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Amyloid aggregation is a key process in amyloidoses and neurodegenerative diseases. Hydrophobicity is one of the major driving forces for this type of aggregation, as an increase in hydrophobicity generally correlates with aggregation susceptibility and rate. However, most experimental systems in vitro and prediction tools in silico neglect the contribution of protective osmolytes present in the cellular environment. Here, we assessed the role of hydrophobic mutations in amyloid aggregation in the presence of osmolytes. To achieve this goal, we used the model protein human muscle acylphosphatase (mAcP) and mutations to leucine that increased its hydrophobicity without affecting its thermodynamic stability. Osmolytes significantly slowed down the aggregation kinetics of the hydrophobic mutants, with an effect larger than that observed on the wild-type protein. The effect increased as the mutation site was closer to the middle of the protein sequence. We propose that the preferential exclusion of osmolytes from mutation-introduced hydrophobic side-chains quenches the aggregation potential of the ensemble of partially unfolded states of the protein by inducing its compaction and inhibiting its self-assembly with other proteins. Our results suggest that including the effect of the cellular environment in experimental setups and predictive softwares, for both mechanistic studies and drug design, is essential in order to obtain a more complete combination of the driving forces of amyloid aggregation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

An understanding of the solution factors contributing to the rate of aggregation of a protein into amyloid oligomers, to the modulation of the conformational state populated prior to aggregation and to the structure/morphology of the resulting oligomers is one of the goals of present research in this field. We have studied the influence of six different salts on the conversion of the N-terminal domain of Escherichiacoli HypF (HypF-N) into amyloid-like oligomers under conditions of acidic pH. Our results show that salts having different anions (NaCl, NaClO(4), NaI, Na(2)SO(4)) accelerate oligomerization with an efficacy that follows the electroselectivity series of the anions (SO(4)(2-)≥ ClO(4)(-)>I(-)>Cl(-)). By contrast, salts with different cations (NaCl, LiCl, KCl) have similar effects. We also investigated the effect of salts on the structure of the final and initial states of HypF-N aggregation. The electroselectivity series does not apply to the effect of anions on the structure of the oligomers. By contrast, it applies to their effect on the content of secondary structure and on the exposure of hydrophobic clusters of the monomeric precursor state. The results therefore indicate that the binding of anions to the positively charged residues of HypF-N at low pH is the mechanism by which salts modulate the rate of oligomerization and the structure of the monomeric precursor state but not the structure of the resulting oligomers. Overall, the data contribute to rationalize the effect of salts on amyloid-like oligomer formation and to explain the role of charged biological macromolecules in protein aggregation processes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A group of diverse human pathologies is associated with proteins unable to retain their native state and convert into prefibrillar and fibrillar amyloid aggregates that are then deposited in the extracellular space. Glycosaminoglycans (GAGs) have been found to physically associate with these deposits and also to promote their formation in vitro. However, the effect of GAGs on the toxicity of these aggregates has been investigated in only one protein system, the amyloid β peptide associated with Alzheimer's disease. In this study, we investigate whether GAGs affect the toxicity of the N-terminal domain of Escherichia coli HypF (HypF-N) oligomers on Chinese hamster ovarian cells and the mechanism by which such suppression is mediated. The results show that heparin and other GAGs inhibit the toxicity observed by HypF-N oligomers in a dose-dependent manner. GAGs were not found to bind preformed HypF-N oligomers, change their morphological and structural characteristics or disaggregate them. Nevertheless, they were found to bind to the cells' surface and prevent the interaction of the oligomers with the cells. Overall, the results indicate that GAGs have a generic ability to inhibit the toxicity of aberrant protein oligomers and that such toxicity suppression can occur through different mechanisms, such as through binding to the oligomers with consequent loss of interaction of the oligomers to the GAGs present on the cell surface, as proposed previously for amyloid β aggregates, or through mechanisms independent of direct GAG-oligomer binding, as shown here for HypF-N aggregates., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Aggregation of peptides and proteins into insoluble amyloid fibrils or related intracellular inclusions is the hallmark of many degenerative diseases, including Alzheimer's disease, Parkinson's disease, and various forms of amyloidosis. In spite of the considerable progress carried out in vitro in elucidating the molecular determinants of the conversion of purified and isolated proteins into amyloid fibrils, very little is known on factors governing this process in the complex environment of living organisms. Taking advantage of increasing evidence that bacterial inclusion bodies consist of amyloid-like aggregates, we have expressed in Escherichia coli both wild type and 21 single-point mutants of the N-terminal domain of the E. coli protein HypF. All variants were expressed as folding-incompetent units in a controlled manner, at low and comparable levels. Their solubilities were measured by quantifying the protein amount contained in the soluble and insoluble fractions by Western blot analysis. A significant negative correlation was found between the solubility of the variants in E. coli and their intrinsic propensity to form amyloid fibrils, predicted using an algorithm previously validated experimentally in vitro on a number of unfolded peptides and proteins, and considering hydrophobicity, beta-sheet propensity, and charge as major sequence determinants of the aggregation process. These findings show that the physicochemical parameters previously recognized to govern amyloid formation by fully or partially unfolded proteins are largely applicable in vivo and pave the way for the molecular exploration of a process as complex as protein aggregation in living organisms., ((c) 2010 Elsevier Ltd. All rights reserved.)

