Amyloid formation from HypF-N under conditions in which the protein is initially in its native state

Aggregation of the N-terminal domain of the Escherichia coli HypF (HypF-N) was investigated in mild denaturing conditions, generated by addition of 6-12% (v/v) trifluoroethanol (TFE). Atomic force microscopy indicates that under these conditions HypF-N converts into the same type of protofibrillar aggregates previously shown to be highly toxic to cultured cells. These convert subsequently, after some weeks, into well-defined fibrillar structures. The rate of protofibril formation, monitored by thioflavin T (ThT) fluorescence, depends strongly on the concentration of TFE. Prior to aggregation the protein has far-UV circular dichroism (CD) and intrinsic fluorescence spectra identical with those observed for the native protein in the absence of co-solvent; the quenching of the intrinsic tryptophan fluorescence by acrylamide and the ANS binding properties are also identical in the two cases. These findings indicate that HypF-N is capable of forming amyloid protofibrils and fibrils under conditions in which the protein is initially in a predominantly native-like conformation. The rate constants for folding and unfolding of HypF-N, determined in 10% TFE using the stopped-flow technique, indicate that a partially folded state is in rapid equilibrium with the native state and populated to ca 1%. A kinetic analysis reveals that aggregation results from molecules accessing such a partially folded state. The approach described here shows that it is possible to probe the mechanism of aggregation of a specific protein under conditions in which the protein is initially native and hence relevant to a physiological environment. In addition, the results indicate that toxic protofibrils can be formed from globular proteins under conditions that are only marginally destabilising and in which the large majority of molecules have the native fold. This conclusion emphasises the importance for cells to constantly combat the propensity for even the most stable of these proteins to aggregate.     

Association of thin filaments into thick filaments revealing the structural hierarchy of amyloid fibrils

Beta2-Microglobulin (beta2-m) is a major structural component of dialysis-related amyloid fibrils. Kozhukh et al. [J. Biol. Chem. 277 (2002) 1310] prepared a series of peptide fragments of beta2-m by the protease digestion and examined their ability to form amyloid fibrils in citrate buffer at pH 2.5. Among various peptides, a 22-residue K3 peptide corresponding to Ser20-Lys41 spontaneously formed amyloid fibrils in aqueous solution. This peptide also formed amyloid protofibrils in 20% (v/v) 2,2,2-trifluoroethanol (TFE). To investigate the influence of solvent conditions on fibril formation, we studied their structures by atomic force microscopy. In aqueous solution, fibrils had a diameter of 4 or 8 nm and tended to cluster each other. On the other hand, protofibrils in 20% (v/v) TFE had a diameter of 2 nm with no tendency of clustering. Intriguingly, when the K3 protofibrils were transferred from 20% (v/v) TFE to aqueous solution, some of them associated to form thicker fibrils with a diameter of 4-15 nm and a left-handed helical twist. TFE is a hydrophobic solvent, so that hydrophobic interactions between molecules may be weakened. The results suggest that the fibrils in aqueous conditions are formed by the cooperative association of protofibrils at the growing ends of the fibrils, in which hydrophobic interactions play a major role.     

Reversal of protein aggregation provides evidence for multiple aggregated States

Observations that prefibrillar aggregates from different amyloidogenic proteins can be solubilised under some conditions have raised questions as to the generality of this phenomenon and the nature of the factors that influence it. By studying aggregates formed from human muscle acylphosphatase (AcP) under mild denaturing conditions, and by using a battery of techniques, we demonstrate that disaggregation is possible under conditions close to physiological where the protein is stable in its native state. In the presence of 25% (v/v) trifluoroethanol (TFE) AcP undergoes partial unfolding and globular aggregates (60-200 nm in diameter) that can assemble further into clusters (400-800 nm in diameter) develop progressively. Yet larger superstructures (>5 microm) are formed when the concentration of the globular aggregates exceeds a critical concentration. After diluting the sample to give a solution containing 5% TFE, the fraction of partially unfolded monomeric protein refolds very rapidly, with a rate constant of approximately 1s(-1). The 60-200 nm globular aggregates disaggregate with an apparent rate constant of approximately 2.5 x 10(-3)s(-1) while the 400-800 nm clusters disassembly more slowly with a rate constant of approximately 3.1 x 10(-4)s(-1). The larger (>5 microm) superstructures are not disrupted under the conditions used here. These results suggest that amyloid formation occurs in discrete steps whose reversibility is increasingly difficult, and dependent on the size of the aggregates, and that disaggregation experiments can provide a powerful method of detecting different types of species within the complex process of aggregation. In addition, our work suggests that destabilization of amyloid aggregates resulting in the conversion of misfolded proteins back to their native states could be an important factor in both the onset and treatment of diseases associated with protein aggregation.     

