Disulfide Interchange Research Articles

Proteome and Physiological Characterization of Halotolerant Nodule Endophytes: The Case of Rahnella aquatilis and Serratia plymuthica

Bacterial endophytes were isolated from nodules of pea and fava bean. The strains were identified and characterized for plant beneficial activities (phosphate solubilization, synthesis of indole acetic acid and siderophores) and salt tolerance. Based on these data, four strains of Rahnella aquatilis and three strains of Serratia plymuthica were selected. To shed light on the mechanisms underlying salt tolerance, the proteome of the two most performant strains (Ra4 and Sp2) grown in the presence or not of salt was characterized. The number of proteins expressed by the endophytes was higher in the presence of salt. The modulated proteome consisted of 302 (100 up-regulated, 202 down-regulated) and 323 (206 up-regulated, 117 down-regulated) proteins in Ra4 and Sp2, respectively. Overall, proteins involved in abiotic stress responses were up-regulated, while those involved in metabolism and flagellum structure were down-regulated. The main up-regulated proteins in Sp2 were thiol: disulfide interchange protein DsbA, required for the sulfur binding formation in periplasmic proteins, while in Ra4 corresponded to the soluble fraction of ABC transporters, having a role in compatible solute uptake. Our results demonstrated a conserved response to salt stress in the two taxonomically related species.

Interactions of Nereistoxin and Its Analogs with Vertebrate Nicotinic Acetylcholine Receptors and Molluscan ACh Binding Proteins.

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [3H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [125I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4β2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [3H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [3H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.

Iso-seco-tanapartholide activates Nrf2 signaling pathway through Keap1 modification and oligomerization to exert anti-inflammatory effects

Covalent modification of Keap1 results in reducing ubiquitination and the accumulation of Nrf2, which subsequently initiates the transcription of cellular anti-oxidant and anti-inflammatory genes. Iso-seco-tanapartholide (IST), a sesquiterpene isolated from the traditional Chinese medicine Artemisia argyi, had been reported to possess NF-κB inhibitory activity. However, its deep anti-inflammatory effects and direct target have never been reported. Here we show that IST activated Nrf2 and increased its target gene expression. In particular, LPS-caused inflammation in vitro and in vivo was mitigated by IST-induced Nrf2 activation but aggravated by Nrf2 inhibition. Mechanically, IST targeted Keap1 proteins via alkylating its cysteine residues 151, 273, 288, and so on. Subsequently, the modifying agent IST was displaced by intermolecular sulfhydryl disulfide interchange to lead to a disulfide dimer of Keap1. The resulting conformational change of Keap1 liberated Nrf2 from sequestration and allowed it translocation to the nucleus to activate the transcriptional program. Further studies demonstrated that Keap1 dimer formation contributed to the anti-inflammatory effects of IST. Taken together, our findings reveal a new mechanism for Nrf2 activation and provide a potential lead compound to treat inflammatory diseases through targeting Keap1.

Efficient encapsulation of fish oil: Capitalizing on the unique inherent characteristics of whey cream and hydrolyzed whey protein

The effects of protein concentration and of blending a phospholipid-rich whey coproduct, Procream (Salibra 700 Procream, Glanbia Nutritionals), with intact or hydrolyzed whey protein concentrate, on fish oil microencapsulation efficiency and oxidative stability were assessed. Trypsin and protease M, from Aspergillus oryzae, were used to produce 2 unique hydrolysates. All microcapsules had excellent encapsulation efficiencies (>92%) and good physical properties, regardless of protein content and Procream inclusion. Intact α-lactalbumin and β-lactoglobulin and their peptides were involved in stabilizing oil droplets. Disulfide interchange resulted in formation of protein aggregates, which were more pronounced in samples containing Procream. Although all microcapsules had relatively good oxidative stability, most had better stability at 2 versus 0.5% protein. Protease M hydrolysate + Procream microcapsules had the highest stability, regardless of protein content. Results demonstrated that Procream, at a reduced protein inclusion level, can partially replace more expensive whey protein ingredients in microencapsulation, when blended with a select hydrolysate.