We have performed an extensive mutational analysis of aggregation and disaggregation of amyloid-like protofibrils of human muscle acylphosphatase. Our findings indicate that the regions that promote aggregation in 25% (v/v) 2,2,2 trifluoroethanol (TFE) are different from those that promote disaggregation under milder conditions (5% TFE). Significant changes in the rate of disaggregation of protofibrils in 5% TFE result not only from mutations situated in the regions of the sequence that play a key role in the mechanism of aggregation in 25% TFE, but also from mutations located in other regions. In order to rationalise these results, we have used a modified version of the Zyggregator aggregation propensity prediction algorithm to take into account structural rearrangements of the protofibrils that may be induced by changes in solution conditions. Our results suggest that a wider range of residues contributes to the stability of the aggregates in addition to those that play an important kinetic role in the aggregation process. The mutational approach described here is capable of providing residue-specific information on the structure and dynamics of amyloid protofibrils under conditions close to physiological and should be widely applicable to other systems.

We present a method for predicting the regions of the sequences of peptides and proteins that are most important in promoting their aggregation and amyloid formation. The method extends previous approaches by allowing such predictions to be carried out for conditions under which the molecules concerned can be folded or contain a significant degree of persistent structure. In order to achieve this result, the method uses only knowledge of the sequence of amino acids to estimate simultaneously both the propensity for folding and aggregation and the way in which these two types of propensity compete. We illustrate the approach by its application to a set of peptides and proteins both associated and not associated with disease. Our results show not only that the regions of a protein with a high intrinsic aggregation propensity can be identified in a robust manner but also that the structural context of such regions in the monomeric form is crucial for determining their actual role in the aggregation process.

The acylphosphatase from Escherichia coli (EcoAcP) is the first AcP so far studied with a disulfide bond. A mutational variant of the enzyme lacking the disulfide bond has been produced by substituting the two cysteine residues with alanine (EcoAcP mutational variant C5A/C49A, mutEcoAcP). The native states of the two protein variants are similar, as shown by far-UV and near-UV circular dichroism and dynamic light-scattering measurements. From unfolding experiments at equilibrium using intrinsic fluorescence and far-UV circular dichroism as probes, EcoAcP shows an increased conformational stability as compared with mutEcoAcP. The wild-type protein folds according to a two-state model with a very fast rate constant (k(F)(H2O)=72,600 s(-1)), while mutEcoAcP folds ca 1500-fold slower, via the accumulation of a partially folded species. The correlation between the hydrophobicity of the polypeptide chain and the folding rate, found previously in the AcP-like structural family, is maintained only when considering the mutant but not the wild-type protein, which folds much faster than expected from this correlation. Similarly, the correlation between the relative contact order and the folding rate holds only for mutEcoAcP. The correlation also holds for EcoAcP, provided the relative contact order value is recalculated by considering the disulfide bridge as an alternate path for the backbone to determine the shortest sequence separation between contacting residues. These results indicate that the presence of a disulfide bond in a protein is an important determinant of the folding rate and allows its contribution to be determined in quantitative terms.

