Photoinduced fibrils formation of chicken egg white lysozyme under native conditions

Recent findings showed that transiently accessing structurally native‐like yet energetically higher conformational states is sufficient to trigger the formation of protein fibrils. Typically, these conformational states are made available through changing solvent conditions or introducing mutations. Here we show a novel way to initialize fibril formation for Chicken egg white lysozyme (CEWL) under native conditions via controlled UV illumination. Through a cassette of tryptophan‐based photochemistry, the two terminal disulfide bonds in CEWL can be selectively reduced. The reduced CEWL is then converted to conformational states with the C‐terminal fragment floppy upon thermal fluctuation. These states serve as precursors for the fibrillar aggregation. Intriguingly, the CEWL fibrils are stabilized by intermolecular disulfide bonds instead of noncovalent β‐sheet structures, distinct from the amyloid‐like lysozyme fibrils reported before. Based on the experimental evidences and all‐atom molecular dynamics simulation, we proposed a “runaway domain‐swapping” model for the structure of the CEWL fibrils, in which each CEWL molecule swaps the C‐terminal fragment into the complementary position of the adjacent molecule along the fibrils. We anticipate that this fibrillation mechanism can be extended to many other disulfide‐containing proteins. Our study stands for the first example of formation of protein fibrils under native conditions upon UV illumination and poses the potential danger of low UV dose to organisms at the protein level. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Mapping superoxide dismutase 1 domains of non-native interaction: roles of intra- and intermolecular disulfide bonding in aggregation

Superoxide dismutase 1 (SOD1) proteins harboring mutations linked to familial amyotrophic lateral sclerosis (FALS) uniformly show heightened potential to form high molecular weight structures. Here, we examine the domains of SOD1 that are involved in forming these structures (aggregates) and study the role of intra- and intermolecular disulfide bonds. An analysis of disease mutations identified to date reveals a non-random distribution with predominant occurrence at residues within highly conserved beta-strands or at highly conserved residues in loop domains. Using a cell transfection assay for aggregation, we determined that no single domain in SOD1 is indispensable in the formation of sedimentable aggregates, suggesting multiple potential motifs in the protein mediate non-native interactions. By a cell-free aggregation assay, analysis of transgenic mouse tissues, and mutagenesis approaches, we found evidence that redox conditions may modulate SOD1 aggregation; reduction of the native intramolecular disulfide bonds may predispose SOD1 to unfolding and aggregation, whereas non-native intermolecular disulfide linkages may help stabilize aggregates in vivo. The results suggest a possible mechanism for diversity in the structures formed by different SOD1 mutants, and define a potential contribution of redox conditions to SOD1 aggregation.     

The active site cysteine of the proapoptotic protein glyceraldehyde-3-phosphate dehydrogenase is essential in oxidative stress-induced aggregation and cell death

Recent studies have revealed that the redox-sensitive glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is involved in neuronal cell death that is triggered by oxidative stress. GAPDH is locally deposited in disulfide-bonded aggregates at lesion sites in certain neurodegenerative diseases. In this study, we investigated the molecular mechanism that underlies oxidative stress-induced aggregation of GAPDH and the relationship between structural abnormalities in GAPDH and cell death. Under nonreducing in vitro conditions, oxidants induced oligomerization and insoluble aggregation of GAPDH via the formation of intermolecular disulfide bonds. Because GAPDH has four cysteine residues, including the active site Cys(149), we prepared the cysteine-substituted mutants C149S, C153S, C244A, C281S, and C149S/C281S to identify which is responsible for disulfide-bonded aggregation. Whereas the aggregation levels of C281S were reduced compared with the wild-type enzyme, neither C149S nor C149S/C281S aggregated, suggesting that the active site cysteine plays an essential role. Oxidants also caused conformational changes in GAPDH concomitant with an increase in beta-sheet content; these abnormal conformations specifically led to amyloid-like fibril formation via disulfide bonds, including Cys(149). Additionally, continuous exposure of GAPDH-overexpressing HeLa cells to oxidants produced disulfide bonds in GAPDH leading to both detergent-insoluble and thioflavin-S-positive aggregates, which were associated with oxidative stress-induced cell death. Thus, oxidative stresses induce amyloid-like aggregation of GAPDH via aberrant disulfide bonds of the active site cysteine, and the formation of such abnormal aggregates promotes cell death.     

