Assembly Of Monomers Research Articles

Knockdown of versican 1 blocks cigarette-induced loss of insoluble elastin in human lung fibroblasts

COPD lung is characterized by loss of alveolar elastic fibers and an increase in the chondroitin sulfate (CS) matrix proteoglycan versican V1 (V1). V1 is a known inhibitor of elastic fiber deposition and this study investigates the effects of knockdown of V1, and add-back of CS, on CCL-210 lung fibroblasts treated with cigarette smoke extract (CSE) as a model for COPD. CSE inhibited fibroblast proliferation, viability, tropoelastin synthesis, and elastin deposition, and increased V1 synthesis and secretion. V1 siRNA decreased V1 and constituent CS, did not affect tropoelastin production, but blocked the CSE-induced loss in insoluble elastin. Exogenous CS reduced insoluble elastin, even in the presence of V1 siRNA. These findings confirm that V1 and CS impair the assembly of tropoelastin monomers into insoluble fibers, and further demonstrate that specific knockdown of V1 alleviates the impaired assembly of elastin seen in cultures of pulmonary fibroblasts exposed to CSE, indicating a regulatory role for this protein in the pathophysiology of COPD.

Assembly of oligo(ethylene glycol)- and amine-containing methacrylic esters in water and water–hexane mixtures

Abstract The monomer assembly in aqueous solutions of N,N-dimethylaminoethyl methacrylate (DMAEMA), ethylene glycol methacrylate (EGMA), oligo(ethylene glycol) methacrylate (OEGMA) and a series of methoxy oligo(ethylene glycol) methacrylates (MOEGMAs) with a variable number of ethylene glycol moieties was investigated by viscosity, surface and interfacial tension measurements, IR and NMR spectroscopy. All monomers were shown to exhibit interfacial activity. The isotherms of the specific viscosity for OEGMA and MOEGMAs exhibit the exponential growth which indicates the presence of sufficiently strong intermolecular noncovalent interactions; for DMAEMA and EGMA the isotherms pass through maxima. It is associated with the formation of monomer complexes due to hydrogen bonding. A combination of computer simulations, IR data analysis and viscosity isotherms allowed one to distinguish three concentration ranges in aqueous solutions of MEG and MOEGMA. These ranges are characterized by predominant types of assemblies with the participation of oxygen-containing functional groups of the monomers.

Destabilizing Aquaporin Z Assembly: Effects on Structure, Function and Dynamics

Aquaporins are membrane proteins, that act as water channels in biological membranes. Members of this family form tetrameric, or rarely pentameric, complexes in the membrane. In order to better understand the folding and multimeric assembly of these proteins we have constructed a series of destabilized proteins by modifying the interface between monomers in Aquaporin Z from E. coli. We have characterized these proteins to test the the effects on the folding of the monomeric unit, the assembly of monomers into tetramers. We have also examined the consequences modified structures on the function of the water channel and the dynamics of the protein.Structure and folding has been examined at the level of the protein topology and modifications observable by FTIR spectroscopy. These methods indicate that the surface mutations do not delectably modify the structure of the monomeric aquaporin Z. Assembly into tetramers has been investigated by hydrodynamic methods, DLS, Fluorescence anisotropy and FCS, FCCS. These methods suggest that in some mutants tetramers are not properly formed and monomeric aquaporins predominate. Some of these mutations can be suppressed by compensatory mutations on the opposite side of the interface. We report the consequences of these changes in assembly.

Understanding Nucleotide-Regulated FtsZ Filament Dynamics and the Monomer Assembly Switch with Large-Scale Atomistic Simulations

Bacterial cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for cell division. Here, we study their dynamics using unbiased atomistic molecular simulations from representative filament crystal structures. In agreement with experimental data, we find different filament curvatures that are supported by a nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby burying the nucleotide, the differently curved GDP-FtsZ filaments exhibit a heterogeneous distribution of open and closed interfaces between monomers. We identify a coordinated Mg2+ ion as the key structural element in closing the nucleotide site and stabilizing GTP filaments, whereas the loss of the contacts with loop T7 from the next monomer in GDP filaments leads to open interfaces that are more prone to depolymerization. We monitored the FtsZ monomer assembly switch, which involves opening/closing of the cleft between the C-terminal domain and the H7 helix, and observed the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive conformation. This result validates the proposed switch between the low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ conformation within filaments. Finally, we observed how the antibiotic PC190723 suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces, thus stabilizing the filament. Our studies provide detailed structural and dynamic insights into modulation of both the intrinsic curvature of the FtsZ filaments and the molecular switch coupled to the high-affinity end-wise association of FtsZ monomers.

