Protein Fibril Formation Research Articles

Changes in structure, rheological property and antioxidant activity of soy protein isolate fibrils by ultrasound pretreatment and EGCG

Impacts of ultrasound pretreatment on the formation of soy protein fibrils, interaction of soy protein fibrils and EGCG, rheological property and antioxidant activity of the complex, and bioaccessibility of EGCG were investigated in this study. Fourier-transform infrared (FTIR) spectroscopy, fluorescence spectroscopy, and surface hydrophobicity (H0) indicated that ultrasound caused the unfolding of soy protein structure and exposure of hydrophobic regions. Compared with the un-treated soy protein fibrils, there were greater Thioflavin T fluorescence intensity and β-sheet content in the ultrasound-treated protein fibrils. These results indicated that ultrasound pretreatment could be used for modifying soy protein to improve its fibrils formation. FTIR spectroscopy, fluorescence spectroscopy, X-ray diffraction, and H0 showed that protein fibrils mainly interacted with EGCG through hydrogen bonds and hydrophobic interactions. The stability, viscosity, and antioxidant activity of soy protein fibrils treated with ultrasound-assisted EGCG (U–F-EGCG) were greater than those of other samples. EGCG drove the formation of protein fibrils-EGCG hydrogels. In vitro bioaccessibility of EGCG was significantly improved after its formation of complex with protein fibrils. Hence, the soy protein fibril hydrogels system can extend the application as the delivery carriers of bioactive compounds.

Multiple-State Monitoring of SOD1 Amyloid Formation at Single-Residue Resolution by Rheo-NMR Spectroscopy.

Formation of protein aggregates or fibrils entails the conversion of soluble native protein monomers via multiple molecular states. No spectroscopic techniques have succeeded in capturing the transient molecular-scale events of fibrillation in situ. Here we report residue- and state-specific real-time monitoring of the fibrillation of amyotrophic lateral sclerosis-related SOD1 by rheology NMR (Rheo-NMR) spectroscopy. Under moderately denaturing conditions, where NMR signals of folded and unfolded monomeric SOD1 are simultaneously observable, the cross-peak intensities of folded monomeric SOD1 decreased faster than those of the unfolded species, and a 310-helix in folded SOD1 was deformed prior to global unfolding. Furthermore, real-time protein dynamics analysis identified residues involved in the core structure formation of SOD1 oligomers. Our findings provide insight into local and global unfolding events in SOD1 and fibril formation. This Rheo-NMR analysis will be applicable not only to atomic-level monitoring of other amyloidogenic proteins but also to quantification of shear-induced structural changes of non-amyloidogenic proteins and elucidation of shear-enhanced chemical phenomena such as viscosity increase and crystallization of various solution-state compounds.

Protein Nanofibrils for Sustainable Food–Characterization and Comparison of Fibrils from a Broad Range of Plant Protein Isolates

Protein nanofibrils (PNFs) from plant-based protein sources have great potential for use in new sustainable food applications or biobased materials. Plant-based proteins from seven different sources (fava bean, mung bean, lupin, oat, rapeseed, soybean, and potato) were evaluated here for their ability for forming PNFs and compared with whey protein PNFs. Formation of PNFs was studied under incubation at acidic conditions (pH 2) and heat (85–90 °C) for 24–96 h. Presence of PNFs was detected using thioflavin T, circular dichroism spectroscopy, Fourier-transform infrared spectroscopy, and atomic force microscopy. The results showed that all plant-based proteins were able to form PNFs. For some of the plant protein isolates in this study, nanofibril formation is reported for the first time. We also describe and compare the distinct features associated with each protein source and the morphological alterations of the nanoscale structures. Finally, we describe how PNF production can be enhanced by purifying the fibril-forming protein component. Taken together, our study suggests that the structural and functional variation within plant protein nanofibrils can be exploited in future scaled-up food or material applications.

Stress Response Is the Main Trigger of Sporadic Amyloidoses.