Comparative Fourier transform infrared spectroscopy study of cold-, pressure-, and heat-induced unfolding and aggregation of myoglobin

We studied the cold unfolding of myoglobin with Fourier transform infrared spectroscopy and compared it with pressure and heat unfolding. Because protein aggregation is a phenomenon with medical as well as biotechnological implications, we were interested in both the structural changes as well as the aggregation behavior of the respective unfolded states. The cold- and pressure-induced unfolding both yield a partially unfolded state characterized by a persistent amount of secondary structure, in which a stable core of G and H helices is preserved. In this respect the cold- and pressure-unfolded states show a resemblance with an early folding intermediate of myoglobin. In contrast, the heat unfolding results in the formation of the infrared bands typical of intermolecular antiparallel beta-sheet aggregation. This implies a transformation of alpha-helix into intermolecular beta-sheet. H/2H-exchange data suggest that the helices are first unfolded and then form intermolecular beta-sheets. The pressure and cold unfolded states do not give rise to the intermolecular aggregation bands that are typical for the infrared spectra of many heat-unfolded proteins. This suggests that the pathways of the cold and pressure unfolding are substantially different from that of the heat unfolding. After return to ambient conditions the cold- or pressure-treated proteins adopt a partially refolded conformation. This aggregates at a lower temperature (32 degrees C) than the native state (74 degrees C).     

Non-native aggregation of recombinant human granulocyte-colony stimulating factor under simulated process stress conditions

Effective inhibition of protein aggregation is a major goal in biopharmaceutical production processes optimized for product quality. To examine the characteristics of process-stress-dependent aggregation of human granulocyte colony-stimulating factor (G-CSF), we applied controlled stirring and bubble aeration to a recombinant non-glycosylated preparation of the protein produced in Escherichia coli. We characterized the resulting denaturation in a time-resolved manner using probes for G-CSF conformation and size in both solution and the precipitate. G-CSF was precipitated rapidly from solutions that were aerated or stirred; only small amounts of soluble aggregates were found. Exposed hydrophobic surfaces were a characteristic of both soluble and insoluble G-CSF aggregates. Using confocal laser scanning microscopy, the aggregates presented mainly a circular shape. Their size varied according to incubation time and stress applied. The native intramolecular disulfide bonds in the insoluble G-CSF aggregates were largely disrupted as shown by mass spectrometry. New disulfide bonds formed during aggregation. All involved Cys(18) , which is the only free cysteine in G-CSF; one of them had an intermolecular Cys(18(A)) -Cys(18(B)) crosslink. Stabilization strategies can involve external addition of thiols and extensive reduction of surface exposition during processing.     

Aggregation of the Acylphosphatase from Sulfolobus solfataricus: the folded and partially unfolded states can both be precursors for amyloid formation

Protein aggregation is associated with a number of human pathologies including Alzheimer's and Creutzfeldt-Jakob diseases and the systemic amyloidoses. In this study, we used the acylphosphatase from the hyperthermophilic Archaea Sulfolobus solfataricus (Sso AcP) to investigate the mechanism of aggregation under conditions in which the protein maintains a folded structure. In the presence of 15-25% (v/v) trifluoroethanol, Sso AcP was found to form aggregates able to bind specific dyes such as thioflavine T, Congo red, and 1-anilino-8-naphthalenesulfonic acid. The presence of aggregates was confirmed by circular dichroism and dynamic light scattering. Electron microscopy revealed the presence of small aggregates generally referred to as amyloid protofibrils. The monomeric form adopted by Sso AcP prior to aggregation under these conditions retained enzymatic activity; in addition, folding was remarkably faster than unfolding. These observations indicate that Sso AcP adopts a folded, although possibly distorted, conformation prior to aggregation. Most important, aggregation appeared to be 100-fold faster than unfolding under these conditions. Although aggregation of Sso AcP was faster at higher trifluoroethanol concentrations, in which the protein adopted a partially unfolded conformation, these findings suggest that the early events of amyloid fibril formation may involve an aggregation process consisting of the assembly of protein molecules in their folded state. This conclusion has a biological relevance as globular proteins normally spend most of their lifetime in folded structures.     