Effect of Trifluoroethanol on the Structural and Functional Properties of α-Crystallin

Alpha crystallin is an eye lens protein with a molecular weight of approximately 800 kDa. It belongs to the class of small heat shock proteins. Besides its structural role, it is known to prevent the aggregation of β- and γ-crystallins and several other proteins under denaturing conditions and is thus believed to play an important role in maintaining lens transparency. In this communication, we have investigated the effect of 2,2,2-trifluoroethanol (TFE) on the structural and functional features of the native α-crystallin and its two constituent subunits. A conformational change occurs from the characteristic β-sheet to the α-helix structure in both native α-crystallin and its subunits with the increase in TFE levels. Among the two subunits, αA-crystallin is relatively stable and upon preincubation prevents the characteristic aggregation of αB-crystallin at 20% and 30% (v/v) TFE. The hydrophobicity and chaperone-like activity of the crystallin subunits decrease on TFE treatment. The ability of αA-crystallin to bind and prevent the aggregation of αB-crystallin, despite a conformational change, could be important in protecting the lens from external stress. The loss in chaperone activity of αA-crystallin exposed to TFE and the inability of peptide chaperone—the functional site of αA-crystallin—to stabilize αB-crystallin at 20–30% TFE suggest that the site(s) involved in subunit interaction and chaperone-like function are quite distinct.     

Fluoroalcohol-induced modulation of the pathway of amyloid protofibril formation by barstar

To understand how the conformational heterogeneity of protofibrils formed by any protein, as well as the mechanisms of their formation, are modulated by a change in aggregation conditions, we studied the formation of amyloid protofibrils by barstar at low pH by multiple structural probes in the presence of hexafluoroisopropanol (HFIP). In the presence of 10% HFIP, aggregation proceeds with the transient formation of spherical oligomers and leads to the formation of both protofibrils and fibrils. Curly short protofibrils and fibrils are seen to form early during the aggregation reaction, and both are seen to grow gradually in length during the course of the reaction. Atomic force microscopy images reveal that the HFIP-induced protofibrils are long (～300 nm in length), curly, and beaded and appear to be composed primarily of β-sheet bilayers, with heights of ～2.4 nm. The protofibrils formed in the presence of HFIP differ in both their structures and their stabilities from the protofibrils formed either in the absence of alcohol or in the presence of a related alcohol, trifluoroethanol (TFE). Aggregation appears to proceed via an isodesmic polymerization mechanism. Internal structure in the growing aggregates changes in two stages during protofibril formation. In the first stage, an α-helix-rich oligomeric intermediate is formed. In the second stage, the level of β-sheet structure increases at the expense of some α-helical structure. The second stage itself appears to occur in two distinct steps. The creation of thioflavin T binding sites occurs concomitantly with aggregate elongation and is seen to precede the change in secondary structure. The long straight fibrils with characteristic heights of 8-10 nm, which form in the course of the HFIP-induced aggregation reaction, have not been observed to form either in the absence of alcohol or in the presence of TFE.     

A molecular dynamics study of acylphosphatase in aggregation-promoting conditions: the influence of trifluoroethanol/water solvent

The 98-residue protein acylphosphatase exhibits a high propensity for aggregation under certain conditions. Aggregates formed from wild-type acylphosphatase in the presence of 2,2,2-trifluoroethanol and from highly destabilized mutants are essentially identical in structure. Furthermore, it has been shown by mutational studies that different regions of the protein are important for aggregation and folding. In the present molecular dynamics study, we compare the behavior of the protein in aqueous solution and in a 25% (v/v) 2,2,2-trifluoroethanol/water environment mimicking the experimental conditions. The 2,2,2-trifluoroethanol surrounding affects the structure of the protein mostly in the regions important for aggregation, in good agreement with experimental data. This suggests that the early step of (partly) unfolding, which precedes the aggregation process, has been observed.     

