Beta-sheet Protein Research Articles

Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).

Refolding multi-disulfide bonded proteins expressed in E. coli into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the E. coli expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in E. coli and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the 15N-HSQC NMR spectrum exhibited several 1H-15N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an E. coli expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.

Impact of steam pressure toasting time on the alpha helix to beta sheet ratios, nutritional value, protein and carbohydrate subfractions, and rumen fermentation parameters of Faba bean seeds for dairy cattle

The use of Faba bean (Vicia faba) seeds (FBS) as a potential feed ingredient for ruminants has been previously evaluated. However, the effects on nutrient supply and degradability linked to protein secondary structure ratios after different steam pressure toasting (SPT) times are scarce. This study aimed to determine the role of varying alpha-helix to beta-sheet ratios induced by SPT duration on the nutritional value of Snowbird FBS in dairy cattle. Fifteen samples (1 variety × 3 years × 5 treatments) were analyzed for protein secondary structures and nutrient composition. The dry matter (DM), crude protein (CP), starch, and neutral detergent fibre-assayed with a heat stable amylase and expressed inclusive of residual ash (aNDF) contents in raw seeds were 914, 286, 328, and 179 g/kg DM, respectively. The averaged DM, CP, starch, and aNDF in the SPT-processed seeds were 904, 292, 374, and 405 g/kg DM, respectively. Soluble crude protein was about five times higher in raw seeds (239 g/kg DM) compared to steam-pressure toasted seeds (avg. 51 g/kg DM) (P < 0.01). The total digestible nutrients (TDN1x) and energy values gradually decreased as SPT time increased (P < 0.01). Hence, the TDN1x in the raw seeds was 832 g/kg DM, while the values at 30, 60, 90, and 120 min were lower by 2.78, 3.87, 6.44, and 7.71 units, respectively. The NEL3x reduced from 2.03 in raw seeds to 1.82 Mcal/kg (P < 0.01). The effective degradability (ED) of nutrients decreased with SPT duration from 0 to 120 min. Hence, the EDDM reduced from 692 to 357 g/kg DM (P < 0.01), the EDCP from 224 to 67 g/kg DM (P < 0.01), and the EDST from 237 to 129 g/kg DM (P = 0.01). Steam pressure toasting from 30 and up to 60 min might be the optimal range for processing FBS (Snowbird var). These findings could be supported by a post-ruminal evaluation of the nutrients that escaped ruminal fermentation.

Characterization of the structural forces governing the reversibility of the thermal unfolding of the human acidic fibroblast growth factor

Human acidic fibroblast growth factor (hFGF1) is an all beta-sheet protein that is involved in the regulation of key cellular processes including cell proliferation and wound healing. hFGF1 is known to aggregate when subjected to thermal unfolding. In this study, we investigate the equilibrium unfolding of hFGF1 using a wide array of biophysical and biochemical techniques. Systematic analyses of the thermal and chemical denaturation data on hFGF1 variants (Q54P, K126N, R136E, K126N/R136E, Q54P/K126N, Q54P/R136E, and Q54P/K126N/R136E) indicate that nullification of charges in the heparin-binding pocket can significantly increase the stability of wtFGF1. Triple variant (Q54P/K126N/R136E) was found to be the most stable of all the hFGF1 variants studied. With the exception of triple variant, thermal unfolding of wtFGF1 and the other variants is irreversible. Thermally unfolded triple variant refolds completely to its biologically native conformation. Microsecond-level molecular dynamic simulations reveal that a network of hydrogen bonds and salt bridges linked to Q54P, K126N, and R136E mutations, are responsible for the high stability and reversibility of thermal unfolding of the triple variant. In our opinion, the findings of the study provide valuable clues for the rational design of a stable hFGF1 variant that exhibits potent wound healing properties.

The significant influence of residual feed intake on flavor precursors and biomolecules in slow-growing Korat chicken meat.

