Cutting edge structural protein from the jaws of Nereis virens

The fang-like jaws of the marine polychaete Nereis virens possess remarkable mechanical properties considering their high protein content and lack of mineralization. Hardness and stiffness properties in the jaw tip are comparable to human dentin and are achieved by extensive coordination of Zn (2+) by a histidine-rich protein framework. In the present study, the predominant protein in the jaw tip, Nvjp-1, was purified and characterized by partial peptide mapping and molecular cloning of a partial cDNA from a jaw pulp library. The deduced amino acid sequence revealed an approximately 38 kDa histidine-rich protein rich in glycine and histidine (approximately 36 and 27%, respectively) with no well-defined repetitive motifs. The effects of pH and metal treatment on aggregation, secondary structure, and hydrodynamic properties of recombinant Nvjp-1 are described. Notably, Zn treatment induced the formation of amyloid-like fibers.     

Utilizing peptidic ordering in the design of hierarchical polyurethane/ureas

One of the key design components of nature is the utilization of hierarchical arrangements to fabricate materials with outstanding mechanical properties. Employing the concept of hierarchy, a new class of segmented polyurethane/ureas (PUUs) was synthesized containing either a peptidic, triblock soft segment, or an amorphous, nonpeptidic homoblock block soft segment with either an amorphous or a crystalline hard segment to investigate the effects of bioinspired, multiple levels of organization on thermal and mechanical properties. The peptidic soft segment was composed of poly(benzyl-l-glutamate)-block-poly(dimethylsiloxane)-block-poly(benzyl-l-glutamate) (PBLG-b-PDMS-b-PBLG), restricted to the β-sheet conformation by limiting the peptide segment length to <10 residues, whereas the amorphous soft segment was poly(dimethylsiloxane) (PDMS). The hard segment consisted of either 1,6-hexamethylene diisocyanate (crystalline) or isophorone diisocyanate (amorphous) and chain extended with 1,4-butanediol. Thermal and morphological characterization indicated microphase separation in these hierarchically assembled PUUs; furthermore, inclusion of the peptidic segment significantly increased the average long spacing between domains, whereas the peptide domain retained its β-sheet conformation regardless of the hard segment chemistry. Mechanical analysis revealed an enhanced dynamic modulus for the peptidic polymers over a broader temperature range as compared with the nonpeptidic PUUs as well as an over three-fold increase in tensile modulus. However, the elongation-at-break was dramatically reduced, which was attributed to a shift from a flexible, continuous domain morphology to a rigid, continuous matrix in which the peptide, in conjunction with the hard segment, acts as a stiff reinforcing element.     

Characterization of Axial and Proximal Histidine Mutations of the Decaheme Cytochrome MtrA from Shewanella sp. Strain ANA-3 and Implications for the Electron Transport System

Extracellular respiration of solid-phase electron acceptors in some microorganisms requires a complex chain of multiheme c-type cytochromes that span the inner and outer membranes. In Shewanella species, MtrA, an ∼35-kDa periplasmic decaheme c-type cytochrome, is an essential component for extracellular respiration of iron(III). The exact mechanism of electron transport has not yet been resolved, but the arrangement of the polypeptide chain may have a strong influence on the capability of the MtrA cytochrome to transport electrons. The iron hemes of MtrA are bound to its polypeptide chain via proximal (CXXCH) and distal histidine residues. In this study, we show the effects of mutating histidine residues of MtrA to arginine on protein expression and extracellular respiration using Shewanella sp. strain ANA-3 as a model organism. Individual mutations to six out of nine proximal histidines in CXXCH of MtrA led to decreased protein expression. However, distal histidine mutations resulted in various degrees of protein expression. In addition, the effects of histidine mutations on extracellular respiration were tested using ferrihydrite and current production in microbial fuel cells. These results show that proximal histidine mutants were unable to reduce ferrihydrite. Mutations to the distal histidine residues resulted in various degrees of ferrihydrite reduction. These findings indicate that mutations to the proximal histidine residues affect MtrA expression, leading to loss of extracellular respiration ability. In contrast, mutations to the distal histidine residues are less detrimental to protein expression, and extracellular respiration can proceed.     