Action of carvacrol in Salmonella Typhimurium biofilm: A proteomic study.

Carvacrol presents action in Salmonella Typhimurium biofilms, however the antibiofilm mechanism of this compound has not been fully established yet. In the present study, the aim was to evaluate protein profile changes in S. Typhimurium biofilm treated with carvacrol. Proteomic analysis of treated versus untreated biofilm showed several changes in proteins involved with S. Typhimurium biofilm and antioxidant activity. The proteins DsbA (thiol: disulfide interchange protein DsbA), LuxS (S-ribosylhomocysteine lyase), DksA (RNA polymerase binding transcription factor DksA), and SODs (superoxide dismutases) A, B and C had their synthesis decreased after treatment with carvacrol. These proteins play a key role in S. Typhimurium biofilm formation, demonstrating the dynamic antibiofilm action of carvacrol. The differentially expressed proteins identified provide possible action targets for future studies in order to gain more insight into the mechanism of action of carvacrol on S. Typhimurium biofilm.

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli.

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding‐induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond‐containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild‐type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding‐induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide‐stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra‐cytosolic compartment, like the periplasm.

Structure and Oxidative Folding of AAI, the Major Alfa-Amylase Inhibitor From Amaranth Seeds.

AAI, the major alpha-amylase inhibitor (AAI) present in the seeds of the Mexican crop plant Amaranthus hypocondriacus is a 32-residue-long polypeptide with three disulfide bridges. Its structure is most closely related to the plant amylase inhibitor subfamily of knottins characterized by a topological knot formed by one disulfide bridge threading through a loop formed by the peptide chain as well as a short three-stranded beta sandwich core. AAI is specific against insect amylases and does not act on corresponding human or mammalian enzymes. It was found that the oxidative folding of AAI seems to follow a hirudine-like pathway with many non-native intermediates, but notably it proceeds through a major folding intermediate (MFI) that contains a vicinal disulfide bridge. Based on a review of the pertinent literature, the known vicinal disulfides in native proteins as well as well as the network of disulfide interchanges, we propose that MFI is a kinetic trap corresponding to a compact molten globule-like state which constrains the peptide chain to a smaller number of conformations that in turn can be rapidly funneled toward the native state.

Catalytic site-selective substrate processing within a tubular nanoreactor.

Chemists have long sought the ability to modify molecules precisely when presented with several sites of similar reactivity. We reasoned that the confinement of substrates within nanostructures might permit site-selective reactions unachievable in bulk solution, even with sophisticated reagents. In particular, the stretching and alignment of polymers within nanotubes might allow site-specific cleavage or modification. To explore this proposition, macromolecular disulfide substrates were elongated within members of a collection of tubular protein nanoreactors, which contained cysteine residues positioned at different locations along the length of each tube. For each nanoreactor, we defined the reactive location by using a set of polymer substrates (site-selectivity) and which of the two sulfur atoms was attacked (regioselectivity), and found that disulfide interchange occurs with atomic precision. Our strategy has potential for the selective processing of a wide variety of biomacromolecules, and the chemistry and substrates might be generalised yet further by using alternative nanotubes.

Double‐crosslinked reversible redox‐responsive hydrogels based on disulfide–thiol interchange