ObjectiveThis study investigated the association between feed efficiency, physicochemical properties, flavor precursors and biomolecules in the thigh meat of Korat (KR) chickens.MethodsThe feed intake and body weight of individual male KR chickens were recorded from 1 to 10 weeks old to calculate the individual residual feed intake (RFI) of 75 birds. At 10 weeks of age, chickens with the 10 highest (HRFI) and lowest RFI (LRFI) were slaughtered to provide thigh meat samples. The physicochemical properties (ultimate pH, water holding capacity [WHC], drip loss) and flavor precursors (guanosine monophosphate, inosine monophosphate (IMP), adenosine monophosphate and inosine) were analyzed conventionally, and Fourier transform infrared spectroscopy was used to identify the composition of biomolecules (lipids, ester lipids, amide I, amide II, amide III, and carbohydrates) and the secondary structure of the proteins. A group t-test was used to determine significant differences between mean values and principal component analysis to classify thigh meat samples into LRFI and HRFI KR chickens.ResultsThe physicochemical properties of thigh meat samples from LRFI and HRFI KR chickens were not significantly different but the IMP content, ratios of lipid, lipid ester, protein (amide I, amide II) were significantly different (p<0.05). The correlation loading results showed that the LRFI group was correlated with high ratios of lipids, lipid esters, collagen content (amide III) and beta sheet protein (rg loading >0.5) while the HRFI group was positively correlated with protein (amide I, amide II), alpha helix protein, IMP content, carbohydrate, ultimate pH and WHC (rg loading >0.5).ConclusionThe thigh meat from chickens with different RFI differed in physiochemical properties affecting meat texture, and in the contents of flavor precursors and biomolecules affecting the nutritional value of meat. This information can help animal breeders to make genetic improvements by taking more account of traits related to RFI.

NMR Structure Determinations of Small Proteins Using only One Fractionally 20% 13C- and Uniformly 100% 15N-Labeled Sample.

Uniformly 13C- and 15N-labeled samples ensure fast and reliable nuclear magnetic resonance (NMR) assignments of proteins and are commonly used for structure elucidation by NMR. However, the preparation of uniformly labeled samples is a labor-intensive and expensive step. Reducing the portion of 13C-labeled glucose by a factor of five using a fractional 20% 13C- and 100% 15N-labeling scheme could lower the total chemical costs, yet retaining sufficient structural information of uniformly [13C, 15N]-labeled sample as a result of the improved sensitivity of NMR instruments. Moreover, fractional 13C-labeling can facilitate reliable resonance assignments of sidechains because of the biosynthetic pathways of each amino-acid. Preparation of only one [20% 13C, 100% 15N]-labeled sample for small proteins (<15 kDa) could also eliminate redundant sample preparations of 100% 15N-labeled and uniformly 100% [13C, 15N]-labeled samples of proteins. We determined the NMR structures of a small alpha-helical protein, the C domain of IgG-binding protein A from Staphylococcus aureus (SpaC), and a small beta-sheet protein, CBM64 module using [20% 13C, 100% 15N]-labeled sample and compared with the crystal structures and the NMR structures derived from the 100% [13C, 15N]-labeled sample. Our results suggest that one [20% 13C, 100% 15N]-labeled sample of small proteins could be routinely used as an alternative to conventional 100% [13C, 15N]-labeling for backbone resonance assignments, NMR structure determination, 15N-relaxation analysis, and ligand–protein interaction.

FSHD / OPMD / MYOTONIC DYSTROPHY: P.240 Targeting unfolded protein response to resolve aggresomes accumulation in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a rare late-onset genetic myopathy, affecting mainly eyelid and pharyngeal muscles leading to ptosis and dysphagia, respectively. OPMD is caused by a mutation in Poly-A Binding Protein Nuclear 1 (PABPN1) gene, that codes for a protein essential for proper mRNA biogenesis in nucleus. Accordingly, mutated PABPN1 in OPMD results in PABPN1 intranuclear aggregates in muscle that sequestrate mRNA and proteins and thus induce a wide range of deleterious consequences on muscle homeostasis (mitochondrial dysfunctions, calcium homeostasis disruption, fibrosis, force impairment, atrophy). We previously demonstrated that mouse model of OPMD presents muscle Endoplasmic Reticulum (ER) stress, i.e. an accumulation of misfolded proteins in ER lumen, and that restoring the physiological and resolutive Unfolded Protein Response (UPR) through guanabenz acetate treatment is efficient to decrease PABPN1 nuclear aggregates and improve muscle function in OPMD mouse (Malerba et al Hum Mol Genet 2019). To validate these results in human, we now analyze muscle biopsies from OPMD patients compared to healthy donors and observe a disruption of both key genes and proteins of UPR signalling in OPMD muscles. Interestingly, we find that the alteration is positively correlated with the clinical state of the muscle. Furthermore, we detect GRP78 (BiP), the most important ER chaperone, trapped in PABPN1 nuclear aggregates, suggesting a reduced folding capacity in ER. As a consequence, OPMD muscles present an accumulation of large beta-sheet protein aggregates called aggresomes. Consistently, we observe that human OPMD myoblasts are more sensible to develop aggresomes than healthy myoblasts in response to ER stress. Overall, our results unravel an impairment of ER folding and UPR as well as an aggresomes accumulation in human OPMD muscle. Thus, restoring the protein folding in ER as well as improving the aggresomes clearance could be a promising therapeutic strategy against OPMD.