Interfacing hard and living matter: plasma-assembled proteins on inorganic functional materials for enhanced coupling to cells and tissue.

When bringing functional hard materials to biomedical applications, control of interfaces with cells and tissue poses one of the largest challenges. Assembly of protein monomers within an inert-gas-plasma constitutes a novel approach to synthesize strongly adherent bioactive coatings that dramatically enhance coupling to living matter. As proof of concept this is demonstrated for a Fe-Pd ferromagnetic shape memory transducer that is functionalized with the amino-acid, l-lysine, by plasma-treatment, resulting in flexible, yet ultra-durable coatings. Containing high amounts of NH2 functional groups, they fulfill all requirements for strong adhesion of cells and tissues. This is confirmed by cell tests with living NIH/3T3 embryonic murine fibroblasts that demonstrate excellent biocompatibility, exceeding conventional poly-l-lysine coatings in terms of cells focal adhesion density. The physics and chemistry behind these scenarios are unraveled by employing ab initio computer calculations. This combined approach opens the way for plasma-assisted functionalization strategies for a broad class of metals and semiconductors with polypeptides.

Potential applications of magnetic particles to detect and treat Alzheimer's disease.

Nanotechnology is an exciting and promising scientific discipline. At the nanoscale, a material displays novel physical properties that offer many new and beneficial products and applications. In particular, magnetic nanoparticles - a core/shell nanoparticle - present considerable diagnostic and therapeutic potentials, and superparamagnetic iron oxide nanoparticles (SPIONs) are considered promising theranostic tools. Alzheimer's disease (AD) is a neurodegenerative disorder that predominantly affects people over 65 years of age. The disease is characterized by the presence of extracellular plaques in the brain which are formed by interwoven fibrils composed of variants of the β-amyloid peptide. Medication can temporarily retard worsening of symptoms, but only in the first stages of the disease; early detection is thus of crucial importance. This minireview covers the progress made in research on the use of magnetic nanoparticles for ex vivo and/or in vivo detection and diagnosis of AD by means of magnetic resonance imaging (MRI), or to label peptides and fibrils. Of particular importance is the use of these nanoparticles to detect AD biomarkers in biological fluids. A description is given of the bio-barcode amplification assay using functionalized magnetic particles, as well as the use of such nanoparticles as a system for inhibiting or delaying the assembly of peptide monomers into oligomers and fibrils. Lastly, a brief overview is given of possible future lines of research in this.

Spatiotemporal control of epithelial remodeling by regulated myosin phosphorylation.

Spatiotemporally regulated actomyosin contractility generates the forces that drive epithelial cell rearrangements and tissue remodeling. Phosphorylation of the myosin II regulatory light chain (RLC) promotes the assembly of myosin monomers into active contractile filaments and is an essential mechanism regulating the level of myosin activity. However, the effects of phosphorylation on myosin localization, dynamics, and function during epithelial remodeling are not well understood. In Drosophila, planar polarized myosin contractility is required for oriented cell rearrangements during elongation of the body axis. We show that regulated myosin phosphorylation influences spatial and temporal properties of contractile behavior at molecular, cellular, and tissue length scales. Expression of myosin RLC variants that prevent or mimic phosphorylation both disrupt axis elongation, but have distinct effects at the molecular and cellular levels. Unphosphorylatable RLC produces fewer, slower cell rearrangements, whereas phosphomimetic RLC accelerates rearrangement and promotes higher-order cell interactions. Quantitative live imaging and biophysical approaches reveal that both phosphovariants reduce myosin planar polarity and mechanical anisotropy, altering the orientation of cell rearrangements during axis elongation. Moreover, the localized myosin activator Rho-kinase is required for spatially regulated myosin activity, even when the requirement for phosphorylation is bypassed by the expression of phosphomimetic myosin RLC. These results indicate that myosin phosphorylation influences both the level and the spatiotemporal regulation of myosin activity, linking molecular properties of myosin activity to tissue morphogenesis.

