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).     

Entrapping intermediates of thermal aggregation in alpha-helical proteins with low concentration of guanidine hydrochloride

Aggregation of proteins is a problem with serious medical implications and economic importance. To develop strategies for preventing aggregation, the mechanism(s) and pathways by which proteins aggregate must be characterized. In this study, the thermally induced aggregation processes of three alpha-helix proteins (myoglobin, cytochrome c, and lysozyme) in the presence and absence of 1.0 m guanidine hydrochloride (GdnHCl) were investigated by means of infrared spectroscopy. In the absence of GdnHCl, intensities of the alpha-helix bands (approximately 1656 cm(-1)) decrease as a function of temperature at above 50 degrees C. With myoglobin and cytochrome c, the loss of helix bands was accompanied by the appearance of two new bands at 1694 and 1623 cm(-1), indicative of the formation of intermolecular beta-sheet aggregates. For lysozyme, bands indicative of intermolecular beta-sheet aggregates did not appear in any significant intensity. In the presence of 1.0 m GdnHCl, two major intermediate states rich in 3(10)-helix (represented by the band at 1663 cm(-1)) and beta-turn structure (represented by the band at 1667 cm(-1)), respectively, were observed. These findings demonstrated that IR spectroscopic studies of protein aggregation using a combination of thermal and chemical denaturing factors could provide a means to populate and characterize aggregation intermediates.     

Beware of proteins in DMSO

The effect on the secondary structure of representative alpha-helical, beta-sheet and disordered proteins by varying concentrations of dimethyl sulphoxide (DMSO) in 2H2O has been investigated by Fourier transform infrared spectroscopy. Significant perturbations of protein secondary structure are induced by DMSO and DMSO/2H2O mixtures. For highly structured proteins, such as myoglobin and concanavalin A, the infrared spectra point to a progressive destabilisation of the secondary structure until at moderate DMSO concentrations (around 0.33 mol fraction) intermolecular beta-sheet formation and aggregation are induced, as indicated by the appearance of a strong band at 1621 cm-1. This is a direct consequence of the disruption of intramolecular peptide group interactions by DMSO (partial unfolding). At higher DMSO concentrations (above 0.75 mol fraction), such aggregates are dissociated by disruption of the intermolecular C = O...2H-N deuterium bonds. The presence of a single amide I band at 1662 cm-1 corresponding to free amide C = O groups indicates that at high concentrations and in pure DMSO the proteins are completely unfolded, lacking any secondary structure. While low concentrations of DMSO showed no detectable effect upon the gross secondary structure of myoglobin and concanavalin A, the thermal stability of both proteins was markedly reduced. In alpha-casein, a highly unstructured protein, the situation is one of direct competition. The amide I maximum in 2H2O, at 1645 cm-1, is typical of unordered proteins with C = O groups deuterium-bonded predominantly to 2H2O. Addition of DMSO disrupts such interactions by competing with the peptide C = O group for the deuterium bond donor capacity of the 2H2O, and so progressively increases the amide I maximum until it stabilizes at 1663 cm-1, a position indicative of free C = O groups.     

Differences between the pressure- and temperature-induced denaturation and aggregation of beta-lactoglobulin A, B, and AB monitored by FT-IR spectroscopy and small-angle X-ray scattering.