Variable tensility of the oral arm plate ligaments of the brittlestar Ophiura ophiura (Echinodermata: Ophiuroidea)

The oral arm plates of the brittlestar Ophiura ophiura L. are connected to lateral arm plates at distal and proximal ligamentous junctions. The distal junction is mobile and is disrupted during arm autotomy; the proximal junction is more rigid and does not participate in autotomy. Aspects of the morphology and mechanical properties of the distal and proximal oral arm plate ligaments have been investigated in order to determine if their tensility is under physiological control. By means of creep tests it was found that elevation of the external potassium (K⁺) ion concentration causes a decrease in the viscosity of the distal ligament which is either transient or continues until rupture intervenes. In forced vibration tests the distal ligament often shows a biphasic stiffening then softening response to excess K ^- ions. Anaesthetics block the softening phase but enhance the stiffening component of this response. This ligament is also softened by repetitive electrical stimuli but stiffened by excess calcium ions and by acetylcholine. The proximal ligament appears to have the capacity for only transient changes in mechanical properties. Both ligaments are penetrated by the processes of juxtaligamental cells whose perikarya are arranged in clusters innervated by hyponeural nerves. These cells are thought to modulate the interfibrillar cohesion of the ligaments. It is concluded that the distal and proximal ligaments are mutable collagenous structures which in their stiffened condition help to maintain arm posture without the need for continuous muscular activity, and that at autotomy the distal ligament undergoes a profound loss of tensile strength which facilitates arm detachment.     

X-ray structure and refinement of carbon-monoxy (Fe II)-myoglobin at 1.5 A resolution

The structure of carbon-monoxy (Fe II) myoglobin at 260 K has been solved at a resolution of 1.5 A by X-ray diffraction and a model refined against the X-ray data by restrained least-squares. The CO ligand is disordered and distorted from the linear conformation seen in model compounds. At least two conformations, with Fe--C--O angles of 140 degrees and 120 degrees, are required to model the system. The heme pocket is significantly larger than in deoxy-myoglobin because the distal residues have relaxed around the ligand; the largest displacement occurs for the distal histidine side-chain, which moves more than 1.4 A on ligand binding. The side-chain of Arg45 (CD3) is disordered and apparently exists in two equally populated conformations. One of these does not block the motion of the distal histidine out of the binding pocket, suggesting a mechanism for ligand entry. The heme group is planar (root-mean-square deviation from planarity is 0.08 A) with no doming of the pyrrole groups. The Fe--N epsilon 2 (His93) bond length is 2.2 A and the Fe--C bond length in the CO complex is 1.9 A. The iron is the least-squares plane of the heme, and this leads to the proximal histidine moving by 0.4 A relative to its position in deoxy-myoglobin. This shift correlates with a global structural change, with the proximal part of the molecule translated towards the heme plane.     

The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme

Sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) are two small, monomeric hemoglobins that share a common globin fold but differ widely in many other aspects. Lba has a much higher affinity for most ligands, and the two proteins use different distal and proximal heme pocket regulatory mechanisms to control ligand binding. Removal of the constraint provided by covalent attachment of the proximal histidine to the F-helices of these proteins decreases oxygen affinity in Lba and increases oxygen affinity in Mb, mainly because of changes in oxygen dissociation rate constants. Hence, Mb and Lba use covalent constraints in opposite ways to regulate ligand binding. Swapping the F-helices of the two proteins brings about similar effects, highlighting the importance of this helix in proximal heme pocket regulation of ligand binding. The F7 residue in Mb is capable of weaving a hydrogen-bonding network that holds the proximal histidine in a fixed orientation. On the contrary, the F7 residue in Lba lacks this property and allows the proximal histidine to assume a conformation favorable for higher ligand binding affinity. Geminate recombination studies indicate that heme iron reactivity on picosecond timescales is not the dominant cause for the effects observed in each mutation. Results also indicate that in Lba the proximal and distal pocket mutations probably influence ligand binding independently. These results are discussed in the context of current hypotheses for proximal heme pocket structure and function.     