Amyloidoses are a group of diseases associated with the formation of pathological protein fibrils with cross-β structures. Approximately 5–10% of the cases of these diseases are determined by amyloidogenic mutations, as well as by transmission of infectious amyloids (prions) between organisms. The most common group of so-called sporadic amyloidoses is associated with abnormal aggregation of wild-type proteins. Some sporadic amyloidoses are known to be induced only against the background of certain pathologies, but in some cases the cause of amyloidosis is unclear. It is assumed that these diseases often occur by accident. Here we present facts and hypotheses about the association of sporadic amyloidoses with vascular pathologies, trauma, oxidative stress, cancer, metabolic diseases, chronic infections and COVID-19. Generalization of current data shows that all sporadic amyloidoses can be regarded as a secondary event occurring against the background of diseases provoking a cellular stress response. Various factors causing the stress response provoke protein overproduction, a local increase in the concentration or modifications, which contributes to amyloidogenesis. Progress in the treatment of vascular, metabolic and infectious diseases, as well as cancers, should lead to a significant reduction in the risk of sporadic amyloidoses.

Systematic stability testing of insulins as representative biopharmaceuticals using ATR FTIR-spectroscopy with focus on quality assurance.

.Significance: Bioactive proteins represent the most important component class in biopharmaceutical products for therapeutic applications. Their production is most often biotechnologically realized by genetically engineered microorganisms. For the quality assurance of insulins as representatives of life-saving pharmaceuticals, analytical methods are required that allow more than total protein quantification in vials or batches. Chemical and physical factors such as unstable temperatures or shear rate exposure under storage can lead to misfolding, nucleation, and subsequent fibril forming of the insulins. The assumption is valid that these processes go parallel with a decrease in bioactivity.Aim: Infrared (IR) spectroscopy has been successfully utilized for secondary structure analysis in cases of protein misfolding and fibril formation.Approach: A reliable method for the quantification of the secondary structure changes has been developed using insulin dry-film Fourier-transform IR spectroscopy in combination with the attenuated total reflection (ATR) technique and subsequent data analyses such as band-shift determination, spectral band deconvolution, and principal component analysis.Results: A systematic study of insulin spectra was carried out on model insulin specimens, available either as original formulations or as hormones purified by ultrafiltration. Insulin specimens were stored at different temperatures, i.e., 0°C, 20°C, and 37°C, respectively, for up to three months. Weekly ATR-measurements allowed the monitoring of hormone secondary structure changes, which are supposed to be negatively correlated with insulin bioactivity.Conclusions: It could be shown that IR-ATR spectroscopy offers a fast and reliable analytical method for the determination of secondary structural changes within insulin molecules, as available in pharmaceutical insulin formulations and therefore challenges internationally established measurement techniques for quality control regarding time, costs, and effort of analysis.

Polyphosphates induce amyloid fibril formation of α-synuclein in concentration-dependent distinct manners

Polyphosphates (polyPs), chains of phosphate residues found in species across nature from bacteria to mammals, were recently reported to accelerate the amyloid fibril formation of many proteins. How polyPs facilitate this process, however, remains unknown. To gain insight into their mechanisms, we used various physicochemical approaches to examine the effects of polyPs of varying chain lengths on ultrasonication-dependent α-synuclein (α-syn) amyloid formation. Although orthophosphate and diphosphate exhibited a single optimal concentration of amyloid formation, triphosphate and longer-chain phosphates exhibited two optima, with the second at a concentration lower than that of orthophosphate or diphosphate. The second optimum decreased markedly as the polyP length increased. This suggested that although the optima at lower polyP concentrations were caused by interactions between negatively charged phosphate groups and the positive charges of α-syn, the optima at higher polyP concentrations were caused by the Hofmeister salting-out effects of phosphate groups, where the effects do not depend on the net charge. NMR titration experiments of α-syn with tetraphosphate combined with principal component analysis revealed that, at low tetraphosphate concentrations, negatively charged tetraphosphates interacted with positively charged “KTK” segments in four KTKEGV repeats located at the N-terminal region. At high concentrations, hydrated tetraphosphates affected the surface-exposed hydrophilic groups of compact α-syn. Taken together, our results suggest that long-chain polyPs consisting of 60 to 70 phosphates induce amyloid formation at sub-μM concentrations, which are comparable with the concentrations of polyPs in the blood or tissues. Thus, these findings may identify a role for polyPs in the pathogenesis of amyloid-related diseases.

Organic dots (O-dots) for theranostic applications: preparation and surface engineering.