We examined the temperature- and pressure-induced unfolding and aggregation of beta-lactoglobulin (beta-Lg) and its genetic variants A and B up to temperatures of 90 degrees C in the pressure range from 1 bar to 10 kbar. To achieve information simultaneously on the secondary, tertiary, and quaternary structures, we have applied Synchrotron small-angle X-ray diffraction and Fourier transform infrared spectroscopy. Upon heating a beta-Lg solution at pH 7.0, the radius of gyration Rg first decreases, indicating a partial dissociation of the dimer into the monomers, the secondary structures remaining essentially unchanged. Above 50 degrees C, the infrared spectroscopy data reveal a decrease in intramolecular beta-sheet and alpha-helical structures, whereas the contribution of disordered structures increases. Within the temperature range from 50 to 60 degrees C, the appearance of the pair distance distribution function is not altered significantly, whereas the amount of defined secondary structures declines approximately by 10%. Above 60 degrees C the aggregation process of 1% beta-Lg solutions is clearly detectable by the increase in Rg and intermolecular beta-sheet content. The irreversible aggregation is due to intermolecular S-H/S-S interchange reactions and hydrophobic interactions. Upon pressurization at room temperature, the equilibrium between monomers and dimers is also shifted and dissociation of dimers is induced. At pressures of approximately 1300 bar, the amount of beta-sheet and alpha-helical structures decreases and the content of disordered structures increases, indicating the beginning unfolding of the protein which enables aggregation. Contrary to the thermal denaturation process, intermolecular beta-sheet formation is of less importance in pressure-induced protein aggregation and gelation. The spatial extent of the resulting protein clusters is time- and concentration-dependent. The aggregation of a 1% (w/w) solution of A, B, and the mixture AB results in the formation of at least octameric units as can be deduced from the radius of gyration of about 36 A. No differences in the pressure stability of the different genetic variants of beta-Lg are detectable in our FT-IR and SAXS experiments. Even application of higher pressures (up to 10 kbar) does not result in complete unfolding of all beta-Lg variants.     

Pressure effects on the heat-induced aggregation of equine serum albumin by FT-IR spectroscopic study: Secondary structure, kinetic and thermodynamic properties

Pressure can restrain the heat-induced aggregation and dissociate the heat-induced aggregates. We investigated the aggregation-preventing pressure effect and the aggregates-dissociating pressure effect to characterize the heat-induced aggregation of equine serum albumin (ESA) by Fourier transform infrared spectroscopy. The results suggest that the alpha-helical structure collapses at the beginning of heat-induced aggregation, then the rearrangement of structure from partially unfolded structure to the intermolecular beta-sheet takes place through the activated state. We determined the activation volume for the heat-induced aggregation (DeltaV( not equal)=+92+/-8 ml mol(-1)) and the partial molar volume difference between native state and heat-induced aggregates (DeltaV(N-->HA)=+32 ml mol(-1)). This positive partial molar volume difference suggests that the heat-induced aggregates have larger internal voids than the native structure. Moreover, the positive volume change implies that the formation of the intermolecular beta-sheet is unfavorable under high pressure. We also determined the free energy profile of ESA. This energy profile explains the restriction of the formation of heat-induced aggregates by pressure. These results explain the structural differences between heat-induced aggregates with intermolecular beta-sheet and pressure-induced aggregates without intermolecular beta-sheet.     

Pressure-Temperature Stability, Ca(2+) Binding, and Pressure-Temperature Phase Diagram of Cod Parvalbumin: Gad m 1

Fish allergy is associated with IgE-mediated hypersensitivity reactions to parvalbumins, which are small calcium-binding muscle proteins and represent the major and sole allergens for 95% of fish-allergic patients. We performed Fourier transform infrared and tryptophan fluorescence spectroscopy to explore the pressure-temperature (p-T) phase diagram of cod parvalbumin (Gad m 1) and to elucidate possible new ways of pressure-temperature inactivation of this food allergen. Besides the secondary structure of the protein, the Ca(2+) binding to aspartic and glutamic acid residues was detected. The phase diagram was found to be quite complex, containing partially unfolded and molten globule states. The Ca(2+) ions were essential for the formation of the native structure. A molten globule conformation appears at 50 °C and atmospheric pressure, which converts into an unordered aggregated state at 75 °C. At >200 MPa, only heat unfolding, but no aggregation, was observed. A pressure of 500 MPa leads to a partially unfolded state at 27 °C. The complete pressure unfolding could only be reached at an elevated temperature (40 °C) and pressure (1.14 GPa). A strong correlation was found between Ca(2+) binding and the protein conformation. The partially unfolded state was reversibly refolded. The completely unfolded molecule, however, from which Ca(2+) was released, could not refold. The heat-unfolded protein was trapped either in the aggregated state or in the molten globule state without aggregation at elevated pressures. The heat-treated and the combined heat- and pressure-treated protein samples were tested with sera of allergic patients, but no change in allergenicity was found.     

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.     