Enrichment of metals in the jaws of fossil and extant polychaetes - distribution and function

Jawed polychaete annelids are successful in modern oceans, just as they were in early Palaeozoic oceans: the fossil evidence bears witness to abundant and taxonomically diverse faunas. The jaws are composed of sclerotized proteins and were used for grasping in sediment or capturing prey and therefore needed to be resistant to wear. A nuclear microprobe, Particle-Induced X-ray Emission (PIXE) and Scanning Transmission Ion Microscopy (STIM) techniques revealed an enrichment of metals, commonly Zn, Fe and Cu, concentrated mainly at the tips or delicate parts of the jaws. This suggests that accumulation was regulated by the animal and that these elements had functional significance. Similar enrichment was detected in the jaws of Recent Polychaeta and has also been reported in stress-related, 'tool-like' exoskeletal structures of other animal groups, including arthropods, chaetognaths and molluscs.     

Mismatch in mechanical and adhesive properties induces pulsating cancer cell migration in epithelial monolayer

The mechanical and adhesive properties of cancer cells significantly change during tumor progression. Here we assess the functional consequences of mismatched stiffness and adhesive properties between neighboring normal cells on cancer cell migration in an epithelial-like cell monolayer. Using an in vitro coculture system and live-cell imaging, we find that the speed of single, mechanically soft breast carcinoma cells is dramatically enhanced by surrounding stiff nontransformed cells compared with single cells or a monolayer of carcinoma cells. Soft tumor cells undergo a mode of pulsating migration that is distinct from conventional mesenchymal and amoeboid migration, whereby long-lived episodes of slow, random migration are interlaced with short-lived episodes of extremely fast, directed migration, whereas the surrounding stiff cells show little net migration. This bursty migration is induced by the intermittent, myosin II-mediated deformation of the soft nucleus of the cancer cell, which is induced by the transient crowding of the stiff nuclei of the surrounding nontransformed cells, whose movements depend directly on the cadherin-mediated mismatched adhesion between normal and cancer cells as well as α-catenin-based intercellular adhesion of the normal cells. These results suggest that a mechanical and adhesive mismatch between transformed and nontransformed cells in a cell monolayer can trigger enhanced pulsating migration. These results shed light on the role of stiff epithelial cells that neighbor individual cancer cells in early steps of cancer dissemination.     

Structure and function of the horn shark (Heterodontus francisci) cranium through ontogeny: development of a hard prey specialist

The horn sharks (Heterodontidae: Chondrichthyes) represent one of four independent evolutions of durophagy in the cartilaginous fishes. We used high-resolution computed tomography (CT scanning) to visualize and quantify the mineralized tissue of an ontogenetic series of horn sharks. CT scanning of neonatal through adult California horn sharks (Heterodontus francisci) confirmed that this technique is effective for examining mineralized tissue in even small (<10 mm) specimens. The jaw joint is among the first areas to become mineralized and is the most heavily mineralized area in the cranium of a neonatal horn shark. The hyoid is also well mineralized, although the poorly mineralized molariform teeth indicate that the neonatal animal may be a suction feeder on softer prey. The symphysis of the jaws never mineralizes, in sharp contrast to the condition in the hard prey-crushing stingrays. Digitally reslicing the CT scans along the jaws allowed measurement of the second moment of area (Ina). Assuming that the jaws are made of the same material at all ages, Ina is an indicator of the flexural stiffness of the jaws. In all sizes of shark the lower jaws were stiffer than the upper and the stiffness increased in the area of the molariform teeth. The central region of the jaws, where the rami meet, support cuspidate grasping teeth and has the lowest Ina. The spotted eagle ray (Aetobatus narinari), a hard prey-crushing stingray, shows a different pattern of flexural stiffness, with the peak at the central part of the jaws where the prey is reduced between flattened tooth plates. Although the eagle ray jaws have a higher Ina than the horn shark, they are also far more heavily mineralized. When the relative amounts of mineralization are taken into account, horn sharks do better with what mineral they have than does the eagle ray. With a tight jaw joint and loose mandibular symphysis, as well as nearly opposite patterns of stiffness in the jaws, it is clear that two of the clades of hard prey specialists use very different methods for cracking the hard prey problem.     