Organic dots is a term used to represent materials including graphene quantum dots and carbon quantum dots because they rely on the presence of other atoms (O, H, and N) for their photoluminescence or fluorescence properties. They generally have a small size (as low as 2.5 nm), and show good photostability under prolonged irradiation. The excitation and emission wavelengths of O-dots can be tailored according to their synthetic procedure, where although their quantum yield is quite low compared with organic dyes, this is partly compensated by their large absorption coefficients. A wide range of strategies have been used to modify the surface of O-dots for passivation, improving their solubility and biocompatibility, and allowing the attachment of targeting moieties and therapeutic cargos. Hybrid nanostructures based on O-dots have been used for theranostic applications, particularly for cancer imaging and therapy. This review covers the synthesis, physics, chemistry, and characterization of O-dots. Their applications cover the prevention of protein fibril formation, and both controlled and targeted drug and gene delivery. Multifunctional therapeutic and imaging platforms have been reported, which combine four or more separate modalities, frequently including photothermal or photodynamic therapy and imaging and drug release.

Amyloid fibril formation is suppressed in microgravity

In the future, humans may live in space because of global pollution and weather fluctuations. In microgravity, convection does not occur, which may change the amyloidogenicity of proteins. However, the effect of gravity on amyloid fibril formation is unclear and remains to be elucidated. Here, we analyzed the effect of microgravity on amyloid fibril formation of amyloidogenic proteins including insulin, amyloid β42 (Aβ42), and transthyretin (TTR). We produced microgravity (10−3 g) by using the gravity controller Gravite. Human insulin, Aβ42, and human wild-type TTR (TTRwt) were incubated at pH 3.0, 7.0, and 3.5 at 37 °C, respectively, in 1 g on the ground or in microgravity. We measured amyloidogenicity via the thioflavin T (ThT) method and cell-based 1-fluoro-2,5-bis[(E)-3-carboxy-4-hydroxystyryl]benzene (FSB) assay. ThT fluorescence intensity and cell-based FSB assay results for human insulin samples were decreased in microgravity compared with results in 1 g. Aβ42 samples did not differ in ThT fluorescence intensity in microgravity and in 1 g on the ground. However, in the cell-based FSB assay, the staining intensity was reduced in microgravity compared with that on 1 g. Human TTRwt tended to form fewer amyloid fibrils in ThT fluorescence intensity and cell-based FSB assays in microgravity than in 1 g. Human insulin and Aβ42 showed decreased amyloid fibril formation in microgravity compared with that in 1 g. Human TTRwt tended to form fewer amyloid fibrils in microgravity. Our experiments suggest that the earth's gravity may be an accelerating factor for amyloid fibril formation.

Texturized mung bean protein as a sustainable food source: Effects of extrusion on its physical, textural and protein quality

Despite being inexpensive and a sustainable source of plant protein, mung bean remains underutilized due to its hard-to-cook characteristics. The aim of this study is to produce texturized mung bean protein (TMBP) with desirable physical properties, through optimization of extrusion parameters determined as follows: 49.33% feed moisture, 80.66 rpm screw speed and 144.57 °C barrel temperature. Under these conditions, all physical properties (expansion ratio, bulk density, rehydration ratio, water absorption capacity and degree of texturization) are desirably altered. Microstructure analysis reveals fibrous structure that is aligned along the shear flow direction in TMBP while SDS-PAGE shows partial protein unfoldment that is crucial for protein fibril formation during texturization. Amino acid profile shows high retention of amino acids after texturization. Current study successfully demonstrates the optimized production of TMBP from mung bean protein with strong potential as meat extender to serve as a healthier option compared to animal proteins. Industrial relevanceCurrent work is conducted on a pilot-scale basis, from initial protein extraction up to final extrusion step. This allows food industry to readily adapt the processing parameters, as detailed in current work, for customized production of texturized mung bean protein.

Spearmint Extract Containing Rosmarinic Acid Suppresses Amyloid Fibril Formation of Proteins Associated with Dementia.

Neurological dementias such as Alzheimer’s disease and Lewy body dementia are thought to be caused in part by the formation and deposition of characteristic insoluble fibrils of polypeptides such as amyloid beta (Aβ), Tau, and/or α-synuclein (αSyn). In this context, it is critical to suppress and remove such aggregates in order to prevent and/or delay the progression of dementia in these ailments. In this report, we investigated the effects of spearmint extract (SME) and rosmarinic acid (RA; the major component of SME) on the amyloid fibril formation reactions of αSyn, Aβ, and Tau proteins in vitro. SME or RA was added to soluble samples of each protein and the formation of fibrils was monitored by thioflavin T (ThioT) binding assays and transmission electron microscopy (TEM). We also evaluated whether preformed amyloid fibrils could be dissolved by the addition of RA. Our results reveal for the first time that SME and RA both suppress amyloid fibril formation, and that RA could disassemble preformed fibrils of αSyn, Aβ, and Tau into non-toxic species. Our results suggest that SME and RA may potentially suppress amyloid fibrils implicated in the progression of Alzheimer’s disease and Lewy body dementia in vivo, as well.

