Β-sheet Fibrils Research Articles

Primary Fibril Nucleation of Aggregation Prone Tau Fragments PHF6 and PHF6.

We performed replica exchange molecular dynamics and forward flux sampling simulations of hexapeptide VQIINK and VQIVYK systems, also known as, respectively, fragments PHF6* and PHF6 from the tau protein. Being a part of the microtubule binding region, these fragments are known to be aggregation prone, and at least one of them is a prerequisite for fibril formation of the tau protein. Using a coarse-grained force field, we establish the phase behavior of both fragments, and investigate the nucleation kinetics for the conversion into a β-sheet fibril. As the conversion is, in principle, a reversible process, we predict the rate constants for both the fibril formation and melting, and examine the corresponding mechanisms. Our simulations indicate that, while both fragments form disordered aggregates, only PHF6 is able to form β-sheet fibrils. This observation provides a possible explanation for the lack of available steric zipper crystal structures for PHF6*.

Human Islet Amyloid Polypeptide Assembly: The Key Role of the 8-20 Fragment.

The aggregation of human islet amyloid polypeptide (hIAPP) has been closely associated with the pathogeny of type 2 diabetes mellitus (T2DM) and destruction of pancreatic islet β-cells. Several amyloidogenic domains within the hIAPP sequence capable of self-association have been identified. Among them is the 8-20 region of hIAPP, which has formed β-sheet fibrils despite being contained within an α-helical region of full-length hIAPP. To further understand the propensity of this region for self-assembly, two peptide fragments were compared, one consisting of the residues 8-20 (WT8-20) and a mutant fragment with a His18Pro substitution (H18P8-20). The conformational distribution and aggregation propensity of these peptides was determined using a combination of ion mobility mass spectrometry and atomic force microscopy. Our results reveal that the two peptide fragments have vastly differing assembly pathways. WT8-20 produces a wide range of oligomers up to decamer whereas the H18P8-20 mutant produces only low order oligomers. This study confirms the propensity of the 8-20 region to aggregate from its native α-helical structure into amyloid β-sheet oligomers and highlights the significance of the charged His18 in the aggregation process.

Receptor for Advanced Glycation End Products (RAGE) in Type 1 Diabetes Pathogenesis.

The receptor for advanced glycation end products (RAGE) is a novel protein increasingly studied in the pathogenesis of type 1 diabetes (T1D). RAGE is expressed by several immune cell types, including T cells, antigen-presenting cells, endothelial cells, and the endocrine cells of the pancreatic islets. RAGE binds various ligands including advanced glycation end products (AGEs), high-mobility group box protein 1 (HMGB1), S100 proteins, β-amyloid, β-sheet fibrils, and lipopolysaccharide. AGEs are a particularly interesting ligand because their exogenous introduction into the body can be accelerated by the consumption of AGE-rich processed foods. This review will detail RAGE isoforms and its ligands and discuss how RAGE binding on the aforementioned cells could be linked to T1D pathogenesis.

Structural and Mechanical Properties of Amyloid Beta Fibrils: A Combined Experimental and Theoretical Approach.

In this combined experimental (deep ultraviolet resonance Raman (DUVRR) spectroscopy and atomic force microscopy (AFM)) and theoretical (molecular dynamics (MD) simulations and stress-strain (SS)) study, the structural and mechanical properties of amyloid beta (Aβ40) fibrils have been investigated. The DUVRR spectroscopy and AFM experiments confirmed the formation of linear, unbranched and β-sheet rich fibrils. The fibrils (Aβ40)n, formed using n monomers, were equilibrated using all-atom MD simulations. The structural properties such as β-sheet character, twist, interstrand distance, and periodicity of these fibrils were found to be in agreement with experimental measurements. Furthermore, Young's modulus (Y) = 4.2 GPa computed using SS calculations was supported by measured values of 1.79 ± 0.41 and 3.2 ± 0.8 GPa provided by two separate AFM experiments. These results revealed size dependence of structural and material properties of amyloid fibrils and show the utility of such combined experimental and theoretical studies in the design of precisely engineered biomaterials.