Tumor-derived extracellular mutations of PTPRT /PTPrho are defective in cell adhesion

Receptor protein tyrosine phosphatase T (PTPRT/PTPrho) is frequently mutated in human cancers including colon, lung, gastric, and skin cancers. More than half of the identified tumor-derived mutations are located in the extracellular part of PTPrho. However, the functional significance of those extracellular domain mutations remains to be defined. Here we report that the extracellular domain of PTPrho mediates homophilic cell-cell aggregation. This homophilic interaction is very specific because PTPrho does not interact with its closest homologue, PTPmu, in a cell aggregation assay. We further showed that all five tumor-derived mutations located in the NH(2)-terminal MAM and immunoglobulin domains impair, to varying extents, their ability to form cell aggregates, indicating that those mutations are loss-of-function mutations. Our results suggest that PTPrho may play an important role in cell-cell adhesion and that mutational inactivation of this phosphatase could promote tumor migration and metastasis.     

Cosolvents Induced Unfolding and Aggregation of Keyhole Limpet Hemocyanin

The objective of this study was to examine the effects of 2,2,2 trifluoroethanol (TFE) and acetonitrile (ACN) on the stability, behavior, and structural characteristics of giant multimeric protein Keyhole Limpet hemocyanin (KLH) by combining the circular dichroism (CD) and fluorescence measurements of KLH solution. In concentration range 20–50 % (v/v) TFE, protein at pH 7.4 shows visible aggregation while no aggregation was observed in the entire concentration range of TFE at molten globule (MG) state (pH 2.8) and resulted in stable α-helix. Our result shows that in the presence of 80 % (v/v) and 40 % (v/v) TFE, at native (pH 7.4) and MG state (pH 2.8) occurred in a highly helical state referred to as TFE denatured state I and II, respectively. However, in case of ACN, aggregation starts above 40 % (v/v) for pH 7.4 and at 80 % (v/v) for acid-induced MG (pH 2.8) state, which was dominated by β-sheet structure and referred to as ACN denatured state III and IV. An important object of our investigation is to get more detail study of efficiency of cosolvents in inducing structural changes in KLH. The dependence of scattering intensity and the R _h on alcohol concentrations was investigated at 25 °C.     

Fluoroalcohols induced unfolding of Succinylated Con A: native like beta-structure in partially folded intermediate and alpha-helix in molten globule like state

Concanavalin A (Con A) exists in dimeric state at pH 5. In concentration range 20-60% (v/v) 2,2,2-trifluoroethanol (TFE) and 2-40% (v/v) 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), Con A at pH 5.0 shows visible aggregation. However, when succinyl Con A was used, no aggregation was observed in the entire concentration range of fluoroalcohols (0-90% v/v TFE and HFIP) and resulted in stable alpha-helix formation. Temperature-induced concentration-dependent aggregation in Con A was also found to be prevented/reduced in succinylated form. Possible role of electrostatic repulsion among residues in the prevention of hydrophobically driven aggregation has been discussed. Results indicate that succinylation of a protein resulted in greater stability (in both beta-sheet and alpha-helical forms) against alcohol-induced and temperature-induced concentration-dependent aggregation and this observation may play significant role in amyloid-forming proteins. Effect of TFE and HFIP on the conformation of a dimeric protein, Succinylated Con A, has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of hydrophobic dye ANS (8-anilinonaphthalene-1-sulfonic acid). Far UV-CD, a probe for secondary structure shows loss of native secondary structure in the presence of low concentration of both the alcohols, TFE (10% v/v) and HFIP (4% v/v). Upon addition of higher concentration of these alcohols, Succinylated Con A exhibited transformation from beta-sheet to alpha-helical structure. Intrinsic tryptophan fluorescence studies, ANS binding and near UV-CD experiments indicate the protein is more expanded, have more exposed hydrophobic surfaces and highly disrupted tertiary structure at 60% (v/v) TFE and 30% (v/v) HFIP concentrations. Taken together, these results it might be concluded that TFE and HFIP induce two intermediate states at their low and high concentrations in Succinyl Con A.     

Hsp104 targets multiple intermediates on the amyloid pathway and suppresses the seeding capacity of Abeta fibrils and protofibrils