Collagen and Endothelial Cell Coculture Improves β-Cell Functionality and Rescues Pancreatic Extracellular Matrix.

The use of biomaterials and biomaterial functionalization is a promising approach to support pancreatic islet viability posttransplantation in an effort to reduce insulin dependence for patients afflicted with diabetes mellitus type 1. Extracellular matrix (ECM) proteins are known to impact numerous reparative functions in the body. Assessing how endogenously expressed pancreatic ECM proteins are affected by posttransplant-like hypoxic conditions may provide significant insights toward the development of tissue-engineered therapeutic strategies to positively influence β-cell survival, proliferation, and functionality. Here, we investigated the expression of three relevant groups of pancreatic ECM proteins in human native tissue, including basement membrane (BM) proteins (collagen type 4 [COL4], laminins [LAM]), proteoglycans (decorin [DCN], nidogen-1 [NID1]), and fibril-forming proteins (fibronectin [FN], collagen type 1 [COL1]). In an in vitro hypoxia model, we identified that ECM proteins were differently affected by hypoxic conditions, contributing to an overall loss of β-cell functionality. The use of a COL1 hydrogel as carrier material demonstrated a protective effect on β-cells mitigating the effect of hypoxia on proteoglycans as well as fibril-forming protein expression, supporting β-cell functionality in hypoxia. We further showed that providing endothelial cells (ECs) into the COL1 hydrogel improves β-cell response as well as the expression of relevant BM proteins. Our data show that β-cells benefit from a microenvironment composed of structure-providing COL1 with the incorporation of ECs to withstand the harsh conditions of hypoxia. Such hydrogels support β-cell survival and can serve as an initial source of ECM proteins to allow cell engraftment while preserving cell functionality posttransplantation. Impact statement Expression analysis identifies hypoxia-induced pathological changes in extracellular matrix (ECM) homeostasis as potential targets to support β-cell transplants by encapsulation in biomaterials for the treatment of diabetes mellitus. A collagen-1 hydrogel is shown to attenuate the effect of hypoxia on β-cells and their ECM expression. The functionalization of the hydrogel with endothelial cells increases the β-cell response to glucose and rescues essential basement membrane proteins.

Effect of the air-water interface on the conformation of amyloid beta.

It has long been recognized that liquid interfaces, such as the air-water interface (AWI), can enhance the formation of protein fibrils. This makes liquid interfaces attractive templates for fibril formation but fully realizing this requires knowledge of protein behavior at interfaces, which is currently lacking. To address this, molecular dynamics simulation is used to investigate fragments of amyloid beta, a model fibril forming protein, at the air-water interface. At the air-water interface, the enrichment of aggregation-prone helical conformations provides a mechanism for the enhancement of fibrillation at interfaces. The conformational ensemble at the air-water interface was also considerably reduced compared to bulk solution due to the tendency of hydrophobic side chains partitioning into the air restricting the range of conformations. Little overlap between the conformational ensembles at the AWI and in the bulk solution was found, suggesting that AWI induces the formation of a different set of structures compared to bulk solution. The smaller Aβ(16-22) and Aβ(25-35) fragments show an increase in the propensity for an ordered secondary structure at the air-water interface but with a increased propensity for turn over other motifs, illustrating the importance of intra-protein interactions for stabilizing helical and extended conformations.

Effect of O-glycosylation on amyloid fibril formation of the variable domain in the Vλ6 light chain mutant Wil

Glycosylation is one of the major post-translational modifications in eukaryotic cells and has been reported to affect the amyloid fibril formation in several amyloidogenic proteins and peptides. In this study, we expressed a Vλ6 light chain mutant, Wil, which is an amyloidogenic mutant in AL amyloidosis, by the yeast Pichia pastoris. After separation by cation exchange chromatography, we obtained the O-glycosylated and non-glycosylated Wil mutants in high yield. The structures of these Wil mutants were identical except with respect to glycosylation, and the stabilities were also identical. On the other hand, the O-glycosylation retarded the amyloid fibril formation in a sugar size-dependent manner. From these results, we discussed the role of covalently attached glycan in the retardation of amyloid fibril formation.