The mechanical properties of hydrated intermediate filaments: insights from hagfish slime threads

Intermediate filaments (IFs) impart mechanical integrity to cells, yet IF mechanics are poorly understood. It is assumed that IFs in cells are as stiff as hard alpha-keratin, F-actin, and microtubules, but the high bending flexibility of IFs and the low stiffness of soft alpha-keratins suggest that hydrated IFs may be quite soft. To test this hypothesis, we measured the tensile mechanics of the keratin-like threads from hagfish slime, which are an ideal model for exploring the mechanics of IF bundles and IFs because they consist of tightly packed and aligned IFs. Tensile tests suggest that hydrated IF bundles possess low initial stiffness (E(i) = 6.4 MPa) and remarkable elasticity (up to strains of 0.34), which we attribute to soft elastomeric IF protein terminal domains in series with stiffer coiled coils. The high tensile strength (180 MPa) and toughness (130 MJ/m(3)) of IF bundles support the notion that IFs lend mechanical integrity to cells. Their long-range elasticity suggests that IFs may also allow cells to recover from large deformations. X-ray diffraction and congo-red staining indicate that post-yield deformation leads to an irreversible alpha-->beta conformational transition in IFs, which leads to plastic deformation, and may be used by cells as a mechanosensory cue.     

Passive dynamics change leg mechanics for an unexpected surface during human hopping.

Humans running and hopping maintain similar center-of-mass motions, despite large changes in surface stiffness and damping. The goal of this study was to determine the contributions of anticipation and reaction when human hoppers encounter surprise, expected, and random changes from a soft elastic surface (27 kN/m) to a hard surface (411 kN/m). Subjects encountered the expected hard surface on every fourth hop and the random hard surface on an average of 25% of the hops in a trial. When hoppers on a soft surface were surprised by a hard surface, the ankle and knee joints were forced into greater flexion by passive interaction with the hard surface. Within 52 ms after subjects landed on the surprise hard surface, joint flexion increased, and the legs became less stiff than on the soft surface. These mechanical changes occurred before electromyography (EMG) first changed 68-188 ms after landing. Due to the fast mechanical reaction to the surprise hard surface, center-of-mass displacement and average leg stiffness were the same as on expected and random hard surfaces. This similarity is striking because subjects anticipated the expected and random hard surfaces by landing with their knees more flexed. Subjects also anticipated the expected hard surface by increasing the level of EMG by 24-76% during the 50 ms before landing. These results show that passive mechanisms alter leg stiffness for unexpected surface changes before muscle EMG changes and may be critical for adjustments to variable terrain encountered during locomotion in the natural world.     