Blo t 2: Group 2 allergen from the dust mite Blomia tropicalis

Blomia tropicalis has been recognized as a cause of allergic diseases in the tropical and subtropical regions. Here we report the immuno-characterization of its group 2 allergen, Blo t 2. Allergen Blo t 2 was amplified from the cDNA of B. tropicalis using degenerate primers, expressed in Escherichia coli as a recombinant protein and purified to homogeneity. The mature protein of Blo t 2 was 126 amino acids long with 52% sequence identity to Der p 2 and apparent molecular mass of 15 kDa. Circular dichroism spectroscopy showed that Blo t 2 is mainly a beta-sheeted protein. We confirmed the presence of three disulfide bonds in recombinant (r) Blo t 2 protein using electrospray mass spectrometry. Thirty-four percent of dust-mite allergic individuals from the Singapore showed specific IgE binding to rBlo t 2 as tested using immuno dot-blots. IgE-cross reactivity assays showed that Blo t 2 had between 20–50% of unique IgE-epitopes compared to Der p 2. IgE binding of native and recombinant forms of Blo t 2 were highly concordant (r2 = 0.77, p < 0.0001) to rBlo t 2. Dose-dependent in vitro histamine was observed when rBlo t 2 was incubated with whole blood of Blo t 2 sensitized individuals, demonstrating that it is a functional allergen. Nine naturally occurring isoforms of Blo t 2 were identified in this study, each having between 1–3 amino acid variations compared to the reference clone. Blo t 2 is a clinically relevant allergen of B. tropicalis as it has unique IgE epitopes compared to major group 2 allergens from Dermatophagoides spp.

Investigating the Effect of Chain Connectivity on the Folding of a Beta-Sheet Protein On and Off the Ribosome

Determining the relationship between protein folding pathways on and off the ribosome remains an important area of investigation in biology. Studies on isolated domains have shown that alteration of the separation of residues in a polypeptide chain, while maintaining their spatial contacts, may affect protein stability and folding pathway. Due to the vectorial emergence of the polypeptide chain from the ribosome, chain connectivity may have an important influence upon cotranslational folding. Using MATH, an all β-sandwich domain, we investigate whether the connectivity of residues and secondary structure elements is a key determinant of when cotranslational folding can occur on the ribosome. From Φ-value analysis, we show that the most structured region of the transition state for folding in MATH includes the N and C terminal strands, which are located adjacent to each other in the structure. However, arrest peptide force-profile assays show that wild-type MATH is able to fold cotranslationally, while some C-terminal residues remain sequestered in the ribosome, even when destabilized by 2–3 kcal mol−1. We show that, while this pattern of Φ-values is retained in two circular permutants in our studies of the isolated domains, one of these permutants can fold only when fully emerged from the ribosome. We propose that in the case of MATH, onset of cotranslational folding is determined by the ability to form a sufficiently stable folding nucleus involving both β-sheets, rather than by the location of the terminal strands in the ribosome tunnel.

Structural studies of the N-terminal fragments of the WW domain: Insights into co-translational folding of a beta-sheet protein.