Local retention of antibodies in vivo with an injectable film embedded with a fluorogen-activating protein

Herein we report an injectable film by which antibodies can be localized in vivo. The system builds upon a bifunctional polypeptide consisting of a fluorogen-activating protein (FAP) and a β-fibrillizing peptide (βFP). The FAP domain generates fluorescence that reflects IgG binding sites conferred by Protein A/G (pAG) conjugated with the fluorogen malachite green (MG). A film is generated by mixing these proteins with molar excess of EAK16-II, a βFP that forms β-sheet fibrils at high salt concentrations. The IgG-binding, fluorogenic film can be injected in vivo through conventional needled syringes. Confocal microscopic images and dose–response titration experiments showed that loading of IgG into the film was mediated by pAGMG bound to the FAP. Release of IgG in vitro was significantly delayed by the bioaffinity mechanism; 26% of the IgG were released from films embedded with pAGMG after five days, compared to close to 90% in films without pAGMG. Computational simulations indicated that the release rate of IgG is governed by positive cooperativity due to pAGMG. When injected into the subcutaneous space of mouse footpads, film-embedded IgG were retained locally, with distribution through the lymphatics impeded. The ability to track IgG binding sites and distribution simultaneously will aid the optimization of local antibody delivery systems.

Contribution of domain 30 of tropoelastin to elastic fiber formation and material elasticity

Elastin is a fibrous structural protein of the extracellular matrix that provides reversible elastic recoil to vertebrate tissues such as arterial vessels, lung, and skin. The elastin monomer, tropoelastin, contains a large proportion of intrinsically disordered and flexible hydrophobic sequences that collectively are responsible for the initial phase separation of monomers during assembly, and are essential for driving elastic recoil. While structural disorder of hydrophobic sequences is controlled by a high proline and glycine residue composition, hydrophobic domain 30 of human tropoelastin is atypically proline-poor, and forms β-sheet amyloid-like fibrils as an individual peptide. We explored the contribution of confined regions of secondary structure at the location of domain 30 in human tropoelastin to fiber assembly and mechanical properties using a set of mutations designed to inhibit or enhance the propensity of β-sheet formation at this location. Our data support a dual role for confined β-sheet secondary structure in domain 30 of tropoelastin in guiding the formation of fibers, and as a determinant of stiffness and viscoelastic properties of cross-linked materials. Together, these results suggest a mechanism for specificity in fiber assembly, and elucidate structure-function relationships for the rational design of elastomeric biomaterials with defined mechanical properties.

Structural remodeling during amyloidogenesis of physiological Nα-acetylated α-synuclein

The misfolding and aggregation of the presynaptic protein α-synuclein (AS) into amyloid fibrils is pathognomonic of Parkinson's disease, though the mechanism by which this structural conversion occurs is largely unknown. Soluble oligomeric species that accumulate as intermediates in the process of fibril formation are thought to be highly cytotoxic. Recent studies indicate that oligomer-to-fibril AS transition plays a key role in cell toxicity and progression of neurodegeneration. We previously demonstrated that a subgroup of oligomeric AS species are ordered assemblies possessing a well-defined pattern of intermolecular contacts which are arranged into a distinctive antiparallel β-sheet structure, as opposed to the parallel fibrillar fold. Recently, it was demonstrated that the physiological form of AS is N-terminally acetylated (Ac-AS). Here, we first showed that well-characterized conformational ensembles of Ac-AS, namely monomers, oligomers and fibrils, recapitulate many biophysical features of the nonacetylated protein, such as hydrodynamic, tinctorial, structural and membrane-leakage properties. Then, we relied on ATR-FTIR spectroscopy to explore the structural reorganization during Ac-AS fibrillogenesis. We found that antiparallel β-sheet transient intermediates are built-up at early stages of aggregation, which then evolve to parallel β-sheet fibrils through helix-rich/disordered species. The results are discussed in terms of regions of the protein that might participate in this structural rearrangement. Our work provides new insights into the complex conformational reorganization occurring during Ac-AS amyloid formation.