The conversion of specific proteins or protein fragments into insoluble, ordered fibrillar aggregates is a fundamental process in protein chemistry, biology, medicine and biotechnology. As this structural conversion seems to be a property shared by many proteins, understanding the mechanism of this process will be of extreme importance. Here we present a structural characterisation of a conformational state populated at low pH by the N-terminal domain of Escherichia coli HypF. Combining different biophysical and biochemical techniques, including near- and far-UV circular dichroism, intrinsic and 8-anilinonaphthalene-1-sulfonate-derived fluorescence, dynamic light scattering and limited proteolysis, we will show that this state is largely unfolded but contains significant secondary structure and hydrophobic clusters. It also appears to be more compact than a random coil-like state but less organised than a molten globule state. Increase of the total ionic strength of the solution induces aggregation of such a pre-molten globule state into amyloid-like protofibrils, as revealed by thioflavin T fluorescence and atomic force microscopy. These results show that a pre-molten globule state can be, among other possible conformational states, one of the precursor states of amyloid formation. In addition, the possibility of triggering aggregation by modulating the ionic strength of the solution provides one a unique opportunity to study both the initial precursor state and the aggregation process.

The N-terminal fragment 1-29 of horse heart apomyoglobin (apoMb(1-29)) is highly prone to form amyloid-like fibrils at low pH. Fibrillogenesis at pH 2.0 occurs following a nucleation-dependent growth mechanism, as evidenced by the thioflavin T (ThT) assay. Transmission electron microscopy (TEM) confirms the presence of regular amyloid-like fibrils and far-UV circular dichroism (CD) spectra indicate the acquisition of a high content of beta-sheet structure. ThT assay, TEM and CD highlight fast and complete disaggregation of the fibrils, if the pH of a suspension of mature fibrils is increased to 8.3. It is of interest that amyloid-like fibrils form again if the pH of the solution is brought back to 2.0. While apoMb(1-29) fibrils obtained at pH 2.0 are resistant to proteolysis by pepsin, the disaggregated fibrils are easily cleaved at pH 8.3 by trypsin and V8 protease, and some of the resulting fragments aggregate very quickly in the proteolysis mixture, forming amyloid-like fibrils. We show that the increase of amyloidogenicity of apoMb(1-29) following acidification or proteolysis at pH 8.3 can be attributed to the decrease of the peptide net charge following these alterations. The results observed here for apoMb(1-29) provide an experimental basis for explaining the effect of charge and pH on amyloid fibril formation by both unfolded and folded protein systems.

The aggregation of the alpha/beta protein acylphosphatase from Sulfolobus solfataricus has been studied under conditions in which the protein maintains a native-like, although destabilised, conformation and that therefore bear resemblance to a physiological medium. Static and dynamic light-scattering measurements indicate that under these conditions the protein aggregates rapidly, within two minutes. The initial aggregates are enzymatically active and have a secondary structure that is not yet characterized by the high content of cross-beta structure typical of amyloid, as inferred from Fourier transform infra-red and circular dichroism measurements. These species then convert slowly into enzymatically inactive aggregates that bind thioflavin T and Congo red, characteristic of amyloid structures, and contain extensive beta-sheet structure. Transmission electron microscopy reveals the presence in the latter aggregates of spherical species and thin, elongated protofibrils, both with diameters of 3-5 nm. Kinetic tests reveal that this process occurs without the need for dissolution and re-nucleation of the aggregates. Formation of thioflavin T-binding and beta-structured aggregates is substantially more rapid than unfolding of the native state, indicating that the initial aggregation process promotes formation of amyloid structures. Taken together, these findings suggest a mechanism of amyloid formation that may have physiological relevance and in which the amyloid structures result from reorganisation of the molecular interactions within the initially formed non-amyloid aggregates.

Increasing evidence indicates that many peptides and proteins can be converted in vitro into highly organised amyloid structures, provided that the appropriate experimental conditions can be found. In this work, we define intrinsic propensities for the aggregation of individual amino acids and develop a method for identifying the regions of the sequence of an unfolded peptide or protein that are most important for promoting amyloid formation. This method is applied to the study of three polypeptides associated with neurodegenerative diseases, Abeta42, alpha-synuclein and tau. In order to validate the approach, we compare the regions of proteins that are predicted to be most important in driving aggregation, either intrinsically or as the result of mutations, with those determined experimentally. The knowledge of the location and the type of the "sensitive regions" for aggregation is important both for rationalising the effects of sequence changes on the aggregation of polypeptide chains and for the development of targeted strategies to combat diseases associated with amyloid formation.

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.

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.