Structural and hydration properties of the partially unfolded states of the prion protein

Misfolding and aggregation of the prion protein (PrP) is responsible for the development of transmissible spongiform encephalopathies (TSE). To gain insights into possible aggregation-prone intermediate states, we construct the free energy surface of the C-terminal globular domain of the PrP from enhanced sampling of replica exchange molecular dynamics. This cellular domain is characterized by three helices H1-H3 and a small beta-sheet. In agreement with experimental studies, the partially unfolded states display a stable core built from the central portions of helices H2 and H3 and a high mobility of helix H1 from the core. Among all identified conformational basins, a marginally populated state appears to be a very good candidate for aggregation. This intermediate is stabilized by four TSE-sensitive key interactions, displays a longer helix H1 with both a dry and solvated surface, and is featured by a significant detachment of helix H1 from the PrP-core.     

Counteracting effects of thiocyanate and sucrose on chymotrypsinogen secondary structure and aggregation during freezing, drying, and rehydration.

Studies of numerous proteins with infrared spectroscopy have documented that unfolding is a general response of unprotected proteins to freeze-drying. Some proteins that are unfolded in the dried solid aggregate during rehydration, whereas others refold. It has been proposed for the latter case that aggregation is avoided because refolding kinetically outcompetes intermolecular interactions. In contrast, with proteins that normally aggregate after rehydration, minimizing unfolding during freeze-drying with stabilizer has been shown to be needed to favor the recovery of native protein molecules after rehydration. The purpose of the current study was to examine first the opposite situation, in which a denaturant is used to foster additional unfolding in the protein population during freeze-drying. If the protein is not intrinsically resistant to aggregation under the study conditions (e.g., because of intermolecular charge repulsion) and the denaturant does not disrupt intermolecular interactions during rehydration, this treatment should favor aggregation upon rehydration. With infrared spectroscopy we found that at concentrations of the denaturant Na thiocyanate (NaSCN) that only slightly perturbed chymotrypsinogen secondary structure in solution before freeze-drying, there was a large increase in protein unfolding in the dried solid and in protein aggregation measured after rehydration. Bands assigned to intermolecular beta sheet were present in the spectra of samples dried with NaSCN, indicating that aggregation could also arise in the dried solid. By examining the protein structure in the frozen state, we determined that in the absence of NaSCN the protein remains native. NaSCN caused structural perturbations during freezing, without the formation of intermolecular beta sheet, that were intermediate to structural changes noted after freeze-drying. In contrast, samples treated in the presence of NaSCN and sucrose had native-like spectra in the frozen and dried states, and much reduced aggregation after rehydration. These results indicate that during freezing and drying the sugar can counteract and mostly reverse the structural perturbations induced by NaSCN before and during these treatments.     

Heat capacity of protein folding.

We construct a Hamiltonian for a single domain protein where the contact enthalpy and the chain entropy decrease linearly with the number of native contacts. The hydration effect upon protein unfolding is included by modeling water as ideal dipoles that are ordered around the unfolded surfaces, where the influence of these surfaces, covered with an "ice-like" shell of water, is represented by an effective field that directs the water dipoles. An intermolecular pair interaction between water molecules is also introduced. The heat capacity of the model exhibits, the common feature of small globular proteins, two peaks corresponding to cold and warm unfolding, respectively. By introducing ad hoc vibrational modes, we obtain quantitatively good accordance with experiments on myoglobin.     

Molecular basis of co-operativity in protein folding.

The folding/unfolding transition of proteins is a highly co-operative process characterized by the presence of very few or no thermodynamically stable partially folded intermediate states. The purpose of this paper is to present a thermodynamic formalism aimed at describing quantitatively the co-operative folding behavior of proteins. In order to account for this behavior, a hierarchical algorithm aimed at evaluating the folding/unfolding partition function has been developed. This formalism defines the partition function in terms of multiple levels of interacting co-operative folding units. A co-operative folding unit is defined as a protein structural element that exhibits two-state folding/unfolding behavior. At the most fundamental level are those structural elements that behave co-operatively as a result of purely local interactions. Higher-order co-operative folding units are formed through interactions between different structural elements. The hierarchical formalism utilizes the crystallographic structure of the protein as a template to generate partially folded conformations defined in terms of co-operative folding units. The Gibbs free energy of those states and their corresponding statistical weights are then computed using experimental energetic parameters determined calorimetrically. This formalism has been applied to the case of myoglobin. It is shown that the hierarchical partition function correctly predicts the presence, energetics and co-operativity of the heat and cold denaturation transitions. The major contribution to the co-operative folding behavior arises from the solvent exposure of non-polar residues located in regions complementary to those that have undergone unfolding. This entropically uncompensated and energetically unfavorable solvent exposure characterizes all partially folded states but not the unfolded state, thus minimizing the population of partially folded intermediates throughout the folding/unfolding transition.     