ABSTRACTWe present novel redox‐responsive hydrogels based on poly(N‐isopropylacrylamide) or poly(acrylamide), consisting of a reversible disulfide crosslinking agent N,N′‐bis(acryloyl)cystamine and a permanent crosslinking agent N,N′‐methylenebisacrylamide for microfluidic applications. The mechanism of swelling/deswelling behavior starts with the cleavage and reformation of disulfide bonds, leading to a change of crosslinking density and crosslinking points. Raman and ultraviolet‐visible spectroscopy confirm that conversion efficiency of thiol–disulfide interchange up to 99%. Rheological analysis reveals that the E modulus of hydrogel is dependent on the crosslinking density and can be repeatedly manipulated between high‐ and low‐stiffness states over at least 5 cycles without significant decrease. Kinetic studies showed that the mechanical strength of the gels changes as the redox reaction proceeds. This process is much faster than the autonomous diffusion in the hydrogel. Moreover, cooperative diffusion coefficient (Dcoop) indicates that the swelling process of the hydrogel is affected by the reduction reaction. Finally, this reversibly switchable redox behavior of bulky hydrogel could be proven in microstructured hydrogel dots through short‐term photopatterning process. These hydrogel dots on glass substrates also showed the desired short response time on cyclic swelling and shrinking processes known from downsized hydrogel shapes. Such stimuli‐responsive hydrogels with redox‐sensitive crosslinkers open a new pathway in exchanging analytes for sensing and separating in microfluidics applications. © 2019 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2019, 57, 2590–2601

Vapor-Deposited Reactive Coating with Chemically and Topographically Erasable Properties.

An erasable coating was prepared to modify material surfaces with accessibilities, including specific conjugation, elimination of the conjugated chemistry/function, and the reactivation of a second new chemistry/function. The coating was realized based on a vapor-deposited functional poly-p-xylylene coating composed of an integrated 3-((3-methylamido)-disulfanyl)propanoic acid functional group, resulting in not only chemical reactivity, but also a disulfide interchange mechanism. Mechanically, the coating was robust in terms of the thermal stability and adhesive property on a variety of substrate materials. Chemically, the anchoring site of carboxylic acid was accessible for specific conjugation, and a disulfide bridge moiety was used to disengage already installed functions/properties. In addition, the homogeneous nature of the vapor-phased coating technique is known for its morphology/thickness and distribution of the functional moiety, which allowed precision to address the installation or erasure of functions and properties. Characterization of the precisely confined hydrophilic/hydrophobic wetting property and the alternating reversibility of this wetting property on the same surface was achieved.

High Homogenization Speeds for Preparing Unstable Myofibrillar Protein-Olive Oil Emulsions.

Natural protein-based oil-in-water emulsions have recently attracted a lot of attention because of their potential as a synthetic emulsifier replacer. It is, however, unclear how the emulsification process and protein concentration may affect the stability of such emulsions. Therefore, this study investigated the effects of homogenization speeds (4,000, 8,000, 12,000, and 16,000 rpm/min) and myofibrillar protein (MP) concentrations (10, 20, 30, 40, and 50 mg/mL) on the stability of MP-olive oil emulsion. The emulsifying creaming index, emulsifying activity index (EAI), droplet size, microstructure, free sulfhydryl content, and zeta potential of the emulsion were measured. The results showed that with the condition of sufficient emulsifier (at least 20 mg/mL), the EAI increased, and the droplet size and zeta potential of emulsions decreased with the increase of homogenization speed. Emulsions were stable at 4,000 and 8,000 rpm/min (20, 30, 40, and 50 mg/mL) within 48 hr, and they were unstable at 12,000 and 16,000 rpm/min (20, 30, 40, and 50 mg/mL) within 48 hr. This result is mainly attributed to the fact that sulfhydryl-disulfide interchange leads the excessive aggregate of MP at the oil-water interface. PRACTICAL APPLICATION: Myofibrillar protein-olive oil emulsions (oil-in-water) may be used to deliver nutrients into food products. In this study, myofibrillar protein-olive oil emulsions stabilized with the optimizing emulsification conditions. This study may have important implications to produce food-grade myofibrillar protein-olive oil emulsions to deliver nutrients.