“Native-like aggregation” of the acylphosphatase from Sulfolobus solfataricus and its biological implications

Studies in vitro show that globular proteins can experience the formation of native-like conformational states able to self-assemble with no need of transitions across the energy barrier for unfolding, and that such processes can lead eventually to the formation of amyloid-like species. Circumstantial evidence collected in vivo suggests that aggregation of native-like states can be a concrete possibility for living organisms and thus more relevant than previously thought. In this review we summarize the key observations collected on the “native-like aggregation” of the acylphosphatase from Sulfolobus solfataricus, a protein that has allowed the direct monitoring and analysis of native-like aggregates for its propensity to form rapidly native-like aggregates and their slow conversion into amyloid-like aggregates.     

Nanofibers made of globular proteins

Strong nanofibers composed entirely of a model globular protein, namely, bovine serum albumin (BSA), were produced by electrospinning directly from a BSA solution without the use of chemical cross-linkers. Control of the spinnability and the mechanical properties of the produced nanofibers was achieved by manipulating the protein conformation, protein aggregation, and intra/intermolecular disulfide bonds exchange. In this manner, a low-viscosity globular protein solution could be modified into a polymer-like spinnable solution and easily spun into fibers whose mechanical properties were as good as those of natural fibers made of fibrous protein. We demonstrate here that newly formed disulfide bonds (intra/intermolecular) have a dominant role in both the formation of the nanofibers and in providing them with superior mechanical properties. Our approach to engineer proteins into biocompatible fibrous structures may be used in a wide range of biomedical applications such as suturing, wound dressing, and wound closure.     

Amyloid fibril formation can proceed from different conformations of a partially unfolded protein

Protein misfolding and aggregation are interconnected processes involved in a wide variety of nonneuropathic, systemic, and neurodegenerative diseases. More generally, if mutations in sequence or changes in environmental conditions lead to partial unfolding of the native state of a protein, it will often aggregate, sometimes into well-defined fibrillar structures. A great deal of interest has been directed at discovering the characteristic features of metastable partially unfolded states that precede the aggregated states of proteins. In this work, human muscle acylphosphatase (AcP) has been first destabilized, by addition of urea or by means of elevated temperatures, and then incubated in the presence of different concentrations of 2,2,2, trifluoroethanol ranging from 5% to 25% (v/v). The results show that AcP is able to form both fibrillar and nonfibrillar aggregates with a high beta-sheet content from partially unfolded states with very different structural features. Moreover, the presence of alpha-helical structure in such a state does not appear to be a fundamental determinant of the ability to aggregate. The lack of ready aggregation under some of the conditions examined here is attributable primarily to the intrinsic properties of the solutions rather than to specific structural features of the partially unfolded states that precede aggregation. Aggregation appears to be favored when the solution conditions promote stable intermolecular interactions, particularly hydrogen bonds. In addition, the structures of the resulting aggregates are largely independent of the conformational properties of their soluble precursors.     

Kinetics control preferential heterodimer formation of platelet-derived growth factor from unfolded A- and B-chains

The folding and assembly of platelet-derived growth factor (PDGF), a potent mitogen involved in wound-healing processes and member of the cystine knot growth factor family, was studied. The kinetics of the formation of disulfide-bonded dimers were investigated under redox reshuffling conditions starting either from unfolded and reduced PDGF-A- or B-chains or an equimolar mixture of both chains. It is shown that in all cases the formation of disulfide-bonded dimers is a very slow process occurring in the time scale of hours with a first-order rate-determining step. The formation of disulfide-bonded PDGF-AA or PDGF-BB homodimers displayed identical kinetics, indicating that both monomeric forms as well as the dimerized homodimer have similar folding and assembly pathways. In contrast, the formation of the heterodimer occurred three times more rapidly compared with the formation of the homodimers. As both monomeric forms revealed similar renaturation kinetics, it can be concluded that the first-order rate-determining folding step does not occur during monomer folding but must be attributed to conformational rearrangements of the dimerized, not yet disulfide-bonded protein. These structural rearrangements allow a more rapid formation of intermolecular disulfide bonds between the two different monomers of a heterodimer compared with the formation of the disulfide bonds between two identical monomers. The preferential formation of disulfide-bonded heterodimers from an equimolar mixture of unfolded A- and B-chains is thus a kinetically controlled process. Moreover, similar activation enthalpies for the formation of all different isoforms suggest that faster heterodimerization is controlled by entropic factors.     