Isomeric routes to Schiff-base single-layered covalent organic frameworks.

With graphene-like topology and designable functional moieties, single-layered covalent organic frameworks (sCOFs) have attracted enormous interest for both fundamental research and application prospects. As the growth of sCOFs involves the assembly and reaction of precursors in a spatial defined manner, it is of great importance to understand the kinetics of sCOFs formation. Although several large families of sCOFs and bulk COF materials based on different coupling reactions have been reported, the synthesis of isomeric sCOFs by exchanging the coupling reaction moieties on precursors has been barely explored. Herein, a series of isomeric sCOFs based on Schiff-base reaction is designed to understand the effect of monomer structure on the growth kinetics of sCOFs. The distinctly different local packing motifs in the mixed assemblies for the two isomeric routes closely resemble to those in the assemblies of monomers, which affect the structural evolution process for highly ordered imine-linked sCOFs. In addition, surface diffusion of monomers and the molecule-substrate interaction, which is tunable by reaction temperature, also play an important role in structural evolutions. This study highlights the important roles of monomer structure and reaction temperature in the design and synthesis of covalent bond connected functional nanoporous networks.

Increased C-telopeptide Cross-linking of Tendon Type I Collagen in Fibromodulin-deficient Mice

The controlled assembly of collagen monomers into fibrils, with accompanying intermolecular cross-linking by lysyl oxidase-mediated bonds, is vital to the structural and mechanical integrity of connective tissues. This process is influenced by collagen-associated proteins, including small leucine-rich proteins (SLRPs), but the regulatory mechanisms are not well understood. Deficiency in fibromodulin, an SLRP, causes abnormal collagen fibril ultrastructure and decreased mechanical strength in mouse tendons. In this study, fibromodulin deficiency rendered tendon collagen more resistant to nonproteolytic extraction. The collagen had an increased and altered cross-linking pattern at an early stage of fibril formation. Collagen extracts contained a higher proportion of stably cross-linked α1(I) chains as a result of their C-telopeptide lysines being more completely oxidized to aldehydes. The findings suggest that fibromodulin selectively affects the extent and pattern of lysyl oxidase-mediated collagen cross-linking by sterically hindering access of the enzyme to telopeptides, presumably through binding to the collagen. Such activity implies a broader role for SLRP family members in regulating collagen cross-linking placement and quantity.

The tyrosine sulfate domain of fibromodulin represents a new binding site to collagen enhancing the interaction and fibril formation