Contribution of elastin and collagen to the inflation response of the pig thoracic aorta: assessing elastin's role in mechanical homeostasis

This study was undertaken to understand elastin's role in the mechanical homeostasis of the arterial wall. The mechanical properties of elastin vary along the aorta, and we hypothesized this maintained a uniform mechanical environment for the elastin, despite regional variation in loading. Elastin's physiological loading was determined by comparing the inflation response of intact and autoclave purified elastin aortas from the proximal and distal thoracic aorta. Elastin's stretch and stress depend on collagen recruitment. Collagen recruitment started in the proximal aorta at systolic pressures (13.3 to 14.6 kPa) and in the distal at sub-diastolic pressures (9.3 to 10.6 kPa). In the proximal aorta collagen did not contribute significantly to the stress or stiffness, indicating that elastin determined the vessel properties. In the distal aorta, the circumferential incremental modulus was 70% higher than in the proximal aorta, half of which (37%) was due to a stiffening of the elastin. Compared to the elastin tissue in the proximal aorta, the distal elastin suffered higher physiological circumferential stretch (29%, P=0.03), circumferential stress (39%, P=0.02), and circumferential stiffness (37%, P=0.006). Elastin's physiological axial stresses were also higher (67%, P=0.003). These findings do not support the hypothesis that the loading on elastin is constant along the aorta as we expected from homeostasis.     

Crystal structures of myoglobin-ligand complexes at near-atomic resolution.

We have used x-ray crystallography to determine the structures of sperm whale myoglobin (Mb) in four different ligation states (unligated, ferric aquomet, oxygenated, and carbonmonoxygenated) to a resolution of better than 1.2 A. Data collection and analysis were performed in as much the same way as possible to reduce model bias in differences between structures. The structural differences among the ligation states are much smaller than previously estimated, with differences of <0.25 A root-mean-square deviation among all atoms. One structural parameter previously thought to vary among the ligation states, the proximal histidine (His-93) azimuthal angle, is nearly identical in all the ferrous complexes, although the tilt of the proximal histidine is different in the unligated form. There are significant differences, however, in the heme geometry, in the position of the heme in the pocket, and in the distal histidine (His-64) conformations. In the CO complex the majority conformation of ligand is at an angle of 18 +/- 3 degrees with respect to the heme plane, with a geometry similar to that seen in encumbered model compounds; this angle is significantly smaller than reported previously by crystallographic studies on monoclinic Mb crystals, but still significantly larger than observed by photoselection. The distal histidine in unligated Mb and in the dioxygenated complex is best described as having two conformations. Two similar conformations are observed in MbCO, in addition to another conformation that has been seen previously in low-pH structures where His-64 is doubly protonated. We suggest that these conformations of the distal histidine correspond to the different conformational substates of MbCO and MbO(2) seen in vibrational spectra. Full-matrix refinement provides uncertainty estimates of important structural parameters. Anisotropic refinement yields information about correlated disorder of atoms; we find that the proximal (F) helix and heme move approximately as rigid bodies, but that the distal (E) helix does not.     

Adhesion dynamics and durotaxis in migrating cells

When tissue cells are plated on a flexible substrate, durotaxis, the directed migration of cells toward mechanically stiff regions, has been observed. Environmental mechanical signals are not only important in cell migration but also seem to influence all aspects of cell differentiation and development, including the metastatic process in cancer cells. Based on a theoretical model suggesting that this mechanosensation has a mechanical basis, we introduce a simple model of a cell by considering the contraction of F-actin bundles containing myosin motors (stress fibers) mediated by the movement of adhesions. We show that, when presented with a linear stiffness gradient, this simple model exhibits durotaxis. Interestingly, since stress fibers do not form on soft surfaces and since adhesion sliding occurs very slowly on hard surfaces, the model predicts that the expected cell velocity reaches a maximum at an intermediate stiffness. This prediction can be experimentally tested. We therefore argue that stiffness-dependent cellular adaptations (mechanosensation) and durotaxis are intimately related and may share a mechanical basis. We therefore identify the essential physical ingredients, which combined with additional biochemical mechanisms can explain durotaxis and mechanosensation in cells.     