Protein substrate discrimination in the quiescin sulfhydryl oxidase (QSOX) family

This work explores the substrate specificity of the quiescin sulfhydryl oxidase (QSOX) family of disulfide-generating flavoenzymes to provide enzymological context for investigation of the physiological roles of these facile catalysts of oxidative protein folding. QSOX enzymes are generally unable to form disulfide bonds within well-structured proteins. Use of a temperature-sensitive mutant of ubiquitin-conjugating enzyme 4 (Ubc4') as a model substrate shows that QSOX activity correlates with the unfolding of Ubc4' monitored by circular dichroism. Fusion of Ubc4' with the more stable glutathione-S-transferase domain demonstrates that QSOX can selectively introduce disulfides into the less stable domain of the fusion protein. In terms of intermolecular disulfide bond generation, QSOX is unable to cross-link well-folded globular proteins via their surface thiols. However, the construction of a septuple mutant of RNase A, retaining a single cysteine residue, demonstrates that flexible protein monomers can be directly coupled by the oxidase. Steady- and pre-steady-state kinetic experiments, combined with static fluorescence approaches, indicate that while QSOX is an efficient catalyst for disulfide bond formation between mobile elements of structure, it does not appear to have a significant binding site for unfolded proteins. These aspects of protein substrate discrimination by QSOX family members are rationalized in terms of the stringent steric requirements for disulfide exchange reactions.     

Characterization of heat-induced aggregates of globulin from common buckwheat (Fagopyrum esculentum Moench)

Some physicochemical properties and the microstructure of heat-induced aggregates of globulin from common buckwheat (Fagopyrum esculentum Moench) (BWG) formed at 100 °C in 0.01 M phosphate buffer containing 1.0 M NaCl, pH 7.4 were studied. Differential scanning calorimetric (DSC) analysis shows a re-distribution of native and extensively denatured proteins in the heat-induced aggregates of BWG, particularly in the ISA fraction. Sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis suggests the occurrence of both dissociation and association of molecules and the involvement of intermolecular disulfide linkages during thermal aggregation. Transmission electron microscopy (TEM) reveals that native BWG appeared as uniform compact globules with diameters ranging between 11.7 and 12.5 nm. TEM examination of the buffer-soluble aggregates, fractionated by sucrose density gradient ultracentrifugation, demonstrates the formation of strand-like small aggregates and large compact globular soluble macroaggregates.     

Conformational specificity of the chaperonin GroEL for the compact folding intermediates of alpha-lactalbumin.

The chaperonin GroEL binds unfolded polypeptides, preventing aggregation, and then mediates their folding in an ATP-dependent process. To understand the structural features in non-native polypeptides recognized by GroEL, we have used alpha-lactalbumin (alpha LA) as a model substrate. alpha LA (14.2 kDa) is stabilized by four disulfide bonds and a bound Ca2+ ion, offering the possibility of trapping partially folded disulfide intermediates between the native and the fully unfolded state. The conformers of alpha LA with high affinity for GroEL are compact, containing up to three disulfide bonds, and have significant secondary structure, but lack stable tertiary structure and expose hydrophobic surfaces. Complex formation requires almost the complete alpha LA sequence and is strongly dependent on salts that stabilize hydrophobic interactions. Unfolding of alpha LA to an extended state as well as the burial of hydrophobic surface upon formation of ordered tertiary structure prevent the binding to GroEL. Interestingly, GroEL interacts only with a specific subset of the many partially folded disulfide intermediates of alpha LA and thus may influence in vitro the kinetics of the folding pathways that lead to disulfide bonds with native combinations. We conclude that the chaperonin interacts with the hydrophobic surfaces exposed by proteins in a flexible compact intermediate or molten globule state.     