Grafting of organic sulfur-containing functional groups on activated carbon for gold(I) adsorption from thiosulfate solution

In the present study, sulfur-containing functional groups were grafted onto activated carbon and adsorption behavior for [Au(S2O3)2]3− was studied. Activated carbon grafted with sulfur-containing functional groups (S-AC) was developed with MBT (2-Mercaptobenzothiazole). The effects of MBT dosage and contact time in the preparation process were optimized; solution pH, thiosulfate ion concentration, and temperature of the surroundings in the adsorption stage over gold adsorption were studied. With a contact time of 24 h and MBT dosage of 0.7 g, S-AC had better gold adsorption ability and the prepared adsorbents worked well at a pH of about 9.0 and thiosulfate concentration of about 0.1 mol/L. The Freundlich model and pseudo-second order kinetics model fit the [Au(S2O3)2]3− adsorption process well. A total gold amount of 18.402 kg/t for five stages was attained with S-AC. The presence of thiol and disulfide linkages on the surface of activated carbon was proved using XPS, FT-IR, BET, and SEM. The adsorption of [Au(S2O3)2]3− onto S-AC involved a gold(I) thiolate and disulfide interchange mechanism. This study made the adsorption of [Au(S2O3)2]3− on activated carbon possible via a simple and feasible method, and this is of great significance for future research in leaching gold with thiosulfate.

Thermodynamic aspects of bis(3‑sulfopropyl) disulfide and 3‑mercapto‑1‑propanesulfonic acid in Cu electrodeposition

Organic additives play an important role in regulating film growth and properties in Cu electrodeposition. 3‑Mercapto‑1‑propane sulfonate (MPS) and its dimer bis(3‑sulfopropyl) disulfide (SPS) are representative accelerators that promote Cu electrodeposition rate. In this study, the formal reduction potential of SPS to MPS was determined by measuring the equilibrium constants of thiol–disulfide interchange reactions with the reference thiol/disulfide pairs by 1H nuclear magnetic resonance (1H NMR) spectroscopy. The calculated formal reduction potential of SPS to MPS was 0.153 V (vs. standard hydrogen electrode). Based on this calculation, the cell potentials of Cu(I)(thiolate) formation reaction with either SPS or MPS, which is a key reaction in accelerating mechanism, were also estimated by ultraviolet–visible absorption and 1H NMR spectroscopies: they were found to be 0.202 V and 0.214 V for SPS and MPS, respectively. The positive cell potential indicates that the formation of Cu(I)(thiolate) complexes is thermodynamically favorable in the presence of additives.

Effects of thermal and mechanical treatments on the physicochemical properties of wheat flour

Planetary ball milling is a possible way to improve the technological properties of wheat flour by thermal and mechanical modifications. In the present study, roller milled common wheat flours (Triticum aestivum L.) were additionally modified in a ball mill by varying mill parameters such as rotation speed and grinding time. As a result of ball milling, the flours experienced temperatures of up to 100 °C, the range of particle sizes strongly increased as well as the content of damaged starch. The treated flours were also analyzed for the protein distribution by SDS-polyacrylamide-gel electrophoresis and proteins were quantitated by an extraction/high-performance liquid chromatography (Osborne fractionation) and an extraction/gel permeation chromatography procedure (SDSL/GMP fractionation). Intense mechanical/thermal treatment led to a decrease of the content of extractable protein and shifted ethanol-soluble gliadins to the glutenin fraction. Cysteine-containing alpha- and gamma-gliadins initially soluble in 60% ethanol were bound to ethanol-insoluble glutenins by thiol–disulfide interchange reactions. Linked to that, a high level of modification also significantly decreased the gliadin/glutenin ratio and increased the glutenin macropolymer content. Cysteine-free omega5- and omega1,2-gliadin types were only weakly affected by milling. Heating induced by mechanical treatment is supposed to be mainly responsible for these effects. Thiol–disulfide interchange can also explain the increase of the particle size after intense ball milling, because flour particles could stick together via disulfide bonds between proteins located on the surface of different particles.