Protein aggregation is associated with a variety of pathological conditions, including Alzheimer's and Creutzfeldt-Jakob diseases and type II diabetes. Such degenerative disorders result from the conversion of the normal soluble state of specific proteins into aggregated states that can ultimately form the characteristic amyloid fibrils found in diseased tissue. Under appropriate conditions it appears that many, perhaps all, proteins can be converted in vitro into amyloid fibrils. The aggregation propensities of different polypeptide chains have, however, been observed to vary substantially. Here, we describe an approach that uses the knowledge of the amino acid sequence and of the experimental conditions to reproduce, with a correlation coefficient of 0.92 and over five orders of magnitude, the in vitro aggregation rates of a wide range of unstructured peptides and proteins. These results indicate that the formation of protein aggregates can be rationalised to a considerable extent in terms of simple physico-chemical parameters that describe the properties of polypeptide chains and their environment.

Much information has appeared in the last few years on the low resolution structure of amyloid fibrils and on their non-fibrillar precursors formed by a number of proteins and peptides associated with amyloid diseases. The fine structure and the dynamics of the process leading misfolded molecules to aggregate into amyloid assemblies are far from being fully understood. Evidence has been provided in the last five years that protein aggregation and aggregate toxicity are rather generic processes, possibly affecting all polypeptide chains under suitable experimental conditions. This evidence extends the number of model proteins one can investigate to assess the molecular bases and general features of protein aggregation and aggregate toxicity. We have used tapping mode atomic force microscopy to investigate the morphological features of the pre-fibrillar aggregates and of the mature fibrils produced by the aggregation of the hydrogenase maturation factor HypF N-terminal domain (HypF-N), a protein not associated to any amyloid disease. We have also studied the aggregate-induced permeabilization of liposomes by fluorescence techniques. Our results show that HypF-N aggregation follows a hierarchical path whereby initial globules assemble into crescents; these generate large rings, which evolve into ribbons, further organizing into differently supercoiled fibrils. The early pre-fibrillar aggregates were shown to be able to permeabilize synthetic phospholipid membranes, thus showing that this disease-unrelated protein displays the same amyloidogenic behaviour found for the aggregates of most pathological proteins and peptides. These data complement previously reported findings, and support the idea that protein aggregation, aggregate structure and toxicity are generic properties of polypeptide chains.

Formation of misfolded aggregates is an essential part of what proteins can do. The process of protein aggregation is central to many human diseases and any aggregating event needs to be prevented within a cell and in protein design. In order to aggregate, a protein needs to unfold its native state, at least partially. The conformational state that is prone to aggregate is difficult to study, due to its aggregating potential and heterogeneous nature. Here, we use a systematic approach of limited proteolysis, in combination with electrospray ionisation mass spectrometry, to investigate the regions that are most flexible and solvent-exposed within the native, ligand-bound and amyloidogenic states of muscle acylphosphatase (AcP), a protein previously shown to form amyloid fibrils in the presence of trifluoroethanol. Seven proteases with different degrees of specificity have been used for this purpose. Following exposure to the aggregating conditions, a number of sites along the sequence of AcP become susceptible to proteolytic digestion. The pattern of proteolytic cleavages obtained under these conditions is considerably different from that of the native and ligand-bound conformations and includes a portion within the N-terminal tail of the protein (residues 6-7), the region of the sequence 18-23 and the position 94 near the C terminus. There is a significant overlap between the regions of the sequence found to be solvent-exposed from the present study and those previously identified to be critical in the rate-determining steps of aggregation from protein engineering approaches. This indicates that a considerable degree of solvent exposure is a feature of the portions of a protein that initiate the process of aggregation.

The SH3 domains are small protein modules of 60-85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The SH3 domain of the p85alpha subunit of bovine phosphatidylinositol 3'-kinase (PI3-SH3) under acidic solution adopts a compact denatured state from which amyloid fibrils are readily formed. This aggregation process has been found to be modulated substantially by solution conditions. Here, we have analyzed the conformational features of the native and acid denatured states of PI3-SH3 by limited proteolysis experiments using proteinase K and pepsin, respectively. Moreover, we have analyzed the propensity of PI3-SH3 to be hydrolyzed by pepsin at different stages in the process of aggregation and amyloid formation at pH 1.2 and 2.0 and compared the sites of proteolysis under these conditions with the conformational features of both native and aggregated PI3-SH3. The results demonstrate that the denatured state of PI3-SH3 formed at low pH is relatively resistant to proteolysis, indicating that it is partially folded. The long loop connecting beta-strands b and c in the native protein is the region in this structure most susceptible to proteolysis. Remarkably, aggregates of PI3-SH3 that are formed initially from this denatured state in acid solution display enhanced susceptibility to proteolysis of the long loop, suggesting that the protein becomes more unfolded in the early stages of aggregation. By contrast, the more defined amyloid fibrils that are formed over longer periods of time are completely resistant to proteolysis. We suggest that the protein aggregates formed initially are relatively dynamic species that are able readily to reorganize their interactions to enable formation of very well ordered fibrillar structures. In addition, the disordered and non-native character of the polypeptide chains in the early aggregates could be important in determining the high cytotoxicity that has been revealed in previous studies of these species.