The heat shock protein Hsp104 has been reported to possess the ability to modulate protein aggregation and toxicity and to “catalyze” the disaggregation and recovery of protein aggregates, including amyloid fibrils, in yeast, Escherichia coli, mammalian cell cultures, and animal models of Huntington's disease and Parkinson's disease. To provide mechanistic insight into the molecular mechanisms by which Hsp104 modulates aggregation and fibrillogenesis, the effect of Hsp104 on the fibrillogenesis of amyloid beta (Aβ) was investigated by characterizing its ability to interfere with oligomerization and fibrillogenesis of different species along the amyloid-formation pathway of Aβ. To probe the disaggregation activity of Hsp104, its ability to dissociate preformed protofibrillar and fibrillar aggregates of Aβ was assessed in the presence and in the absence of ATP. Our results show that Hsp104 inhibits the fibrillization of monomeric and protofibrillar forms of Aβ in a concentration-dependent but ATP-independent manner. Inhibition of Aβ fibrillization by Hsp104 is observable up to Hsp104/Aβ stoichiometric ratios of 1:1000, suggesting a preferential interaction of Hsp104 with aggregation intermediates (e.g., oligomers, protofibrils, small fibrils) on the pathway of Aβ amyloid formation. This hypothesis is consistent with our observations that Hsp104 (i) interacts with Aβ protofibrils, (ii) inhibits conversion of protofibrils into amyloid fibrils, (iii) arrests fibril elongation and reassembly, and (iv) abolishes the capacity of protofibrils and sonicated fibrils to seed the fibrillization of monomeric Aβ. Together, these findings suggest that the strong inhibition of Aβ fibrillization by Hsp104 is mediated by its ability to act at different stages and target multiple intermediates on the pathway to amyloid formation.     

Interactions of trifluroethanol with the Trp-cage peptide

It has been suggested that aggregation of fluorinated alcohols in water solutions is involved with the abilities of these alcohols to provoke conformational changes in peptides and proteins. The extent of fluoroalcohol aggregation depends on the degree of fluorination: hexafluoroisopropanol (HFIP) is more extensively aggregated than is TFE. We previously described a study of the interactions of HFIP with the peptide Trp-cage and provided evidence for the formation of long-lived complexes between this fluoroalcohol and the peptide. In the present work, we have examined the interactions of the less-fluorinated TFE with Trp-cage, in order to probe the role of fluoroalcohol aggregation in the phenomena observed. Intermolecular (1)H{(19)F} nuclear Overhauser effects arising from interactions of TFE with the hydrogens of the peptide in a solution containing 42% TFE were determined at sample temperatures from 5 to 45 degrees C. It is shown that the folded state of the peptide under these conditions is essentially the same as that observed in water and in 30% HFIP-water. The observed peptide-solvent NOEs indicate formation of complexes of Trp-cage with TFE that persist for times of the order of 1 ns. The interactions leading to complexes with TFE are somewhat weaker than those involved in complex formation with HFIP. There are no indications that the aggregation of fluoroalcohol is a necessary concomitant of the interactions of TFE or HFIP with Trp-cage. Rather, the stronger and more long-lived interactions of HFIP with Trp-cage appear to be primarily the result of the greater hydrogen-bonding ability and hydrophobicity of this fluoroalcohol.     

Molecular Dynamics Simulations to Gain Insights into the Stability and Morphologies of K3 Oligomers from β2-microglobulin

Abstract

β2-Microglobulin (β2-m) forms amyloid fibrils in patients undergoing long-term hemodialysis. K3 peptide, a Ser20-Lys41 fragment of β2-m, has been known to form fibrils over a wide range of pH and solvent conditions. Recent solid-state NMR has revealed that K3 oligomer adopts a parallel U-shaped β-strand-turn-β-strand motif. In order to investigate the stability and morphologies of K3 oligomers with different sizes (dimer, trimer, and tetrameri and organizations (single and double layers), several all-atom molecular dynamics simulations were conducted at 310 K and pH 2 in water and 2,2,2-trifluoroethanol (TFE). For single-layered organizations, our results show that TFE destabilizes the stacking of K3 peptides due to the fact that TFE weakens the intermolecular hydrophobic interactions of K3 oligomers. In addition, we also identified that the loop region is stabilized by the hydrophobic cluster involving resides Y7, Fll, and I16. Our results further suggest that K3 tetramer is a potential minimal nucleus seed for the formation of K3 protofibrils. For dou-ble-layered organizations in water, our data demonstrate that K3 peptides can form various stable assemblies through different interfacial arrangements, such as NN, NC, and CC, by different driving forces. We further propose that the stacking of different interfaces between two facing β-sheets of K3 peptides could be related to different fibril morphologies, which is in good agreement with the previous experimental results, showing that K3 protofibrils associated to formed mature fibrils with a wide range of diameters from 4 to 15 nm when they were transferred from 20% (v/v) TFE to aqueous solution.     