Purpose: Fibromodulin is a collagen modulating protein belonging to the family of the small leucine rich repeat proteoglycans. The collagen modulating effect is demonstrated by in vitro fibrillogenesis assay, resulting in retardation of turbidity compared to fibrillogenesis by collagen alone. Fibromodulin binds collagen via the LRR domain, but whether the binding via the LRR has any role in the assembly of the collagen is not clear. Fibromodulin null mice have larger population of thinner collagen fibers in ligaments compared to the wild type mice and develop osteoarthritis due to joint instability, indicating a regulatory role by fibromodulin in vivo as well. The N-terminal domain of fibromodulin has a highly negative charge due to the presence of the posttranslational modification O-sulfation on tyrosine residues that binds heparin binding proteins. The aim of this work is to investigate binding of this domain to collagen and its role in the regulation of collagen fibril assembly. Methods: Different variants of fibromodulin (full length, the N-terminal domain of fibromodulin and N-terminal truncated fibromodulin) were used to study interaction to collagen type I in solid phase assay and surface plasmon resonance assay. Fibrillogenesis assay was used to monitor changes in turbidity over time in the presence of the different variants of fibromodulin. Samples were collected from the fibril formation and visualized by EM negative staining. Specific binding sites for fibromodulin on the collagen monomers by detection with a gold-labeled fibromodulin specific antibody was also identified by EM negative staining. Results: In solid phase assay, all three variants of fibromodulin bind to coated collagen type I, showing a novel binding site located in the N-terminal domain of fibromodulin. By surface plasmon resonance a distinct difference in the dissociation phase is observed. The dissociation of full-length fibromodulin is much slower compared to the N-terminal truncated variant of fibromodulin, indicating contribution of both the N-terminal site and the LRR-domain sites in binding to the collagen molecule. In an in vitro fibrillogenesis assay, full-length and N-terminal truncated fibromodulin delays the fibril formation. Adding the N-terminal tyrosin sulfate rich domain instead accelerates the fibril formation, indicating that this domain binds and facilitates collagen monomer assembly. Samples taken after 24 h of fibril formation in the presence of the different fibromodulin variants or PBS were analysed by electron microscopy. Full-length fibromodulin shows the thinnest fibers, but with the most mature cross-striation. Presence of the N-terminal also results in thinner fibers compared to the control and the N-terminal truncated variant but showed less maturation. Further, all three variants bind to three common sites on the collagen type I molecule, visualized by electron microscopy. An informative observation by the electron microscopy is the finding of full-length fibromodulin binding two collagen molecules at the same time. This shows engagement of the N-terminal and one of the binding sites in the LRR region, concomitantly binding different collagen molecules, which can be important for the assembly. Conclusion: This work shows that the tyrosine sulfate rich domain of fibromodulin contributes to the high affinity binding of the fibromodulin molecule to collagen. It influences and contributes to the arrangement of the collagen molecules, which results in well defined, highly organized collagen fibers.

Membrane Translocation of Highly Charged Antimicrobial Peptides via Multi-Microsecond All-Atom Md Simulations

We report the first computational description of the membrane translocation mechanism and pathway of two antimicrobial peptides, PGLa from X. laevis (charge +5), and maculatin from L. genimaculata (charge +2), via multi-microsecond MD simulations. At P/L=1:20-50, assembly of monomers into higher oligomeric aggregates, or the formation of pores, is not observed for either peptide. Instead, individual monomers are transported across the bilayer in a two-stage mechanism in which charged sidechains are separately translocated via assistance of other peptides. Transmembrane inserted water-free intermediate states are observed that are stable over many microseconds. Our proposed mechanism disagrees with earlier models of antimicrobial bilayer penetration, and suggests that translocation of highly charged peptides across a membrane is simpler than previously thought.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Human F1F0 ATP Synthase, Mitochondrial Ultrastructure and OXPHOS Impairment: A (Super-)Complex Matter?

Mitochondrial morphogenesis is a key process of cell physiology. It is essential for the proper function of this double membrane-delimited organelle, as it ensures the packing of the inner membrane in a very ordered pattern called cristae. In yeast, the mitochondrial ATP synthase is able to form dimers that can assemble into oligomers. Two subunits (e and g) are involved in this supramolecular organization. Deletion of the genes encoding these subunits has no effect on the ATP synthase monomer assembly or activity and only affects its dimerization and oligomerization. Concomitantly, the absence of subunits e and g and thus, of ATP synthase supercomplexes, promotes the modification of mitochondrial ultrastructure suggesting that ATP synthase oligomerization is involved in cristae morphogenesis. We report here that in mammalian cells in culture, the shRNA-mediated down-regulation of subunits e and g affects the stability of ATP synthase and results in a 50% decrease of the available functional enzyme. Comparable to what was shown in yeast, when subunits e and g expression are repressed, ATP synthase dimers and oligomers are less abundant when assayed by native electrophoresis. Unexpectedly, mammalian ATP synthase dimerization/oligomerization impairment has functional consequences on the respiratory chain leading to a decrease in OXPHOS activity. Finally these structural and functional alterations of the ATP synthase have a strong impact on the organelle itself leading to the fission of the mitochondrial network and the disorganization of mitochondrial ultrastructure. Unlike what was shown in yeast, the impairment of the ATP synthase oligomerization process drastically affects mitochondrial ATP production. Thus we propose that mutations or deletions of genes encoding subunits e and g may have physiopathological implications.