Molecular differences in the formation and structure of fine-stranded and particulate beta-lactoglobulin gels

In order to reveal at a molecular level differences between fine-stranded and particulate gels, we present an Fourier transform infrared spectroscopic study of the thermal behavior of beta-lactoglobulin (beta-lg) in salt-free D(2)O solutions and low ionic strength at different pDs. Differences are found in the denaturation mechanism, in the unfolded state of the protein, in the aggregate formation, and in the strength of the intermolecular interactions. For fine-stranded gels (pD 2.8 and 7.8), heating induces the dissociation of the dimers into monomers. The protein undergoes extensive structural modifications before aggregation begins. Aggregation is characterized by the appearance of a new band attributed to intermolecular beta-sheets which is located in the 1613-1619 cm(-1) range. For particulate gels (pD 4.4 and 5.4), the protein structure is almost preserved up to 75-80 degrees C with no splitting of the dimers. The band characteristic of aggregation originates from the component initially located at 1623 cm(-1), suggesting that at the beginning of aggregation, globular beta-lg in the dimeric form associate to constitute oligomers with higher molecular mass. Aggregation may result in the association of globular slightly denatured dimers, leading to the formation of spherical particles rather than linear strands. The aggregation band is always located in the 1620-1623 cm(-1) range for particulate gels showing that hydrogen bonds are weaker for these aggregates than for fine-stranded ones. This has been related to a more extensive protein unfolding for fine-stranded gels that allows a closer alignment of the polypeptide chains, and then to the formation of much stronger hydrogen bonds. Small differences are also found in protein organization and in intermolecular hydrogen bond strength vs pD within the same type of gel. Protein conformation and protein-protein interactions in the gel state may be responsible of the specific macroscopic properties of each gel network. A coarse representation of the different modes of gelation is described.     

Disulphide bond formation in food protein aggregation and gelation

In this short review we discuss the role of cysteine residues and cystine bridges for the functional aggregation of food proteins. We evaluate how formation and cleavage of disulphide bonds proceeds at a molecular level, and how inter- and intramolecular disulfide bonds can be detected and modified. The differences between heat-, high-pressure-, and denaturant-induced unfolding and aggregation are discussed. The effect of disulphide bonding between aggregates of proteins and protein mixtures on the functional macroscopic properties of space filling networks in protein gels is briefly presented.     

Pressure effect on the temperature-induced unfolding and tendency to aggregate of myoglobin

This work demonstrates that pressure-induced partially unfolded states play a very important role in the aggregation of proteins. The high-pressure unfolding of horse heart metmyoglobin results in an intermediate form that shows a strong tendency to aggregate after pressure release. These aggregates are similar to those that are usually observed upon temperature denaturation. Infrared spectra in the amide I region indicate the formation of an intermolecular antiparallel beta-sheet stabilized by hydrogen bonding. The formation of the aggregates is temperature-dependent. Below 30 degrees C, no aggregation is taking place as seen from the infrared spectra. At 45 and 60 degrees C, two types of aggregates are formed: one that can be dissociated by moderate pressures and one that is pressure-insensitive. When precompressed at 5 degrees C, temperature-induced aggregation takes place at lower temperature (38 degrees C) than without pressure pretreatment (74 degrees C).     

Influence of native disulfide bonds of globular proteins on their retention behavior on reverse phase supports

The reduction of the disulfide bonds of globular proteins, for example, those of lysozyme or ribonuclease-A, results in an increase in the hydrodynamic volume of the polypeptide chain. This is reflected in an earlier elution of the reduced protein on gel filtration compared to that of the native disulfide-bonded form. The reduction of the four disulfide bonds of ribonuclease-A increased its retention time on reverse phase support, suggesting an increase in the apparent hydrophobicity of the protein molecule on reduction. Performic acid-oxidized ribonuclease-A eluted ahead of native disulfide-bonded ribonuclease on RP HPLC, suggesting a decrease in the hydrophobicity of the molecule. However, the hydrodynamic volume of performic acid-oxidized ribonuclease-A is similar to that of reduced protein as reflected in its gel filtration behavior. Thus, the increased retention of the reduced protein compared to that of native disulfide-bonded protein is not related to the increased hydrodynamic volume, and is a reflection of the stronger interaction of reduced protein with the reverse phase support. Reoxidation of the reduced ribonuclease-A regenerated the original chromatographic behavior of the protein on the reverse phase support. Similar results were also obtained with hen egg white lysozyme. The results of the present study are interpreted as indicating that the native disulfide bonds of a globular protein restrict the exposure of the hydrophobic amino acid residues of the polypeptide chain with a consequent lower retention on the reverse phase support compared to its reduced form.     