Oligomeric Structures of an Aggregation-Triggering Fragment of SOD1 Protein

Abstract: Amyloid fibril formation of copper-zinc superoxide dismutase (SOD1) is linked to familial amyotrophic lateral sclerosis (fALS), a progressive neurodegenerative disease. A recent experimental study has shown that 147GVIGIAQ153 peptide of the SOD1 C-terminal segment can form amyloid fibril and also accelerate the aggregation of full-length SOD1, while substitution of isoleucine at site 149 by proline blocks fibril formation of this peptide. However, the oligomeric structure and aggregation mechanism of this fragment remain elusive. In this study, we have investigated the hexameric structures of GVIGIAQ and its I149P variant by performing extensive atomistic explicit-solvent replica exchange molecular dynamics (REMD) simulations. We found that the GVIGIAQ hexamers adopt various β-sheet-rich conformations, including disordered aggregates and, to a much lesser extent, ordered fibril-like bilayer β-sheets and β-barrels. Structural analysis shows that four-stranded monolayer β-sheets are the smallest β-sheets that can form both bilayer β-sheets and β-barrels. In contrast, substitution of I149 by proline significantly reduces the β-sheet probability of all residues, especially those close to I149 in amino acid sequence, thus resulting in the vanish of bilayer β-sheets, a decrease of β-barrels, and a dramatic increase of disordered aggregates. The lack of bilayer β-sheets provides an explanation for the experimental observation of no fibril formation for I149P variant. MD simulation results on the structural stability of different sizes of experimental bilayer β-sheet fibril models further support the importance of I149 on fibril formation and also suggest that the bilayer β-sheet consisting of eight chains might be the smallest nucleus for 147GVIGIAQ153 fibril formation.

A peptide hydrogel derived from a fragment of human cardiac troponin C.

The human cardiac troponin C peptide fragment H-V(9)EQLTEEQKNEFKAAFDIFVLGA(31)-OH, which covers helix-A in the native protein, self-assembles into β-sheet fibrils in solution. These fibrils further entangle to give a hydrogel. This peptide may therefore serve as a template for development of novel biomaterials.

Tau Aggregation Propensity Engrained in Its Solution State

A peptide fragment of the human tau protein which stacks to form neat cross β-sheet fibrils, resembling that found in pathological aggregation, 273GKVQIINKKLDL284 (here “R2/WT”), was modified with a spin-label at the N-terminus. With the resulting peptide, R2/G273C-SL, we probed events at time scales spanning seconds to hours after aggregation is initiated using transmission electron microscopy (TEM), thioflavin T (THT) fluorescence, ion mobility mass spectrometry (IMMS), electron paramagnetic resonance (EPR), and Overhauser dynamic nuclear polarization (ODNP) to determine if deliberate changes to its conformational states and population in solution influence downstream propensity to form fibrillar aggregates. We find varying solution conditions by adding the osmolyte urea or TMAO, or simply using different buffers (acetate buffer, phosphate buffer, or water), produces significant differences in early monomer/dimer populations and conformations. Crucially, these characteristics of the peptide in solution state before aggregation is initiated dictate the fibril formation propensity after aggregation. We conclude the driving forces that accelerate aggregation, when heparin is added, do not override the subtle intra- or interprotein interactions induced by the initial solvent conditions. In other words, the balance of protein–protein vs protein–solvent interactions present in the initial solution conditions is a critical driving force for fibril formation.

Functional bacterial amyloid increases Pseudomonas biofilm hydrophobicity and stiffness.

The success of Pseudomonas species as opportunistic pathogens derives in great part from their ability to form stable biofilms that offer protection against chemical and mechanical attack. The extracellular matrix of biofilms contains numerous biomolecules, and it has recently been discovered that in Pseudomonas one of the components includes β-sheet rich amyloid fibrils (functional amyloid) produced by the fap operon. However, the role of the functional amyloid within the biofilm has not yet been investigated in detail. Here we investigate how the fap-based amyloid produced by Pseudomonas affects biofilm hydrophobicity and mechanical properties. Using atomic force microscopy imaging and force spectroscopy, we show that the amyloid renders individual cells more resistant to drying and alters their interactions with hydrophobic probes. Importantly, amyloid makes Pseudomonas more hydrophobic and increases biofilm stiffness 20-fold. Deletion of any one of the individual members of in the fap operon (except the putative chaperone FapA) abolishes this ability to increase biofilm stiffness and correlates with the loss of amyloid. We conclude that amyloid makes major contributions to biofilm mechanical robustness.

Rapid assessment of human amylin aggregation and its inhibition by copper(II) ions by laser ablation electrospray ionization mass spectrometry with ion mobility separation.