Thermal unfolding mechanism of lipocalin-type prostaglandin D synthase

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dual-functioning protein in the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant-induced unfolding of L-PGDS follows a four-state pathway, including an activity-enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L-PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L-PGDS was a completely reversible process at pH 4.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L-PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two-state unfolding mechanism, the ratio of van't Hoff enthalpy (DeltaH(vH)) to calorimetric enthalpy (DeltaH(cal)) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three-state unfolding process were performed. The heat capacity curves fit well with a three-state process; and the estimated transition temperature (T(m)) and enthalpy change (DeltaH(cal)) of the N<-->I and I<-->U transitions were 48.2 degrees C and 190 kJ.mol(-1), and 60.3 degrees C and 144 kJ.mol(-1), respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290 nm revealed that L-PGDS unfolded through the intermediate state, where its main chain retained the characteristic beta-sheet structure without side-chain interactions.     

Mechanistic insight of photo-induced aggregation of chicken egg white lysozyme: the interplay between hydrophobic interactions and formation of intermolecular disulfide bonds

Recently, it was reported that ultraviolet (UV) illumination could trigger the unfolding of proteins by disrupting the buried disulfide bonds. However, the consequence of such unfolding has not been adequately evaluated. Here, we report that unfolded chicken egg white lysozyme (CEWL) triggered by UV illumination can form uniform globular aggregates as confirmed by dynamic light scattering, atomic force microscopy, and transmission electron microscopy. The assembling process of such aggregates was also monitored by several other methods, such as circular dichroism, fluorescence spectroscopy, mass spectrometry based on chymotrypsin digestion, ANS-binding assay, Ellman essay, and SDS-PAGE. Our finding is that due to the dissociation of the native disulfide bonds by UV illumination, CEWL undergoes drastic conformational changes resulting in the exposure of some hydrophobic residues and free thiols. Subsequently, these partially unfolded molecules self-assemble into small granules driven by intermolecular hydrophobic interaction. With longer UV illumination or longer incubation time, these granules can further self-assemble into larger globular aggregates. The combined effects from both the hydrophobic interaction and the formation of intermolecular disulfide bonds dominate this process. Additionally, similar aggregation behavior can also be found in other three typical disulfide-bonded proteins, that is, α-lactalbumin, RNase A, and bovine serum albumin. Thus, we propose that such aggregation behavior might be a general mechanism for some disulfide-bonded proteins under UV irradiation.     

Cation binding effects on the pH, thermal and urea denaturation transitions in alpha-lactalbumin

The binding of monovalent (Na+, K+) and divalent (Ca2+, Mg2+) cations to bovine alpha-lactalbumin at 20 and 37 degrees C has been studied by means of intrinsic protein fluorescence. The values of apparent binding constants for these ions obtained at 37 degrees C are about one order of magnitude lower than those measured at 20 degrees C. Urea and alkali (pH greater than 10) induce unfolding transitions which involve stable partially unfolded intermediates for all metal ion-bound forms of alpha-lactalbumin. Heating induces similar partially unfolded states. Nevertheless, the partially unfolded states induced by heating, urea, alkaline or acidic treatments are somewhat different in their tryptophan residue environment properties. The results have been interpreted in terms of a simple scheme of equilibria between metal-free and metal-bound forms in their native, partially unfolded and unfolded states. The scheme provides an approach to the quantitative interpretation of any transition equilibrium shift induced by a low molecular mass species able to be bound by a protein.     