The N-terminal domain of HypF from Escherichia coli (HypF-N) is a 91 residue protein module sharing the same folding topology and a significant sequence identity with two extensively studied human proteins, muscle and common-type acylphosphatases (mAcP and ctAcP). With the aim of learning fundamental aspects of protein folding from the close comparison of so similar proteins, the folding process of HypF-N has been studied using stopped-flow fluorescence. While mAcP and ctAcP fold in a two-state fashion, HypF-N was found to collapse into a partially folded intermediate before reaching the fully folded conformation. Formation of a burst-phase intermediate is indicated by the roll over in the Chevron plot at low urea concentrations and by the large jump of intrinsic and 8-anilino-1-naphtalenesulphonic acid-derived fluorescence immediately after removal of denaturant. Furthermore, HypF-N was found to fold rapidly with a rate constant that is approximately two and three orders of magnitudes faster than ctAcP and mAcP, respectively. Differences between the bacterial protein and the two human counterparts were also found as to the involvement of proline isomerism in their respective folding processes. The results clearly indicate that features that are often thought to be relevant in protein folding are not highly conserved in the evolution of the acylphosphatase superfamily. The large difference in folding rate between mAcP and HypF-N cannot be entirely accounted for by the difference in relative contact order or related topological metrics. The analysis shows that the higher folding rate of HypF-N is in part due to the relatively high hydrophobic content of this protein. This conclusion, which is also supported by the highly significant correlation found between folding rate and hydrophobic content within a group of proteins displaying the topology of HypF-N and AcPs, suggests that the average hydrophobicity of a protein sequence is an important determinant of its folding rate.

beta 2-Microglobulin is a small, major histocompatibility complex class I-associated protein that undergoes aggregation and accumulates as amyloid deposits in human tissues as a consequence of long-term haemodialysis. The folding process of this amyloidogenic protein has been studied in vitro by diluting the guanidine hydrochloride-denatured protein in refolding buffer at pH 7.4 and monitoring the folding process by means of a number of spectroscopic probes that allow the native structure of the protein to be detected as it develops. These techniques include fluorescence spectroscopy, far and near-UV circular dichroism, 8-anilino-1-naphthalenesulfonic acid binding and double jump assays. All spectroscopic probes indicate that a significant amount of structure forms within the dead-time of stopped-flow measurements (<5 ms). The folding reaction goes to completion through a fast phase followed by a slow phase, whose rate constants are ca 5.1 and 0.0030 s(-1) in water, respectively. Unfolding-folding double jump experiments, together with the use of peptidyl prolyl isomerase, reveal that the slow phase of folding of beta 2-microglobulin is not fundamentally determined by cis/trans isomerisation of X-Pro peptide bonds. Other folding-unfolding double jump experiments also suggest that the fast and slow phases of folding are not related to independent folding of different populations of protein molecules. Rather, we provide evidence for a sequential mechanism of folding where denatured beta 2-microglobulin collapses to an ensemble of partially folded conformations (I(1)) which fold subsequently to a more highly structured species (I(2)) and, finally, attain the native state. The partially folded species I(2) appears to be closely similar to previously studied amyloidogenic forms of beta 2-microglobulin, such as those adopted by the protein at mildly acid pH values and by a variant with six residues deleted at the N terminus. Since amyloid formation in vivo originates from partial denaturation of beta 2-microglobulin under conditions favouring the folding process, the long-lived, partially structured species detected here might be significantly populated under some physiological conditions and hence might play an important role in the process of amyloid formation., (Copyright 2001 Academic Press.)