Nascent proteins fold co-translationally because the folding speed and folding pathways are limited by the rate of ribosome biosynthesis in the living cell. In addition, though full-length proteins can fold all their residues during the folding process, nascent proteins initially fold only with the N-terminal residues. However, the transient structure and the co-translational folding pathway are not well understood. Here we report the atomic structures of a series of N-terminal fragments of the WW domain with increasing amino acid length. Unexpectedly, the structures indicate that the intermediate-length fragments take helical conformations even though the full-length protein has no helical regions. The circular dichroism spectra and theoretical calculations also support the crystallographic results. This suggests that the short-range interactions are more decisive in the structure formation than the long-range interactions for short nascent proteins. In the course of the peptide extension, the helical structure change to the structure mediated by the long-range interactions at a particular polypeptide length. Our results will provide unique information for elucidating the nature of co-translational folding.

Raman spectroscopy of fibroblast cells from a Huntington's disease patient

ABSTRACTWe present a Raman spectroscopy case study of living fibroblast (skin) cells from a patient who developed Huntington’s disease, with fibroblasts from a healthy volunteer as a control. Spectra were processed to remove cosmic rays, had a spectrum of the quartz substrate subtracted, and were flattened to remove cellular autofluorescence. We achieved an accuracy of 95% in discriminating individual cells, and assign spectral differences to (i) the reduction of cholesterol, (ii) the reduction of lipids, and (iii) an increase in beta-sheet proteins for fibroblasts with Huntington’s disease. All these biochemical changes have been previously measured by other methods. Averages over all the cells in this study yield a difference which is extremely statistically significant [p < 0.0001].

Kinetic Compensation between Ester-Bond Cleavage, Folding and Release from the Ribosome in Protein Biogenesis

In the healthy cell, bioactive proteins fold without aggregating. However, the mechanism of protein folding and release from the ribosome after completion of protein biosynthesis is still largely unknown. In this work, we focused on two bacterial single-domain monomeric model proteins: the alpha-helical globin ApoHmpH and the beta sheet cold shock protein A (CspA). We studied the kinetics of hydrolysis of the ester bond between the nascent polypeptides and the 3' end of the tRNA. In addition, we characterized the escape and folding of the model proteins from the ribosome. Time-resolved fluorescence depolarization in the frequency domain led to insights into the compaction of ribosome-bound and -released nascent proteins. Chain release was triggered by the antibiotic puromycin at concentrations yielding rates of ester bond cleavage similar to those of the naturally occurring release factors. Surprisingly, we found that departure of apoHmpH from the ribosome is rate-limited by the bond cleavage step and that escape from the ribosomal tunnel and completion of folding occur quickly afterwards. On the other hand, for the smaller beta-sheet protein CspA ester-bond cleavage is faster, and escape from the tunnel and folding occur more slowly. Yet, the overall rate of native protein production is comparable to that of apoHmpH. Overall, the ribosomal context promotes “temporally efficient” folding upon nascent protein release. We propose that the rapid combined rate of ribosome release and folding is tuned to prevent undesirable diffusion- and concentration-dependent aggregation in the cellular environment, at physiologically relevant concentrations.

Folding and Unfolding Simulations of a Three-Stranded Beta-Sheet Protein

Understanding the folding processes of a protein into its three-dimensional native structure only with its amino-acid sequence information is a long-standing challenge in modern science. Two- hundred independent folding simulations (starting from non-native conformations) and two- hundred independent unfolding simulations (starting from the folded native structure) are performed using the united-residue force field and Metropolis Monte Carlo algorithm for betanova (three-stranded antiparallel beta-sheet protein). From these extensive computer simulations, two representative folding pathways and two representative unfolding pathways are obtained in the reaction coordinates such as the fraction of native contacts, the radius of gyration, and the root- mean-square deviation. The folding pathways and the unfolding pathways are similar each other. The largest deviation between the folding pathways and the unfolding pathways results from the root-mean-square deviation near the folded native structure. In general, unfolding computer simulations could capture the essentials of folding simulations.

Label-free identification and characterization of living human primary and secondary tumour cells.