Polymeric Frameworks as Organic Semiconductors with Controlled Electronic Properties

The rational assembly of monomers, in principle, enables the design of a specific periodicity of polymeric frameworks, leading to a tailored set of electronic structure properties in these solid-state materials. The further development of these emerging systems requires a combination of both experimental and theoretical studies. Here, we investigated the electronic structures of two-dimensional polymeric frameworks based on triazine and benzene rings by means of electrochemical techniques. The experimental density of states was obtained from quasi-open-circuit voltage measurements through a galvanostatic intermittent titration technique, which we show to be in excellent agreement with first-principles calculations performed for two- and three-dimensional structures of these polymeric frameworks. These findings suggest that the electronic properties depend not only on the number of stacked layers but also on the ratio of the different aromatic rings.

Technical Advance: Actin CytoFRET, a novel FRET flow cytometry method for detection of actin dynamics in resting and activated T cell

Actin cytoskeleton plays a critical role in regulating T cell motility and activation. However, the lack of a real-time quantitative method to analyze actin assembly has limited the progress toward understanding actin regulation. Here, we describe a novel approach to probe actin dynamics on living T cells using FRET combined with flow cytometry. We have first generated a Jurkat T cell line stably coexpressing EGFP and mOrange FPs fused to actin. The real-time variation of actin monomer assembly or disassembly into filaments was quantified using a ratiometric flow cytometry method measuring changes in the mOrange/EGFP emission ratio. The method was validated on resting T cells by using chemical compounds with known effects on actin filaments and comparison with conventional microscopy imaging. Our method also detected the rapid and transient actin assembly in T cells stimulated by anti-CD3/CD28-coated beads, demonstrating its robustness and high sensitivity. Finally, we provide evidence that lentiviral-mediated transduction of shRNAs in engineered Jurkat cells could be used as a strategy to identify regulators of actin remodeling. In conclusion, the flow cytometric FRET analysis of actin polymerization represents a new technical advance to study the dynamics of actin regulation in intact cells.

Galectin-3 mediates oligomerization of secreted hensin using its carbohydrate-recognition domain

A multidomain, multifunctional 230-kDa extracellular matrix (ECM) protein, hensin, regulates the adaptation of rabbit kidney to metabolic acidosis by remodeling collecting duct intercalated cells. Conditional deletion of hensin in intercalated cells of the mouse kidney leads to distal renal tubular acidosis and to a significant reduction in the number of cells expressing the basolateral chloride-bicarbonate exchanger kAE1, a characteristic marker of α-intercalated cells. Although hensin is secreted as a monomer, its polymerization and ECM assembly are essential for its role in the adaptation of the kidney to metabolic acidosis. Galectin-3, a unique lectin with specific affinity for β-galactoside glycoconjugates, directly interacts with hensin. Acidotic rabbits had a significant increase in the number of cells expressing galectin-3 in the collecting duct and exhibited colocalization of galectin-3 with hensin in the ECM of microdissected tubules. In this study, we confirmed the increased expression of galectin-3 in acidotic rabbit kidneys by real-time RT-PCR. Galectin-3 interacted with hensin in vitro via its carbohydrate-binding COOH-terminal domain, and the interaction was competitively inhibited by lactose, removal of the COOH-terminal domain of galectin-3, and deglycosylation of hensin. Galectin-9, a lectin with two carbohydrate-recognition domains, is also present in the rabbit kidney; galectin-9 partially oligomerized hensin in vitro. Our results demonstrate that galectin-3 plays a critical role in hensin ECM assembly by oligomerizing secreted monomeric hensin. Both the NH₂-terminal and COOH-terminal domains are required for this function. We suggest that in the case of galectin-3-null mice galectin-9 may partially substitute for the function of galectin-3.