Native electrospray ionization (ESI) mass spectrometry (MS) is often used to monitor noncovalent complex formation between peptides and ligands. The relatively low throughput of this technique, however, is not compatible with extensive screening. Laser ablation electrospray ionization (LAESI) MS combined with ion mobility separation (IMS) can analyze complex formation and provide conformation information within a matter of seconds. Islet amyloid polypeptide (IAPP) or amylin, a 37-amino acid residue peptide, is produced in pancreatic beta-cells through proteolytic cleavage of its prohormone. Both amylin and its precursor can aggregate and produce toxic oligomers and fibrils leading to cell death in the pancreas that can eventually contribute to the development of type 2 diabetes mellitus. The inhibitory effect of the copper(II) ion on amylin aggregation has been recently discovered, but details of the interaction remain unknown. Finding other more physiologically tolerated approaches requires large scale screening of potential inhibitors. Here, we demonstrate that LAESI-IMS-MS can reveal the binding stoichiometry, copper oxidation state, and the dissociation constant of human amylin-copper(II) complex. The conformations of hIAPP in the presence of copper(II) ions were also analyzed by IMS, and preferential association between the β-hairpin amylin monomer and the metal ion was found. The copper(II) ion exhibited strong association with the -HSSNN- residues of the amylin. In the absence of copper(II), amylin dimers were detected with collision cross sections consistent with monomers of β-hairpin conformation. When copper(II) was present in the solution, no dimers were detected. Thus, the copper(II) ions disrupt the association pathway to the formation of β-sheet rich amylin fibrils. Using LAESI-IMS-MS for the assessment of amylin-copper(II) interactions demonstrates the utility of this technique for the high-throughput screening of potential inhibitors of amylin oligomerization and fibril formation. More generally, this rapid technique opens the door for high-throughput screening of potential inhibitors of amyloid protein aggregation.

Multiple function fluorescein probe performs metal chelation, disaggregation, and modulation of aggregated Aβ and Aβ-Cu complex.

An exceptional probe comprising indole-3-carboxaldehyde fluorescein hydrazone (FI) performs multiple tasks, namely, disaggregating amyloid β (Aβ) aggregates in different biomarker environments such as cerebrospinal fluid (CSF), Aβ1-40 fibrils, β-amyloid lysozyme aggregates (LA), and U87 MG human astrocyte cells. Additionally, the probe FI binds with Cu(2+) ions selectively, disrupts the Aβ aggregates that vary from few nanometers to micrometers, and prevents their reaggregation, thereby performing disaggregation and modulation of amyloid-β in the presence as well as absence of Cu(2+) ion. The excellent selectivity of probe FI for Cu(2+) was effectively utilized to modulate the assembly of metal-induced Aβ aggregates by metal chelation with the "turn-on" fluorescence via spirolactam ring opening of FI as well as the metal-free Aβ fibrils by noncovalent interactions. These results confirm that FI has exceptional ability to perform multifaceted tasks such as metal chelation in intracellular conditions using Aβ lysozyme aggregates in cellular environments by the disruption of β-sheet rich Aβ fibrils into disaggregated forms. Subsequently, it was confirmed that FI had the ability to cross the blood-brain barrier and it also modulated the metal induced Aβ fibrils in cellular environments by "turn-on" fluorescence, which are the most vital properties of a probe or a therapeutic agent. Furthermore, the morphology changes were examined by atomic force microscopy (AFM), polarizable optical microscopy (POM), fluorescence microscopy, and dynamic light scattering (DLS) studies. These results provide very valuable clues on the Aβ (CSF Aβ fibrils, Aβ1-40 fibrils, β-amyloid lysozyme aggregates) disaggregation behavior via in vitro studies, which constitute the first insights into intracellular disaggregation of Aβ by "turn-on" method thereby influencing amyloidogenesis.

Stabilization of Insulin by Adsorption on a Hydrophobic Silane Self-Assembled Monolayer.