We used three label-free minimally invasive methods to characterize individual cells derived from primary and secondary tumours from the same patient, and of the same type – colorectal. Raman spectroscopy distinguished cells by their biochemical 'fingerprint' in a vibrational spectrum with 100% accuracy, and revealed that the primary cell line contains more lipids and alpha-helix proteins, whereas the secondary cell line contains more porphyrins and beta-sheet proteins. Stimulated Raman scattering (SRS) microscopy distinguished cells in chemically-specific images of CH2 bonds which revealed lipid droplets in secondary tumour cells. Atomic force microscopy (AFM) was used to distinguish cells with 80% accuracy by measuring their elasticity – secondary tumour cells (SW620) are around 3 times softer than primary ones (SW480). As well as characterizing the physical and biochemical differences between cell lines in vitro, these techniques offer three novel methods which could potentially be used for diagnosis – to assign a tumour as primary or secondary.

Computer Simulation Study of Biopolymer Betanova

Betanova is a monomeric, three-stranded antiparallel beta-sheet protein with twenty residues. The pathways between the folded native structure and unfolded conformations of betanova are studied using UNRES force field and the most popular computer simulation method, Metropolis Monte Carlo algorithm. At a fixed temperature, 100 Monte Carlo simulations are performed, starting from the folded native structure, and the pathways are obtained at two different temperatures.

Computational Modeling of Beta-Fibrils

Beta-sheet protein fibrils have been associated with many important pathological conditions such as Alzheimer's disease and type II diabetes. These fibrils are also important in the emerging field of peptide drug delivery since fibrillation is one of the critical degradation pathways for this class of drug molecules. Moreover, these structures have bionanotechnological applications such as formation of bio-nano-tubular scaffolds. Rational design of anti-fibrillation drugs, stable formulation of peptide drug molecules and development of bionanotechnological fibril devices all depend on understanding the structure of these fibrils. Although structures for some of such fibrils are experimentally determined, there are many others for which no structure is available. Hence, a computational method to model the structure of beta-sheet rich fibrils will be very valuable. We have devised such a computational method by combining homology modelling and MD simulation. The process starts with homology modelling of the beta-sheet building blocks using an optimized alignment and modelling scheme. Using those building blocks higher structures of the fibril are constructed. Finally, MD simulations are used to refine the fibril model and validate its energetic stability. We demonstrate the validity of our method by reproducing the experimentally determined structure of a number of beta-sheet fibrils. We also present the structure of glucagon beta-fibril for which no experimental structure is available. Understanding the structure of glucagon fibril can help inhibiting its fibrillation which is a major obstacle facing glucagon drug delivery.

Detection and quantification of soymilk in cow–buffalo milk using Attenuated Total Reflectance Fourier Transform Infrared spectroscopy (ATR–FTIR)

Milk consumption is steadily increasing, especially in India and China, due to rising income. To bridge the gap between supply and demand, unscrupulous milk vendors add milk-like products from vegetable sources (soymilk) to milk without declaration. A rapid detection technique is required to enforce the safety norms of food regulatory authorities. Fourier Transform Infrared (FTIR) spectroscopy has demonstrated potential as a rapid quality monitoring method and was therefore explored for detection of soymilk in milk. In the present work, spectra of milk, soymilk (SM), and milk adulterated with known quantity of SM were acquired in the wave number range of 4000–500cm−1 using Attenuated Total Reflectance (ATR)–FTIR. The acquired spectra revealed differences amongst milk, SM and adulterated milk (AM) samples in the wave number range of 1680–1058cm−1. This region encompasses the absorption frequency of amide-I, amide-II, amide-III, beta-sheet protein, α-tocopherol and Soybean Kunitz Trypsin Inhibitor. Principal component analysis (PCA) showed clustering of samples based on SM concentration at 5% level of significance and thus SM could be detected in milk using ATR–FTIR. The SM was best predicted in the range of 1472–1241cm−1 using multiple linear regression with coefficient of determination (R2) of 0.99 and 0.92 for calibration and validation, respectively.