Cystamine-mediated inhibition of protein disulfide isomerase triggers aggregation of misfolded orexin-A in the Golgi apparatus and prevents extracellular secretion of orexin-A

Orexins (orexin-A and orexin-B) are neuropeptides that are reduced in narcolepsy, a sleep disorder that is characterized by excessive daytime sleepiness, sudden sleep attacks and cataplexy. However, it remains unclear how orexins in the brain and orexin neurons are reduced in narcolepsy. Orexin-A has two closely located intramolecular disulfide bonds and is prone to misfolding due to the formation of incorrect disulfide bonds. Protein disulfide isomerase (PDI) possesses disulfide interchange activity. PDI can modify misfolded orexin-A to its native form by rearrangement of two disulfide bonds. We have previously demonstrated that sleep deprivation and a high fat diet increase nitric oxide in the brain. This increase triggers S-nitrosation and inactivation of PDI, leading to aggregation of orexin-A and reduction of orexin neurons. However, the relationship between PDI inactivation and loss of orexin neurons has not yet been fully elucidated. In the present study, we used a PDI inhibitor, cystamine, to elucidate the precise molecular mechanism by which PDI inhibition reduces the number of orexin neurons. In rat hypothalamic slice cultures, cystamine induced selective depletion of orexin-A, but not orexin-B and melanin-concentrating hormone. Moreover, cystamine triggered aggregation of orexin-A, but not orexin-B in the Golgi apparatus of hypothalamic slice cultures and in vivo mouse brains. However, cystamine did not induce endoplasmic reticulum (ER) stress, and an ER stress inducer did not trigger aggregation of orexin-A in slice cultures. Finally, we demonstrated that cystamine significantly decreased extracellular secretion of orexin-A in AD293 cells overexpressing prepro-orexin. These findings suggest that cystamine-induced PDI inhibition induces selective depletion, aggregation in the Golgi apparatus and impaired secretion of orexin-A. These effects may represent an initial step in the pathogenesis of narcolepsy.

Alkylation of Free Thiol Groups During Extraction: Effects on the Osborne Fractions of Wheat Flour

Wheat proteins are classified according to solubility into the so‐called Osborne fractions. Because wheat flour contains both free thiol and disulfide groups, thiol–disulfide interchange reactions are possible during extraction. Osborne fractionation of 12 different wheat flour samples was performed in the presence of N‐ethylmaleinimide (NEMI) to alkylate free thiol groups and without addition of NEMI (control). The addition of NEMI during extraction tended to decrease the content of gliadins (predominantly α‐gliadins) and caused an increase of the content of glutenins in most flour samples. Thus, alkylation of free thiol groups during extraction led to a decline of the gliadin/glutenin ratio from 2 (control) to approximately 1.5 (NEMI). NEMI and control gliadins were separated by gel‐permeation HPLC into an oligomeric subfraction (high‐molecular‐weight [HMW] gliadins) and two monomeric subfractions. In most flours (8 of 12), the addition of NEMI led to a significant increase of the content of HMW gliadins. HMW gliadins from cultivar Akteur wheat were preparatively isolated from NEMI and control gliadins and characterized by HPLC, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and N‐terminal sequencing. HMW gliadin isolated in the presence of NEMI had a significantly higher content of low‐molecular‐weight glutenin subunits and disulfide‐bound cysteine as well as a lower content of α‐gliadins and disulfide‐bound glutathione compared with the control.

Photo-responsive liquid crystalline epoxy networks with exchangeable disulfide bonds

Disulfide exchange and thiol–disulfide interchange reactions allow for reprocessing and recycling of azobenzene-based liquid crystalline networks.