Investigating the effect of sugar-terminated nanoparticles on amyloid fibrillogenesis of β-lactoglobulin

In vivo tissue deposition of fibrillar protein aggregates is the cause of several degenerative diseases. Evidence suggests that interfering with the pathology-associated amyloid fibrillogenesis by inhibitory molecules is envisaged as the primary therapeutic strategy. Amyloid fibril formation of proteins has been demonstrated to be influenced by nanoparticles/nanomaterials. As compared with their molecular form counterpart, this work examined the effect of sucrose-terminated nanoparticles on the in vitro amyloid fibrillogenesis and structural properties of β-lactoglobulin at pH 2.0 and 80 °C. ThT binding and electron microscopy results demonstrated that sucrose-terminated nanoparticles were able to suppress β-lactoglobulin fibrillogenesis in a concentration-dependent fashion. Importantly, sucrose-terminated nanoparticles showed better β-lactoglobulin fibril-inhibiting ability than sucrose molecules. ANS fluorescence and right-angle light scattering results showed reduced solvent exposure and decreased aggregation, respectively, in the β-lactoglobulin samples upon treatment with sucrose-terminated nanoparticles. Moreover, fluorescence quenching analyses revealed that the static quenching mechanism and formation of a non-fluorescent fluorophore–nanoparticle complex are involved in the nanoparticle-β-lactoglobulin interaction. We believe that the results from this study may suggest that the nanoparticle form of biocompatible sugar-related osmolytes may serve as effective inhibiting/suppressing agents toward protein fibrillogenesis.

Hydrophobic surfaces help reduce harmful protein fibril formation

Molecular dynamics simulation reveals key insights on the formation of amyloid fibril formation and potentially how to develop treatments against it.

Drying mode and hydrothermal treatment conditions govern the formation of amyloid-like protein fibrils in solutions of dried hen egg white

The recipe of some food products contains dried hen egg white (EW). It is convenient in use and available with a range of functionalities as a result of being stored under different time, temperature and moisture conditions following drying. Amyloid fibrils are fibrillary protein structures composed of highly ordered stacking of cross-β sheets. They can contribute to the foaming and gelling capacity of EW proteins. We here for the first time report that dried EW contains amyloid-like fibrils and that such fibrils in spray-dried EW (EWSD) are longer (ca. 100–200 nm) than those in freeze-dried EW [EWFD] (ca. 20–100 nm). In addition, conditions for optimal fibrillation were determined for both EWFD and freeze-dried ovalbumin (OVAFD) using a response surface design. Dilute solutions of OVAFD and EWFD were incubated at different pH values, times and temperatures. After storage at optimal conditions OVAFD [2.0% (wprotein/v), pH 7.0, 23 h, 76 °C] and EWFD [0.5% (wprotein/v), pH 7.0, 24 h, 85 °C], a higher level of cross-β sheet structures and larger worm-like protein fibrils were observed for OVAFD than for EWFD. Lastly, when EWSD stored for one week at either 50 °C and 50% relative humidity (RH) [EWSD 50°C/50%] or at 60 °C and 80% RH (EWSD 60°C/80%) was submitted to heating [0.5% (wprotein/v), pH 7.0, 24 h, 85 °C], a higher extent of fibrillation was found for EWSD 60°C/80% than for EWSD 50°C/50%. Thus, drying induces EW amyloid-like protein fibrillation. Also dry heating EWSD at 60 °C and 80% RH further enhances such fibrillation during heating in excess of water.

Quadruple abnormal protein aggregates in brainstem pathology and exogenous metal-rich magnetic nanoparticles (and engineered Ti-rich nanorods). The substantia nigrae is a very early target in young urbanites and the gastrointestinal tract a key brainstem portal