Trifluoroethanol-induced conformational transitions of proteins: insights gained from the differences between alpha-lactalbumin and ribonuclease A

The trifluoroethanol (TFE)-induced structural changes of two proteins widely used in folding experiments, bovine alpha-lactalbumin, and bovine pancreatic ribonuclease A, have been investigated. The experiments were performed using circular dichroism spectroscopy in the far- and near-UV region to monitor changes in the secondary and tertiary structures, respectively, and dynamic light scattering to measure the hydrodynamic dimensions and the intermolecular interactions of the proteins in different conformational states. Both proteins behave rather differently under the influence of TFE: alpha-lactalbumin exhibits a molten globule state at low TFE concentrations before it reaches the so-called TFE state, whereas ribonuclease A is directly transformed into the TFE state at TFE concentrations above 40% (v/v). The properties of the TFE-induced states are compared with those of equilibrium and kinetic intermediate states known from previous work to rationalize the use of TFE in yielding information about the folding of proteins. Additionally, we report on the properties of TFE/water and TFE/buffer mixtures derived from dynamic light scattering investigations under conditions used in our experiments.     

Role of the helical structure of the N-terminal region of Plasmodium falciparum merozoite surface protein 2 in fibril formation and membrane interaction

Merozoite surface protein 2 (MSP2), an abundant glycosylphosphatidylinositol-anchored protein on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically disordered and forms amyloid-like fibrils in solution under physiological conditions. The 25 N-terminal residues (MSP2(1-25)) play an important role in both fibril formation and membrane binding of the full-length protein. In this study, the fibril formation and solution structure of MSP2(1-25) in the membrane mimetic solvents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and trifluoroethanol (TFE) have been investigated by transmission electronic microscopy, turbidity, thioflavin T fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Turbidity data showed that the aggregation of MSP2(1-25) was suppressed in the presence of membrane mimetic solvents. CD spectra indicated that helical structure in MSP2(1-25) was stabilized in SDS and DPC micelles and in high concentrations of TFE. The structure of MSP2(1-25) in 50% aqueous TFE, determined using NMR, showed that the peptide formed an amphipathic helix encompassing residues 10-24. Low concentrations of TFE favored partially folded helical conformations, as demonstrated by CD and NMR, and promoted MSP2(1-25) fibril formation. Our data suggest that partially folded helical conformations of the N-terminal region of MSP2 are on the pathway to amyloid fibril formation, while higher degrees of helical structure stabilized by high concentrations of TFE or membrane mimetics suppress self-association and thus inhibit fibril formation. The roles of the induced helical conformations in membrane interactions are also discussed.     

Conformational changes and inactivation of bovine carbonic anhydrase II in 2,2,2-trifluoroethanol solutions

Changes in unfolding and enzymatic activity of bovine carbonic anhydrase II (BCA II) in different concentrations of 2,2,2-trifluoroethanol (TFE) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectra, far-UV CD spectra, and enzyme activity. The results showed that the activity and conformation of BCA II changed according to the concentration of TFE. Significant aggregation was observed when BCA II was denatured at TFE concentrations between 10 and 35% (v/v). When the concentration of TFE exceeded 40%, the aggregation of BCA II was not very obvious. The activity of BCA II decreased almost to zero as the TFE concentration reached 26%. The ANS fluorescence spectra indicated the tertiary conformations of BCA II were more stable in solutions with TFE concentrations lower than 15% (v/v) and higher than 40% (v/v). Far-UV CD spectra showed that high concentrations (higher than 25%) of TFE could induce BCA II to form more alpha-helix structures and caused these structures to be in relatively stable states. The native conformation of BCA II being destroyed after its inactivity indicated that the active sites of BCA II is situated in a limited region and has more flexibility than the whole enzyme molecule.     

Conformational changes and inactivation of bovine carbonic anhydrase II in 2,2,2-Trifluoroethanol solutions

Changes in unfolding and enzymatic activity of bovine carbonic anhydrase II (BCA II) in different concentrations of 2,2,2-trifluoroethanol (TFE) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) fluorescence emission spectra, far-UV CD spectra, and enzyme activity. The results showed that the activity and conformation of BCA II changed according to the concentration of TFE. Significant aggregation was observed when BCA II was denatured at TFE concentrations between 10 and 35% (v/v). When the concentration of TFE exceeded 40%, the aggregation of BCA II was not very obvious. The activity of BCA II decreased almost to zero as the TFE concentration reached 26%. The ANS fluorescence spectra indicated the tertiary conformations of BCA II were more stable in solutions with TFE concentrations lower than 15% (v/v) and higher than 40% (v/v). Far-UV CD spectra showed that high concentrations (higher than 25%) of TFE could induce BCA II to form more α-helix structures and caused these structures to be in relatively stable states. The native conformation of BCA II being destroyed after its inactivation indicated that the active site of BCA II is situated in a limited region and has more flexibility than the whole enzyme molecule.     