Catalysis of creatine kinase refolding by protein disulfide isomerase involves disulfide cross-link and dimer to tetramer switch

Protein disulfide isomerase (PDI) functions as an isomerase to catalyze thiol:disulfide exchange, as a chaperone to assist protein folding, and as a subunit of prolyl-4-hydroxylase and microsomal triglyceride transfer protein. At a lower concentration of 0.2 microm, PDI facilitated the aggregation of unfolded rabbit muscle creatine kinase (CK) and exhibited anti-chaperone activity, which was shown to be mainly due to the hydrophobic interactions between PDI and CK and was independent of the cross-linking of disulfide bonds. At concentrations above 1 microm, PDI acted as a protector against aggregation but an inhibitor of reactivation during CK refolding. The inhibition effect of PDI on CK reactivation was further characterized as due to the formation of PDI-CK complexes through intermolecular disulfide bonds, a process involving Cys-36 and Cys-295 of PDI. Two disulfide-linked complexes containing both PDI and CK were obtained, and the large, soluble aggregates around 400 kDa were composed of 1 molecule of tetrameric PDI and 2 molecules of inactive intermediate dimeric CK, whereas the smaller one, around 200 kDa, was formed by 1 dimeric PDI and 1 dimeric CK. To our knowledge this is the first study revealing that PDI could switch its conformation from dimer to tetramer in its functions as a foldase. According to the observations in this research and our previous study of the folding pathways of CK, a working model was proposed for the molecular mechanism of CK refolding catalyzed by PDI.     

Using intramolecular disulfide bonds in tau protein to deduce structural features of aggregation-resistant conformations

Because tau aggregation likely plays a role in a number of neurodegenerative diseases, understanding the processes that affect tau aggregation is of considerable importance. One factor that has been shown to influence the aggregation propensity is the oxidation state of the protein itself. Tau protein, which contains two naturally occurring cysteine residues, can form both intermolecular disulfide bonds and intramolecular disulfide bonds. Several studies suggest that intermolecular disulfide bonds can promote tau aggregation in vitro. By contrast, although there are data to suggest that intramolecular disulfide bond formation retards tau aggregation in vitro, the precise mechanism underlying this observation remains unclear. While it has been hypothesized that a single intramolecular disulfide bond in tau leads to compact conformations that cannot form extended structure consistent with tau fibrils, there are few data to support this conjecture. In the present study we generate oxidized forms of the truncation mutant, K18, which contains all four microtubule binding repeats, and isolate the monomeric fraction, which corresponds to K18 monomers that have a single intramolecular disulfide bond. We study the aggregation propensity of the oxidized monomeric fraction and relate these data to an atomistic model of the K18 unfolded ensemble. Our results argue that the main effect of intramolecular disulfide bond formation is to preferentially stabilize conformers within the unfolded ensemble that place the aggregation-prone tau subsequences, PHF6* and PHF6, in conformations that are inconsistent with the formation of cross-β-structure. These data further our understanding of the precise structural features that retard tau aggregation.     

Formation of amyloid fibrils by bovine carbonic anhydrase

Amyloids are typically characterized by extensive aggregation of proteins where the participating polypeptides are involved in formation of intermolecular cross beta-sheet structures. Alternate structure attainment and amyloid formation has been hypothesized to be a generic property of a polypeptide, the propensities of which vary widely depending on the polypeptide involved and the physicochemical conditions it encounters. Many proteins that exist in the normal form in-vivo have been shown to form amyloid when incubated in partially denaturing conditions. The protein bovine carbonic anhydrase II (BCA II) when incubated in mildly denaturing conditions showed that the partially unfolded conformers assemble together and form ordered amyloid aggregates. The properties of these aggregates were tested using the traditional Congo-Red (CR) and Thioflavin-T (ThT) assays along with fluorescence microscopy, transmission electron microscopy (TEM), and circular dichroism (CD) spectroscopy. The aggregates were found to possess most of the characteristics ascribed to amyloid fibers. Thus, we report here that the single-domain globular protein, BCA II, is capable of forming amyloid fibrils. The primary sequence of BCA II was also analyzed using recurrence quantification analysis in order to suggest the probable residues responsible for amyloid formation.