The interaction between many proteins and hydrophobic functionalized surfaces is known to induce β-sheet and amyloid fibril formation. In particular, insulin has served as a model peptide to understand such fibrillation, but the early stages of insulin misfolding and the influence of the surface have not been followed in detail under the acidic conditions relevant to the synthesis and purification of insulin. Here we compare the adsorption of human insulin on a hydrophobic (-CH3-terminated) silane self-assembled monolayer to a hydrophilic (-NH3(+)-terminated) layer. We monitor the secondary structure of insulin with Fourier transform infrared attenuated total reflection and side-chain orientation with sum frequency spectroscopy. Adsorbed insulin retains a close-to-native secondary structure on both hydrophobic and hydrophilic surfaces for extended periods at room temperature and converts to a β-sheet-rich structure only at elevated temperature. We propose that the known acid stabilization of human insulin and the protection of the aggregation-prone hydrophobic domains on the insulin monomer by adsorption on the hydrophobic surface work together to inhibit fibril formation at room temperature.

Synthesis of chemically-tethered amyloid-β segment trimer possessing amyloidogenic properties.

As amyloid-β (Aβ) undergoes dynamic aggregation, it is impossible to isolate ('hook') the transient Aβ oligomer in an assembly state-pure form (e.g., sole Aβ dimer, trimer, tetramer, etc.). Obtaining such a pure Aβ oligomer would allow us to establish an in vitro system to perform a more detailed investigation of the pathogenic properties of the oligomer. A chemically-tethered Aβ oligomer, constructed only by covalent bonds, could satisfy this demand. Here we designed a chemically-tethered trimer of a pathogenic Aβ fragment (Aβ25-35) (1) and successfully generated it in situ from its precursor (4), a water-soluble and non-aggregative O-acyl isopeptide of 1, in neutral aqueous media. Chemically-tethered 1 possessed stronger amyloidogenic properties, that is, potential for β-sheet structure, fibril formation, and cytotoxicity, than the corresponding monomer Aβ25-35 (6). Trimerization of Aβ25-35 sequence might affect both the aggregative properties and cytotoxicity, based on the present results. This work opens the door for chemical synthesis of oligomers bigger than trimers in an assembly state-pure form, allowing for identification of the most toxic Aβ oligomer.

Hydrogen sulfide inhibits amyloid formation.

Amyloid fibrils are large aggregates of misfolded proteins, which are often associated with various neurodegenerative diseases such as Alzheimer’s, Parkinson’s, Huntington’s, and vascular dementia. The amount of hydrogen sulfide (H2S) is known to be significantly reduced in the brain tissue of people diagnosed with Alzheimer’s disease relative to that of healthy individuals. These findings prompted us to investigate the effects of H2S on the formation of amyloids in vitro using a model fibrillogenic protein hen egg white lysozyme (HEWL). HEWL forms typical β-sheet rich fibrils during the course of 70 min at low pH and high temperatures. The addition of H2S completely inhibits the formation of β-sheet and amyloid fibrils, as revealed by deep UV resonance Raman (DUVRR) spectroscopy and ThT fluorescence. Nonresonance Raman spectroscopy shows that disulfide bonds undergo significant rearrangements in the presence of H2S. Raman bands corresponding to disulfide (RSSR) vibrational modes in the 550–500 cm–1 spectral range decrease in intensity and are accompanied by the appearance of a new 490 cm–1 band assigned to the trisulfide group (RSSSR) based on the comparison with model compounds. The formation of RSSSR was proven further using a reaction with TCEP reduction agent and LC-MS analysis of the products. Intrinsic tryptophan fluorescence study shows a strong denaturation of HEWL containing trisulfide bonds. The presented evidence indicates that H2S causes the formation of trisulfide bridges, which destabilizes HEWL structure, preventing protein fibrillation. As a result, small spherical aggregates of unordered protein form, which exhibit no cytotoxicity by contrast with HEWL fibrils.

Beta-amyloid-acetylcholine structural interaction: evidence for neuroprotective effects of acetylcholine in neural cells

