benzophenone (MABP) using 2, 2'-azobisisobutyronitrile (AIBN) as initiator. A His-tagged coiled coil stem loop peptide containing a tridecapeptide (TDP) epitope (GFLGEDPGFFNVE) in the loop region (CCSL-TDP) was designed and synthesized genetically by expressing an artificial gene in Escherichia coli BL21 (DE3). The peptide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), size-exclusion chromatography (SEC), and circular dichroism (CD) spectroscopy. Surfaces containing self-assembled CCSL-TDP peptide were prepared by first covalently grafting poly(HPMA-co-MABNTA-co-MABP) onto polystyrene (PS) surface by UV irradiation, then charging the surface with nickel through NTA groups, and finally attaching the CCSL-TDP peptide through Ni-histidine chelation. The modified PS surfaces with and without self-assembled CCSL-TDP peptide were characterized by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Cell attachment studies with human Burkitt's lymphoma Raji B cells showed that the cells selectively bound to the self-assembled CCSL-TDP peptide surfaces, but not to the surfaces of PS, PS with grafted copolymer, and PS with grafted copolymer and self-assembled coiled coil peptide with similar structure but without the epitope. This indicates that the cell attachment was mediated by the CCSL-TDP peptide, most probably by the TDP epitope region. The CCSL peptide self-assembly presented here may represent a feasible model of exposing epitopes for biorecognition studies.

Stimuli-sensitive polymer hydrogels, which swell or shrink in response to changes in the environmental conditions, have been extensively investigated and used as 'smart' biomaterials and drug-delivery systems. Most of these responsive hydrogels are prepared from a limited number of synthetic polymers and their derivatives, such as copolymers of (meth)acrylic acid, acrylamide and N-isopropyl acrylamide. Water-soluble synthetic polymers have also been crosslinked with molecules of biological origin, such as oligopeptides and oligodeoxyribonucleotides, or with intact native proteins. Very often there are several factors influencing the relationship between structure and properties in these systems, making it difficult to engineer hydrogels with specified responses to particular stimuli. Here we report a hybrid hydrogel system assembled from water-soluble synthetic polymers and a well-defined protein-folding motif, the coiled coil. These hydrogels undergo temperature-induced collapse owing to the cooperative conformational transition of the coiled-coil protein domain. This system shows that well-characterized water-soluble synthetic polymers can be combined with well-defined folding motifs of proteins in hydrogels with engineered volume-change properties.