Enigmatic polychaete jaws from the Silurian of Gotland, Sweden

The jaws of the new polychaetaspid polychaete, Oenonites? honki, from the Silurian of Gotland, Sweden, differ from most Palaeozoic polychaete jaws. They exhibit enigmatic microstructural features in that they appear rough and give a corroded, or weathered impression. The altered microstructure of the jaws suggests a jaw chemistry and/or composition differing in some way from that of the co‐occurring polychaete taxa. The jaws appear to have limited preservational potential and/or were particularly susceptible to secondary processes, resulting in microstructural alteration. Commonly, a row of distinct pits occurs on the outer face, especially of the first right maxillae (MIr). Because these pits are interpreted as associated with the dentary, the term ‘denticle marks’ is suggested. The pits may be the result of primary or secondary physical wear, or, more probably, secondary chemical alteration of localized mineral deposits. The primary function of such mineral deposits was to harden those parts of the surface that were exposed to great stress. The restricted occurrence of O.? honki, coupled with occasional increases in abundance (especially in the Halla Formation, unit b), indicates a preference for shallow marine, high‐energy environments, particularly in reefal pockets with calcilutitic sediments. Highest frequency coincides with faunas characteristically containing a few labidognath species also displaying high frequencies.     

1H-NMR investigation of the oxygenation of hemoglobin in intact human red blood cells.

Using improved selective excitation methods for protein nuclear magnetic resonance (NMR), we have conducted measurements of the oxygenation of hemoglobin inside intact human red blood cells. The selective excitation methods use pulse shape-insensitive suppression of the water signal, while producing uniform phase excitation in the region of interest and, thus, are suitable for a wide variety of applications in vivo. We have measured the areas of 1H-NMR resonances of the hyperfine-shifted, exchangeable N delta H protons of the proximal histidine residues of the alpha- and beta-chains in deoxyhemoglobin (63 and 76 ppm downfield from the proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), respectively), which are sensitive to the paramagnetic state of the iron, and for which the alpha- and beta-chain resonances are resolved, and from the ring current-shifted gamma 2-CH3 protons of the distal valine residues in oxyhemoglobin (2.4 ppm upfield from DSS), which are sensitive to the conformation of the heme pocket in the oxy state. We have found that the proximal histidine resonances are directly correlated with the degree of oxygenation of hemoglobin, whereas the distal valine resonances appear to be correlated with the conformation in the heme pocket that occurs after the binding of oxygen, in both the presence and absence of 2,3-diphosphoglycerate. In addition, from the proximal histidine resonances, we have observed a preference for the binding of oxygen to the alpha-chain (up to about 10%) of hemoglobin over the beta-chain in both the presence and absence of 2,3-diphosphoglycerate. These new results obtained in intact erythrocytes are consistent with our previous 1H-NMR studies on purified human normal adult hemoglobin. A unique feature of our 1H-NMR method is the ability to monitor the binding of oxygen specifically to the alpha- and beta-chains of hemoglobin both in solution and in intact red blood cells. This information is essential to our understanding of the molecular basis for the hemoglobin molecule serving as the oxygen carrier in vertebrates.     

Stiffness gradients in vascular bundles of the palm Washingtonia robusta

Palms can grow at sites exposed to high winds experiencing large dynamic wind and gust loads. Their stems represent a system of stiff fibrous elements embedded in the soft parenchymatous tissue. The proper design of the interface of the stiffening elements and the parenchyma is crucial for the functioning of the stem. The strategy of the palm to compromise between stiff fibre caps and the soft parenchymatous tissue may serve as a model system for avoiding stress discontinuities in inhomogeneous and anisotropic fibre-reinforced composite materials. We investigated the mechanical, structural and biochemical properties of the fibre caps of the palm Washingtonia robusta at different levels of hierarchy with high spatial resolution. A gradual decrease in stiffness across the fibre cap towards the surrounding parenchymatous tissue was observed. Structural adaptations at the tissue level were found in terms of changes in cell cross sections and cell wall thickness. At the cell wall level, gradients across the fibre cap were found in the degree of orientation of the microfibrils and in the lignin level and composition. The impact of these structural variations in the local material stiffness distribution is discussed.