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.     

Kinetic studies of protein L aggregation and disaggregation

We have investigated the aggregation of protein L in 25% (vol/vol) TFE and 10 mM HCl. Under both conditions, aggregates adopt a fibrillar structure and bind dyes Congo Red and Thioflavin T consistent with the presence of amyloid fibrils. The kinetics of aggregation in 25% TFE suggest a linear-elongation mechanism with critical nucleus size of either two or three monomers. Aggregation kinetics in 10 mM HCl show a prolonged lag phase prior to a rapid increase in aggregation. The lag phase is time-dependent, but the time dependence can be eliminated by the addition of pre-formed seeds. Disaggregation studies show that for aggregates formed in TFE, aggregate stability is a strong function of aggregate age. For example, after 200 min of aggregation, 40% of the aggregation reaction is irreversible, while after 3 days over 60% is irreversible. When the final concentration of the denaturant, TFE, is reduced from 5% to 0, the amount of reversible aggregation doubles. Disaggregation studies of aggregates formed in TFE and 10 mM HCl reveal a complicated effect of pH on aggregate stability.     

Reactivation and refolding of rabbit muscle creatine kinase denatured in 2,2,2-trifluoroethanol solutions

The unfolding and refolding of creatine kinase (ATP:creatine N-phosphotransferase (CK), EC 2.7.3.2) during denaturation and reactivation by trifluoroethanol (TFE) have been studied. Significant aggregation was observed when CK was denatured at TFE concentrations between 10% and 40% (v/v). 50% TFE (v/v) was used to study the denaturation and unfolding of CK. The activity loss of CK was a very quick process, as was the marked conformational changes during denaturation followed by fluorescence emission spectra and far-ultraviolet CD spectra. DTNB modification and size exclusion chromatography were used to find that CK dissociated and was in its monomer state after denaturation with 50% TFE. Reactivation and refolding were observed after 80-fold dilution of the denatured CK into 0.05 M Tris-HCl buffer, pH 8.0. The denatured CK recovered about 38% activity following a two phase course (k(1)=4.82+/-0.41x10(-3) s(-1), k(2)=0.60+/-0.01x10(-3) s(-1)). Intrinsic fluorescence maximum intensity changes showed that the refolding process also followed biphasic kinetics (k(1)=4.34+/-0.27x10(-3) s(-1), k(2)=0.76+/-0.02x10(-3) s(-1)) after dilution into the proper solutions. The far-ultraviolet CD spectra ellipticity changes at 222 nm during the refolding process also showed a two phase course (k(1)=4.50+/-0.07x10(-3) s(-1), k(2)=1.13+/-0.05x10(-3) s(-1)). Our results suggest that TFE can be used as a reversible denaturant like urea and GuHCl. The 50% TFE induced CK denaturation state, which was referred to as the 'TFE state', and the partially refolded CK are compared with the molten globule state. The aggregation caused by TFE during denaturation is also discussed in this paper.     

Alcohol induced structural and dynamic changes in β-lactoglobulin in aqueous solution: A neutron scattering study

Structural and dynamic properties of β-lactoglobulin (β-LG) were revealed as a function of alcohol concentration in ethanol- and trifluoroethanol(TFE)-water mixtures with circular dichroism (CD), small-angle neutron scattering (SANS) and quasi-elastic neutron scattering (QENS). The CD spectra showed that an increase in TFE concentration promotes the formation of the β-sheet structure of β-LG. The SANS-intensities were fitted using form factors for two attached spheres for the native and native-like states of the protein. At higher alcohol concentrations, where aggregation takes place, a form factor modelling diffusion limited colloidal aggregation (DLCA) was employed. The QENS-data were analyzed in terms of internal motions for all alcohol concentrations. While low concentrations of TFE (10% (v/v)) lead to an increase of the mean square amplitudes of vibrations and a retention of a native-like structure - but not to an increase of the characteristic radius of proton diffusion processes a. Addition of 20% (v/v) of TFE induces aggregation, going along with a further increase of . Further increase of TFE concentration to 30% (v/v) changes the nanoscale structure of the oligomeric nucleate, but induces no further significant changes in . The present study underlines the necessity of methods sensitive to the dynamics of a system to obtain a complete picture of a molecular process.