Fourier transform infrared spectroscopy provides a fingerprint for the tetramer and for the aggregates of transthyretin

Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for several familial amyloid diseases. Here, we use FTIR to describe the secondary structural changes that take place when wt TTR undergoes heat- or high-pressure-induced denaturation, as well as fibril formation. Upon thermal denaturation, TTR loses part of its intramolecular beta-sheet structure followed by an increase in nonnative, probably antiparallel beta-sheet contacts (bands at 1,616 and 1,686 cm(-1)) and in the light scattering, suggesting its aggregation. Pressure-induced denaturation studies show that even at very elevated pressures (12 kbar), TTR loses only part of its beta-sheet structure, suggesting that pressure leads to a partially unfolded species. On comparing the FTIR spectrum of the TTR amyloid fibril produced at atmospheric pressure upon acidification (pH 4.4) with the one presented by the native tetramer, we find that the content of beta-sheets does not change much upon fibrillization; however, the alignment of beta-sheets is altered, resulting in the formation of distinct beta-sheet contacts (band at 1,625 cm(-1)). The random-coil content also decreases in going from tetramers to fibrils. This means that, although part of the tertiary- and secondary-structure content of the TTR monomers has to be lost before fibril formation, as previously suggested, there must be a subsequent reorganization of part of the random-coil structure into a well-organized structure compatible with the amyloid fibril, as well as a readjustment of the alignment of the beta-sheets. Interestingly, the infrared spectrum of the protein recovered from a cycle of compression-decompression at pD 5, 37 degrees C, is quite similar to that of fibrils produced at atmospheric pressure (pH 4.4), which suggests that high hydrostatic pressure converts the tetramers of TTR into an amyloidogenic conformation.     

Oligomerization and aggregation of bovine pancreatic ribonuclease A: backbone hydration probed by infrared band-shift

Protein hydration plays a crucial role in almost all aspects of biomolecular processes. In this research, we studied the hydration/dehydration-induced infrared amide I band-shift by using poly-L-lysine and bovine pancreas ribonuclease A as model polypeptides. It was found that a 1-4 cm(-1) shift could be clearly distinguished for all regular secondary structures during protein thermal unfolding. This shift was proven to be due to backbone hydration but not from experimental error, temperature effect or possible incomplete hydrogen/deuterium exchange of the samples. Moreover, we also found that protein aggregation was closely associated with the backbone hydration/dehydration status of proteins. In conditions favoring aggregation, a significant shift to a higher wavenumber of the band from the intermolecular beta-sheet structures in aggregates was observed. The present study suggested that the changes of the amounts of regular secondary structures could be monitored by the intensity changes, while the changes of the hydration status could be monitored by the shift of the infrared bands.     

Unfolding Simulations of Holomyoglobin from Four Mammals: Identification of Intermediates and β-Sheet Formation from Partially Unfolded States

Myoglobin (Mb) is a centrally important, widely studied mammalian protein. While much work has investigated multi-step unfolding of apoMb using acid or denaturant, holomyoglobin unfolding is poorly understood despite its biological relevance. We present here the first systematic unfolding simulations of holoMb and the first comparative study of unfolding of protein orthologs from different species (sperm whale, pig, horse, and harbor seal). We also provide new interpretations of experimental mean molecular ellipticities of myoglobin intermediates, notably correcting for random coil and number of helices in intermediates. The simulated holoproteins at 310 K displayed structures and dynamics in agreement with crystal structures (R _g ∼1.48–1.51 nm, helicity ∼75%). At 400 K, heme was not lost, but some helix loss was observed in pig and horse, suggesting that these helices are less stable in terrestrial species. At 500 K, heme was lost within 1.0–3.7 ns. All four proteins displayed exponentially decaying helix structure within 20 ns. The C- and F-helices were lost quickly in all cases. Heme delayed helix loss, and sperm whale myoglobin exhibited highest retention of heme and D/E helices. Persistence of conformation (RMSD), secondary structure, and ellipticity between 2–11 ns was interpreted as intermediates of holoMb unfolding in all four species. The intermediates resemble those of apoMb notably in A and H helices, but differ substantially in the D-, E- and F-helices, which interact with heme. The identified mechanisms cast light on the role of metal/cofactor in poorly understood holoMb unfolding. We also observed β-sheet formation of several myoglobins at 500 K as seen experimentally, occurring after disruption of helices to a partially unfolded, globally disordered state; heme reduced this tendency and sperm-whale did not display any sheet propensity during the simulations.     