Alzheimer’s disease (AD) is regarded as a multifactorial disease characterized by a complex pathogenesis including a cholinergic deficit - due to degeneration of cholinergic projections from the basal forebrain - and the extracellular accumulation of amyloid beta (Aβ) peptide. Aβ containing 39 to 42 amino acids is the predominant component of the senile plaques that, together with neurofibrillary tangles, are regarded as the neuropathological hallmarks of AD (Sorrentino et al. 2014). Aβ may assume different conformations changing from random coil or α-helical monomers to β-sheet structures forming toxic oligomers and/or β-sheet mature fibrils. In this framework, we studied the effect of acetylcholine (ACh) on the conformation of Aβ by circular dichroism analysis. Moreover we investigated the ability of ACh to protect neuronal cells from the toxic action of amyloid peptide and to modulate the neuroinflammatory response occurring via the phospholipase A2 (PLA2). Results show that the amount of Aβ(25-35) β-strand raised linearly in absence of ACh, whereas it remained almost constant in presence of ACh. In addition, in a micelle solution mimicking the membrane environment ACh was found effective in increasing and stabilizing the soluble and not toxic helical content of Aβ(25-35) suggesting that ACh is capable to preserve the soluble form of Aβ(25-35), reducing the incipit of Aβ aggregation. In order to assess the neuro-protective ability of ACh against toxic Aβ(25-35) accumulation, we used neural cell (NCC) cultures containing both astrocytes and glial cells prepared from brains embryos from timed pregnant Wistar rats and infused ACh for 48h. By immunostaining, we observed that ACh reduced Aβ(25-35)-induced cell death. Then, we tested the protective effect of ACh on inflammation induced by Aβ administration. NCC were challenged with Aβ(25-35) in the presence and absence of ACh and immunostained for astroglial and neuronal markers: results showed a reduction of the morphological features of astrogliosys in ACh treated cells. PLA2 expression analysis corroborated these data also underlying that ACh can negatively regulate inflammation pathways in glial cells.

A Study in Semenogelin I Hydrogel Aggregation Kinetics

In earlier investigations, we have shown that a peptide sequence, Sg I 38-48, derived from the protein human semenogelin I forms a hydrogel. In this work, we describe approaches to examine the kinetics of aggregation of both Sg I 38-48 and an extended sequence, Sg I 37-49. The kinetics experiments are based on using the chromophore Thioflavin T. When bound to extended β-sheet fibrils the intensity of Thioflavin T fluorescence increases dramatically and this can be used to monitor fibril formation. The fibrils have been shown previously by CD and IR spectroscopy to consist of extended β-sheets. In the kinetics experiments, the peptide is first isolated as a monomer employing gel filtration under acidic conditions when it does not aggregate. The pH is then adjusted to pH 8 to trigger fibril formation and the kinetics of aggregation is monitored using a plate reader, with an excitation and an emission wavelength of 440 nm and 480 nm, respectively. We have studied the effect of concentration, pH, and salt on the kinetics of aggregation. We have also carried out disc centrifugation studies in order to determine the size and homogeneity of the aggregates formed. The next step will be to fit our data to a mechanism. For this purpose we are using Global Fitter, and our preliminary data suggest an initial nucleation step from which the fibril extends. We have also carried out rheology experiments in order to characterize the behavior of the Sg I 38-48 sequence, which have confirmed that this peptide forms a hydrogel. These have also shown that the gel is able to recover its original strength after a strain sweep has broken the gel.

How spiders spin silk.