The isolation of fibrinogen monomer dramatically influences fibrin polymerization

Fibrin polymerization begins with the thrombin-catalyzed cleavage of fibrinopeptides from fibrinogen and proceeds through several assembly steps to form an insoluble fibrin clot. Using dynamic light scattering (DLS), we found that purified fibrinogens are polydisperse, containing small amounts of fibrinogen complexes. In order to characterize the impact of these complexes, we used gel filtration chromatography to isolate monomers from three fibrinogens: plasma, recombinant, and recombinant variant Aα251. SDS-PAGE analysis showed that the polypeptides in the monomers were indistinguishable from those in the initial fibrinogen. DLS showed the fibrinogen monomers were monodisperse. We used turbidity to follow polymerization and found the polymerization of fibrinogen monomers was markedly different from the polymerization of the initial fibrinogen; the final optical density (OD) was significantly higher for monomers. Moreover, the polymerization curve for fibrinogen monomers was independent of the polymerization curves of the fibrinogen samples without gel filtration. For example, monomers isolated from two recombinant fibrinogen preparations polymerized similarly even though the final OD increased 2-fold for one preparation and 3-fold for the other. Scanning electron microscopy of the fibrin clots verified the turbidity data; monomer clots had thicker fibers. We conclude that fibrinogen complexes alter the kinetics of polymerization and impair the assembly of monomers into protofibrils and fibers.

The role of charge compensation on the nucleation of α and γ polymorphs of glycine from aqueous solution

The formation of α and γ nucleation in the aqueous solution of glycine is discussed in the context of self-charge compensation among the molecular clusters. Self-charge compensation is the main process responsible for the creation of dimers needed for the initiation of α nucleation in the solution which is more probable at most environmental conditions, whereas, the creation of γ nucleation is due to the assembly of monomers which requires sufficient/more number of monomers in the solution and has less probability to occur at normal circumstances. In the present work, a new induced charge compensation mechanism was adopted by adding selected externally Induced Charge Compensator (ICC) species by which the dimer formation was completely arrested into the solution and this paved a way for the nucleation of γ. The effect of concentration of the added species on the induction time, nucleation, growth and morphology of the nucleated polymorphs was studied. Structural affirmation of the nucleated polymorphs was confirmed by X-ray diffraction and their functional groups by FTIR analysis. Results reveal that, the changeover of the charge compensation mechanism from the self-charge compensation to the induced one occurs only at a critical concentration of the charged species. This approach is fruitful in the control of either only α or only γ nucleation in the system.

Structural polymorphism and possible pathways of amyloid fibril formation on the example of insulin protein

In this review we analyze the main works on amyloid formation of insulin. There are many environmental factors affecting the formation of insulin amyloid fibrils (and other amyloidogenic proteins) such as: protein concentration, pH, ionic strength of solution, medium composition (anions, cations), presence of denaturants (urea, guanidine chloride) or stabilizers (saccharose), temperature regime, agitation. Since polymorphism is potentially crucial for human diseases and may underlie the natural variability of some amyloid diseases, in this review we focus attention on polymorphism that is an important biophysical difference between native protein folding suggesting correspondence between the amino acid sequence and unique folding state, and formation of amyloid fibrils, when the same amino acid sequence can form amyloid fibrils of different morphology. At present, according to the literature data, we can choose three ways of polymerization of insulin molecules depending on the nucleus size. The first suggests that fibrillogenesis can occur through assembly of insulin monomers. The second suggests that precursors of fibrils are dimers, and the third assumes that precursors of fibrils are oligomers. Additional experimental works and new methods of investigation and assessment of results are needed to clarify the general picture of insulin amyloid formation.

Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology, including intracellular transport, cell motility, cell division, determining cellular shape, and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as "polymerization" and "stiffness" sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments.

Native-sized spider silk proteins synthesized in planta via intein-based multimerization

The synthesis of native-sized proteins is a pre-requisite for exploiting the potential of spider silk as a bio-based material. The unique properties of spider silk, such as extraordinary tensile strength and elasticity, result from the highly repetitive nature of spider silk protein motifs. The present report describes the combination of spider silk flagelliform protein (FLAG) production in the endoplasmic reticulum of tobacco plant leaf cells with an intein-based posttranslational protein fusion technology. The repeated ligation of FLAG monomers resulted in the formation of large multimers. This method avoids the need for highly repetitive transgenes, which may result in a higher genetic and transcriptional stability. Here we show, for the first time, the production of synthetic, high molecular weight spider silk proteins larger than 250kDa based on the assembly of protein monomers via intein-mediated trans-splicing in planta. The resulting multimeric structures form microfibers, thereby demonstrating their great potential as a biomaterial.