Probing folding and fluorescence quenching in human gammaD crystallin Greek key domains using triple tryptophan mutant proteins

Human gammaD crystallin (HgammaD-Crys), a major component of the human eye lens, is a 173-residue, primarily beta-sheet protein, associated with juvenile and mature-onset cataracts. HgammaD-Crys has four tryptophans, with two in each of the homologous Greek key domains, which are conserved throughout the gamma-crystallin family. HgammaD-Crys exhibits native-state fluorescence quenching, despite the absence of ligands or cofactors. The tryptophan absorption and fluorescence quenching may influence the lens response to ultraviolet light or the protection of the retina from ambient ultraviolet damage. To provide fluorescence reporters for each quadrant of the protein, triple mutants, each containing three tryptophan-to-phenylalanine substitutions and one native tryptophan, have been constructed and expressed. Trp 42-only and Trp 130-only exhibited fluorescence quenching between the native and denatured states typical of globular proteins, whereas Trp 68-only and Trp 156-only retained the anomalous quenching pattern of wild-type HgammaD-Crys. The three-dimensional structure of HgammaD-Crys shows Tyr/Tyr/His aromatic cages surrounding Trp 68 and Trp 156 that may be the source of the native-state quenching. During equilibrium refolding/unfolding at 37 degrees C, the tryptophan fluorescence signals indicated that domain I (W42-only and W68-only) unfolded at lower concentrations of GdnHCl than domain II (W130-only and W156-only). Kinetic analysis of both the unfolding and refolding of the triple-mutant tryptophan proteins identified an intermediate along the HgammaD-Crys folding pathway with domain I unfolded and domain II intact. This species is a candidate for the partially folded intermediate in the in vitro aggregation pathway of HgammaD-Crys.     

Amyloid formation of a protein in the absence of initial unfolding and destabilization of the native state

In 5% (v/v) trifluoroethanol, pH 5.5, 25 degrees C one of the acylphosphatases from Drosophila melanogaster (AcPDro2) forms fibrillar aggregates that bind thioflavin T and Congo red and have an extensive beta-sheet structure, as revealed by circular dichroism. Atomic force microscopy indicates that the fibrils and their constituent protofilaments have diameters compatible with those of natural amyloid fibrils. Spectroscopic and biochemical investigation, carried out using near- and far-UV circular dichroism, intrinsic and 1-anilino-8-naphthalenesulfonic acid-derived fluorescence, dynamic light scattering, and enzymatic activity assays, shows that AcPDro2 has, before aggregation, a secondary structure content packing around aromatic and hydrophobic residues, hydrodynamic diameter, and catalytic activity indistinguishable from those of the native protein. The native protein was found to have the same conformational stability under native and aggregating conditions, as determined from urea-induced unfolding. The kinetic analysis supports models in which AcPDro2 aggregates initially without need to unfold and subsequently undergoes a conformational change into amyloid-like structures. Although fully or partially unfolded states have a higher propensity to aggregate, the residual aggregation potential that proteins maintain upon complete folding can be physiologically relevant and be directly involved in the pathogenesis of some protein deposition diseases.     

The thermal denaturation of ribonuclease A in aqueous-methanol solvents.

Circular dichroism was used to monitor the thermal unfolding of ribonuclease A in 50% aqueous methanol. The spectrum of the protein at temperatures below -10 degrees C (pH* 3.0) was essentially identical to that of native ribonuclease A in aqueous solution. The spectrum of the thermally denatured material above 70 degrees C revealed some residual secondary structure in comparison to protein unfolded by 5 M Gdn.HCl at 70 degrees C in the presence or absence of methanol. The spectra as a function of temperature were deconvoluted to determine the contributions of different types of secondary structure. The position of the thermal unfolding transition as monitored by alpha-helix, with a midpoint at 38 degrees C, was at a much higher temperature than that monitored by beta-sheet, 26 degrees C, which also corresponded to that observed by delta A286, tyrosine fluorescence and hydrodynamic radius (from light scattering measurements). Thus, the loss of beta-sheet structure is decoupled from that of alpha-helix, suggesting a step-wise unfolding of the protein. The transition observed for loss of alpha-helix coincides with the previously measured transition for His-12 by NMR from a partially folded state to the unfolded state, suggesting that the unfolding of the N-terminal helix in RNase A is lost after unfolding of the core beta-sheet during thermal denaturation. The thermally denatured protein was relatively compact, as measured by dynamic light scattering.