Fine particulate air pollution (PM2.5) exposures are linked with Alzheimer's and Parkinson's diseases (AD,PD). AD and PD neuropathological hallmarks are documented in children and young adults exposed lifelong to Metropolitan Mexico City air pollution; together with high frontal metal concentrations (especially iron)–rich nanoparticles (NP), matching air pollution combustion- and friction-derived particles. Here, we identify aberrant hyperphosphorylated tau, ɑ synuclein and TDP-43 in the brainstem of 186 Mexico City 27.29 ± 11.8y old residents. Critically, substantia nigrae (SN) pathology seen in mitochondria, endoplasmic reticulum and neuromelanin (NM) is co-associated with the abundant presence of exogenous, Fe-, Al- and Ti-rich NPs.The SN exhibits early and progressive neurovascular unit damage and mitochondria and NM are associated with metal-rich NPs including exogenous engineered Ti-rich nanorods, also identified in neuroenteric neurons. Such reactive, cytotoxic and magnetic NPs may act as catalysts for reactive oxygen species formation, altered cell signaling, and protein misfolding, aggregation and fibril formation. Hence, pervasive, airborne and environmental, metal-rich and magnetic nanoparticles may be a common denominator for quadruple misfolded protein neurodegenerative pathologies affecting urbanites from earliest childhood. The substantia nigrae is a very early target and the gastrointestinal tract (and the neuroenteric system) key brainstem portals. The ultimate neural damage and neuropathology (Alzheimer's, Parkinson's and TDP-43 pathology included) could depend on NP characteristics and the differential access and targets achieved via their portals of entry. Thus where you live, what air pollutants you are exposed to, what you are inhaling and swallowing from the air you breathe,what you eat, how you travel, and your occupational longlife history are key. Control of NP sources becomes critical.

NSs amyloid formation is associated with the virulence of Rift Valley fever virus in mice

Amyloid fibrils result from the aggregation of host cell-encoded proteins, many giving rise to specific human illnesses such as Alzheimer’s disease. Here we show that the major virulence factor of Rift Valley fever virus, the protein NSs, forms filamentous structures in the brain of mice and affects mortality. NSs assembles into nuclear and cytosolic disulfide bond-dependent fibrillary aggregates in infected cells. NSs structural arrangements exhibit characteristics typical for amyloids, such as an ultrastructure of 12 nm-width fibrils, a strong detergent resistance, and interactions with the amyloid-binding dye Thioflavin-S. The assembly dynamics of viral amyloid-like fibrils can be visualized in real-time. They form spontaneously and grow in an amyloid fashion within 5 hours. Together, our results demonstrate that viruses can encode amyloid-like fibril-forming proteins and have strong implications for future research on amyloid aggregation and toxicity in general.

RNA phase separation-mediated direction of molecular trafficking under conditions of molecular crowding.

Living cells are highly crowded with large and small biomolecules. The total concentration of biomolecules can reach 400mg/ml, and 40% of the cell volume is occupied by biomolecules. Droplet formation in cells via liquid-liquid phase separation may play a role in controlling biochemical reactions in this complex molecular environment. Liquid-liquid phase separation generally involves nucleic acids and proteins as anionic and cationic components, respectively. Significant characteristics of droplets, which make them different from protein aggregation or fibril formation, are reversibility of formation and responsiveness to the molecular environment. In this review, we quantitatively describe the molecular environment inside cells and droplets that participate in controlling central dogma reactions. Finally, we discuss the importance of droplets under conditions of molecular crowding within living cells.

Amyloid assembly is dominated by misregistered kinetic traps on an unbiased energy landscape

Atomistic description of protein fibril formation has been elusive due to the complexity and long time scales of the conformational search. Here, we develop a multiscale approach combining numerous atomistic simulations in explicit solvent to construct Markov State Models (MSMs) of fibril growth. The search for the in-register fully bound fibril state is modeled as a random walk on a rugged two-dimensional energy landscape defined by β-sheet alignment and hydrogen-bonding states, whereas transitions involving states without hydrogen bonds are derived from kinetic clustering. The reversible association/dissociation of an incoming peptide and overall growth kinetics are then computed from MSM simulations. This approach is applied to derive a parameter-free, comprehensive description of fibril elongation of Aβ16-22 and how it is modulated by phenylalanine-to-cyclohexylalanine (CHA) mutations. The trajectories show an aggregation mechanism in which the peptide spends most of its time trapped in misregistered β-sheet states connected by weakly bound states twith short lifetimes. Our results recapitulate the experimental observation that mutants CHA19 and CHA1920 accelerate fibril elongation but have a relatively minor effect on the critical concentration for fibril growth. Importantly, the kinetic consequences of mutations arise from cumulative effects of perturbing the network of productive and nonproductive pathways of fibril growth. This is consistent with the expectation that nonfunctional states will not have evolved efficient folding pathways and, therefore, will require a random search of configuration space. This study highlights the importance of describing the complete energy landscape when studying the elongation mechanism and kinetics of protein fibrils.