Persistence length and stochastic fragmentation of supramolecular nanotubes under mechanical force

Cyclic peptide nanotubes (CPNs) exhibit impressive structural, mechanical and chemical properties in resemblance to beta-sheet proteins found in silks and amyloids, and show potential as supramolecular nanotubes that can be utilized to generate novel nanocomposites and nanoporous thin films. Quantifying the persistence length and thermomechanical fragmentation of CPNs is of great importance for establishing a theoretical basis of how to generate rectilinear nanostructures with controlled aspect ratio and rigidity. However, factors governing the elasticity and dynamical breaking of these supramolecular nanostructures remain to be fully understood. Here we present a statistical analysis of the Young’s modulus and persistence length of CPNs using fully-atomistic molecular dynamic simulations in explicit solvent. We show that the measured properties exhibit a dependence on the magnitude of the shear force applied, and extrapolation to the quasi-static deformation case yields 0.46 μm for the persistence length and 7.8 GPa for the Young’s modulus, in agreement with our experimental observations from TEM images. We establish a theoretical model for the spatial and temporal distribution of stochastic fracture, which we use to explain the simulation-based observations of spontaneous fragmentation under an applied shear force. Our methodology, blending theory, simulation and experiments provide a framework that can be utilized to investigate the mechanical behavior of self-assembling protein materials, paving the way for their design towards biological and industrial applications.

Nanoconfinement of spider silk fibrils begets superior strength, extensibility and toughness

AbstractSilk is an exceptionally strong, extensible and tough material made from simple protein building blocks. The molecular structure of dragline spider silk repeat units consists of semi-amorphous and nanocrystalline beta-sheet protein domains. Here we show by a series of computational experiments how the nanoscale properties of silk repeat units are scaled up to create macroscopic silk fibers with outstanding mechanical properties despite the presence of cavities, tears and cracks. We demonstrate that the geometric confinement of silk fibrils to diameters of 50±30 nm width is critical to facilitate a powerful mechanism by which hundreds of thousands of protein domains synergistically resist deformation and failure to provide enhanced strength, extensibility and toughness at the macroscale, closely matching experimentally measured mechanical properties. Through this mechanism silk fibers exploit the full potential of the nanoscale building blocks, regardless of the details of microscopic loading conditions and despite the presence of large defects. Experimental results confirm that silk fibers are composed of silk fibril bundles with diameters in the range of 20-150 nm, in agreement with our predicted length-scale. Our study reveals a general mechanism to map nanoscale properties to the macroscale and provides a potent design strategy towards novel fiber and bulk nanomaterials.

Performance assessment of different constraining potentials in computational structure prediction for disulfide-bridged proteins

The presence of disulfide bonds in proteins has very important implications on the three-dimensional structure and folding of proteins. An adequate treatment of disulfide bonds in de-novo protein simulations is therefore very important. Here we present a computational study of a set of small disulfide-bridged proteins using an all-atom stochastic search approach and including various constraining potentials to describe the disulfide bonds. The proposed potentials can easily be implemented in any code based on all-atom force fields and employed in simulations to achieve an improved prediction of protein structure. Exploring different potential parameters and comparing the structures to those from unconstrained simulations and to experimental structures by means of a scoring function we demonstrate that the inclusion of constraining potentials improves the quality of final structures significantly. For some proteins (1KVG and 1PG1) the native conformation is visited only in simulations in presence of constraints. Overall, we found that the Morse potential has optimal performance, in particular for the β-sheet proteins.

Flaw-tolerance in silk fibrils explains strength, extensibility and toughness of spider silk

AbstractSilk is an ancient but remarkably strong, extensible and tough material made from simple protein building blocks. Earlier work has shown that the particular molecular geometry of silk with a composite of semi-amorphous and nanocrystalline beta-sheet protein domains provides the structural basis for its characteristic softening-stiffening behavior and remarkable strength at the nanoscale. Yet, an open question remains as to how these nanoscale properties are upscaled so effectively to create strong, extensible and tough silk fibers. Here we discover that the geometric confinement of fibrils to ≈50-100 nm width and arranged in bundles to form larger-scale silk fibers, is the key to explaining the upscaling of the mechanical properties of silk from the atomistic scale upwards. We find that under this geometric confinement, hundreds of thousands of protein domains unfold simultaneously and thereby act synergistically to resist deformation and failure, providing access to enhanced large-scale strength, extensibility and toughness. Moreover, since the material is in a flaw-tolerant state under this geometric confinement, structural inhomogeneities such as cavities or tears that typically act as stress concentrators do not compromise the material performance. Indeed, experimental work showed that the diameter of silk fibrils that make up larger-scale silk fibers are on the order of 20-100 nm, in agreement with our findings. The exploitation of this mechanism in engineering design enables the synthesis of hierarchical fiber materials for superior performance despite limited and inferior building blocks.