The effects of stabilising mutations on the folding process of common-type acylphosphatase have been investigated. The mutations were designed to increase the helical propensity of the regions of the polypeptide chain corresponding to the two alpha-helices of the native protein. Various synthetic peptides incorporating the designed mutations were produced and their helical content estimated by circular dichroism. The most substantial increase in helical content is found for the peptide carrying five mutations in the second alpha-helix. Acylphosphatase variants containing the corresponding mutations display, to different extents, enhanced conformational stabilities as indicated by equilibrium urea denaturation experiments monitored by changes of intrinsic fluorescence. All the protein variants studied here refold with apparent two-state kinetics. Mutations in the first alpha-helix are responsible for a small increase in the refolding rate, accompanied by a marked decrease in the unfolding rate. On the other hand, multiple mutations in the second helix result in a considerable increase in the refolding rate without any significant effect on the unfolding rate. Addition of trifluoroethanol was found to accelerate the folding of the acylphosphatase variants, the extent of the acceleration being inversely proportional to the intrinsic rate of folding of the corresponding mutant. The trifluoroethanol-induced acceleration is far less marked for those variants whose alpha-helical structure is efficiently stabilised by amino acid replacements. This observation suggests that trifluoroethanol acts in a similar manner to the stabilising mutations in promoting native-like secondary structure. Analysis of the kinetic data indicates that the second helix is fully consolidated in the transition state for folding of acylphosphatase, whereas the first helix is only partially formed. These data suggest that the second helix is an important element in the folding process of the protein., (Copyright 2000 Academic Press.)

The folding of a 98 residue protein, muscle acylphosphatase (AcP), has been studied using a variety of techniques including circular dichroism, fluorescence and NMR spectroscopy following transfer of chemically denatured protein into refolding conditions. A low-amplitude phase, detected in concurrence with the main kinetic phase, corresponds to the folding of a minor population (13%) of molecules with one or both proline residues in a cis conformation, as shown from the sensitivity of its rate to peptidyl prolyl isomerase. The major phase of folding has the same kinetic characteristics regardless of the technique employed to monitor it. The plots of the natural logarithms of folding and unfolding rate constants versus urea concentration are linear over a broad range of urea concentrations. Moreover, the initial state formed rapidly after the initiation of refolding is highly unstructured, having a similar circular dichroism, intrinsic fluorescence and NMR spectrum as the protein denatured at high concentrations of urea. All these results indicate that AcP folds in a two-state manner without the accumulation of intermediates. Despite this, the folding of the protein is extremely slow. The rate constant of the major phase of folding in water, kfH2O, is 0.23 s-1 at 28 degreesC and, at urea concentrations above 1 M, the folding process is slower than the cis-trans proline isomerisation step. The slow refolding of this protein is therefore not the consequence of populated intermediates that can act as kinetic traps, but arises from a large intrinsic barrier in the folding reaction., (Copyright 1998 Academic Press.)

The transition state for folding of a small protein, muscle acylphosphatase, has been studied by measuring the rates of folding and unfolding under a variety of solvent conditions. A strong dependence of the folding rate on the concentration of urea suggests the occurrence in the transition state of a large shielding of those groups that are exposed to interaction with the denaturant in the unfolded state (mainly hydrophobic moieties and groups located on the polypeptide backbone). The heat capacity change upon moving from the unfolded state to the transition state is small and is indicative of a substantial solvent exposure of hydrophobic groups. The solvent-accessibility of such groups in the transition state has also been found to be significant by measuring the rates of folding and unfolding in the presence of sugars. These rates have also been found to be accelerated by the addition of small quantities of alcohols. Trifluoroethanol and hexafluoroisopropanol were particularly effective, suggesting that stabilisation of local hydrogen bonds lowers the energy of the transition state relative to the folded and unfolded states. Finally, a study with a competitive inhibitor of acylphosphatase has provided evidence for the complete loss of ligand binding affinity in the transition state, indicating that specific long-range interactions at the level of the active site are not yet formed at this stage of the folding reaction. A model of the transition state for acylphosphatase folding, in which beta-turns and one or both alpha-helices are formed to a significant extent but in which the persistent long-range interactions characteristic of the folded state are largely absent, accounts for all our data. These results are broadly consistent with models of the transition states for folding of other small proteins derived from mutagenesis studies, and suggest that solvent perturbation methods can provide complementary information about the transition region of the energy surfaces for protein folding., (Copyright 1998 Academic Press.)