Effects of Microwave Radiation and Water Bath Heating on the Physicochemical Properties of Actomyosin from Silver Carp (Hypophthalmichthys molitrix)during Setting

Changes in the physical and chemical characteristics of actomyosin, isolated from silver carp (Hypophthalmichthys molitrix), induced by heating in a water bath and microwave oven were investigated. Ca2+-ATPase showed a heating-time-dependent decrease in activity, suggesting denaturation of myosin and dissociation of the actomyosin complex. The decrease in actomyosin solubility indicates the formation of protein aggregates. In addition, reactive sulfhydryl (R-SH) groups increased and total SH (T-SH) groups decreased with heating, indicating the formation of disulfide bonds through SH oxidation or disulfide interchanges. The differences of surface hydrophobicity (S0) showed microwave heating has an inhibitory effect on protein conformation changes. Fourier transform infrared confirmed the conformational changes of silver carp actomyosin. This study demonstrated that microwave heating can effectively depressed actomyosin structural changes during heating and provided useful information for surimi production. Practical Applications At present, there is lot of research about heat-induced gel from fish protein, but the way they used are traditional water bath or ohmic heating. Though reports of microwave heating on structure and the characteristics of fish protein are few, it is important for surimi gelation and microwave sterilization. We supposed microwave heating could influence the structure of fish protein and improve the quality of surimi. A preliminary study of microwave heating on actomyosin not only can enrich theory of heated induced protein gel also has important scientific value.

Probing the molecular forces involved in binding of selected volatile flavour compounds to salt-extracted pea proteins

Molecular interactions between heterologous classes of flavour compounds with salt-extracted pea protein isolates (PPIs) were determined using various bond disrupting agents followed by GC/MS analysis. Flavour bound by proteins decreased in the order: dibutyl disulfide>octanal>hexyl acetate>2-octanone=benzaldehyde. Benzaldehyde, 2-octanone and hexyl acetate interacted non-covalently with PPIs, whereas octanal bound PPIs via covalent and non-covalent forces. Dibutyl disulfide reacted with PPIs covalently, as its retention was not diminished by urea and guanidine hydrochloride. Using propylene glycol, H-bonding and ionic interactions were implicated for hexyl acetate, benzaldehyde, and 2-octanone. A protein-destabilising salt (Cl3CCOONa) reduced bindings for 2-octanone, hexyl acetate, and benzaldehyde; however, retention for octanal and dibutyl disulfide increased. Conversely, a protein-stabilising salt (Na2SO4) enhanced retention for benzaldehyde, 2-octanone, hexyl acetate and octanal. Formation of a volatile flavour by-product, 1-butanethiol, from dibutyl disulfide when PPIs were treated with dithiothreitol indicated occurrence of sulfhydryl–disulfide interchange reactions.

Preformed Seeds Modulate Native Insulin Aggregation Kinetics.

Insulin aggregates under storage conditions via disulfide interchange reaction. It is also known to form aggregates at the site of repeated injections in diabetes patients, leading to injection amyloidosis. This has fueled research in pharmaceutical and biotechnology industry as well as in academia to understand factors that modulate insulin stability and aggregation. The main aim of this study is to understand the factors that modulate aggregation propensity of insulin under conditions close to physiological and measure effect of "seeds" on aggregation kinetics. We explored the aggregation kinetics of insulin at pH 7.2 and 37 °C in the presence of disulfide-reducing agent dithiothreitol (DTT), using spectroscopy (UV-visible, fluorescence, and Fourier transform infrared spectroscopy) and microscopy (scanning electron microscopy, atomic force microscopy) techniques. We prepared insulin "seeds" by incubating disulfide-reduced insulin at pH 7.2 and 37 °C for varying lengths of time (10 min to 12 h). These seeds were added to the native protein and nucleation-dependent aggregation kinetics was measured. Aggregation kinetics was fastest in the presence of 10 min seeds suggesting they were nascent. Interestingly, intermediate seeds (30 min to 4 h incubation) resulted in formation of transient fibrils in 4 h that converted to amorphous aggregates upon longer incubation of 24 h. Overall, the results show that insulin under disulfide reducing conditions at pH and temperature close to physiological favors amorphous aggregate formation and seed "maturity" plays an important role in nucleation dependent aggregation kinetics.