Spider silk is wonderful stuff—light as the breeze and stretchy yet stronger than steel. People can manufacture synthetic fibers, such as Kevlar, that come close but can't begin to match the process spiders use. Their silk proteins, called spidroins, rapidly convert from the soluble form to solid fibers at ambient temperatures and with water as the solvent. Not only is this beyond us, we don't even know how spiders do it. Now, in this issue of PLOS Biology, new research by Anna Rising, Jan Johansson, and colleagues shows that silk formation involves structural shifts at either end of the spidroin and that these shifts are completely different, overturning the hypothesis that these protein terminals play similar roles. Spidroins are big proteins of up to 3,500 amino acids that contain mostly repetitive sequences, and the nonrepetitive N- and C-terminal domains at opposite ends are thought to regulate conversion to silk. These terminal domains are unique to spider silk and are highly conserved among spiders. Spidroins have a helical and unordered structure when stored as soluble proteins in silk glands, but, when converted to silk, they contain β-sheets that lend mechanical stability. We know that there is a pH gradient across the spider silk gland, which narrows from a tail to a sac to a slender duct, and that silk forms at a precise site in the duct. However, further details of spider silk production have been elusive. To discover the basis for the pH gradient in the silk gland, the researchers studied the orb weaver spider Nephila clavipes, which reaches 7.6 centimeters wide and spins webs up to 3 meters across. Poking ion-selective microelectrodes into the silk gland revealed that the pH fell from 7.6 to 5.7 between the base of the tail and halfway down the duct, which was as far as the electrodes fit. This pH gradient is much steeper than previously thought. The microelectrodes also showed that bicarbonate ions rise between the base of the tail and the far end of the sac. Taken together, these ion patterns suggest that the pH gradient is due to carbonic anhydrase, an enzyme that converts carbon dioxide and water to bicarbonate and hydrogen ions. In support of this idea, a carbonic anhydrase inhibitor called methazolamide collapsed the pH gradient. Next, the researchers investigated how the pH range across the silk gland affects silk proteins, using N- and C-terminal domains isolated from the spidroins of another orb weaver, Araneus ventricosus (Figure 1). They found that both domains undergo structural changes at the pH found in the duct. Importantly, this is also where carbonic anhydrase activity is concentrated. Figure 1 An Araneus spider in its orb web. The researchers also found pH had opposite effects on the two domains' stability to temperature and urea, which was a surprise given that the domains had been suggested to have a similar impact on silk formation. The N-terminal dimerized at pH 6—which is found in the beginning of the duct—and became increasingly stable as the pH dropped along the duct. In contrast, the C-terminal domain destabilized as the pH dropped, gradually unfolding until it formed the β-sheets characteristic of silk at pH 5.5. What makes the C-terminal domain unfold? This end of the spidroin has a salt bridge joining two helices, and mutations that interfere with this bridge also destabilize the C-terminal domain. Underscoring its importance, the salt bridge is conserved in spidroins from several spider species. The researchers believe that protonating amino acids in the salt bridge would also destabilize the C-terminal domain, facilitating conversion of spidroins from helices to β-sheet fibrils. These findings led the researchers to propose a new “lock and trigger” model for spider silk formation. Gradual dimerization of the N-terminal domains could lock spidroins into multimers, while the β-sheet fibrils at the C-terminals could serve as nuclei that trigger rapid polymerization of spidroins into fibers. Interestingly, the C-terminal β-sheets are similar to those in the amyloid fibrils characteristic of diseases such as Alzheimer disease. This work brings us closer to unraveling the mystery of spider silk, explaining how it can form so quickly—faster than a meter per second—as well as how its formation can be confined to the spinning duct. Moreover, because the N- and C-terminal domains of spidroins are found nowhere else, this lock and trigger formation is likely unique to spider silk. Besides being essential to producing biomimetic spidroin fibers, knowing how spiders spin silk could give insights into natural ways of hindering the amyloid fibrils associated with disease. Andersson M, Chen G, Otikovs M, Landreh M, Nordling K, et al. (2014) Carbonic Anhydrase Generates CO2 and H+ That Drive Spider Silk Formation Via Opposite Effects on the Terminal Domains. doi:10.1371/journal.pbio.1001921

Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains.

Author SummaryThe spinning process of spider silk is crucial for making webs or other complex constructions to catch spider's prey. The main components of the silk are spidroins, which are large and repetitive proteins that have conserved nonrepetitive terminal domains (NT and CT). Spiders manage both to store the highly aggregation-prone spidroins in solution at extreme concentrations in the silk glands and then to rapidly convert these spidroins into a solid fiber within fractions of a second as they spin fibres. This process has been extensively studied and is thought to involve a pH gradient, but how this pH gradient is generated and maintained was not resolved. Here, we measured the pH at locations along the ampullate gland and determined that the pH decreases to 5.7 in the middle of the spinning duct. We also observed that the carbon dioxide pressure is simultaneously increased and that its accumulation may affect the stability of CT. We find that active carbonic anhydrase (CA) is crucial to maintain the pH gradient along the gland. Detailed molecular studies of NT and CT under the disparate conditions present along the gland revealed a lock and trigger mechanism whereby in more neutral pH conditions, precocious spidroin aggregation is prevented, and when in more acidic pH conditions, NT dimers firmly interconnect the spidroins and the CT unfolds into β-sheet nuclei that can trigger rapid polymerization of the spidroins. We conclude that this mechanism enables temporal and spatial control of silk formation and may be harnessed in attempts to produce artificial silk replicas.