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Christopher M. Dobson - One of the best experts on this subject based on the ideXlab platform.
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direct conversion of an enzyme from native like to amyloid like aggregates within inclusion bodies
Biophysical Journal, 2017Co-Authors: Francesco Elia, Fabrizio Chiti, Christopher M. Dobson, Francesca Cantini, Francesco BemporadAbstract:Abstract The Acylphosphatase from Sulfolobus solfataricus (Sso AcP) is a globular protein able to aggregate in vitro from a native-like conformational ensemble without the need for a transition across the major unfolding energy barrier. This process leads to the formation of assemblies in which the protein retains its native-like structure, which subsequently convert into amyloid-like aggregates. Here, we investigate the mechanism by which Sso AcP aggregates in vivo to form bacterial inclusion bodies after expression in E. coli . Shortly after the initiation of expression, Sso AcP is incorporated into inclusion bodies as a native-like protein, still exhibiting small but significant enzymatic activity. Additional experiments revealed that this overall process of aggregation is enhanced by the presence of the unfolded N-terminal region of the sequence and by destabilization of the globular segment of the protein. At later times, the Sso AcP molecules in the inclusion bodies lose their native-like properties and convert into β -sheet-rich amyloid-like structures, as indicated by their ability to bind thioflavin T and Congo red. These results show that the aggregation behavior of this protein is similar in vivo to that observed in vitro, and that, at least for a predominant part of the protein population, the transition from a native to an amyloid-like structure occurs within the aggregate state.
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2002a. Determination of a transition state at atomic resolution from protein engineering data
2015Co-Authors: Emanuelel Paci, Christopher M. Dobson, Michele Vendruscolo, Martin Karplus, Biophysique Isis UniversitéAbstract:We present a method for determining the structure of the transition state ensemble (TSE) of a protein by using f values derived from protein engin-eering experiments as restraints in molecular dynamics simulations employing a realistic all-atom molecular mechanics energy function. The method uses a biasing potential to select an ensemble of structures having f values in agreement with the experimental data set. An application to Acylphosphatase (AcP), a protein for which f values have been measured for 24 out of 98 residues, illustrates the approach. The properties of the TSE determined in this way are compared with those of a coarse-grained model obtained using a Monte Carlo (MC) sampling method based on a Ca representation of the structure. The two TSEs determined at different structural resolution are consistent and complementary. While the Ca model allows better sampling of the conformation space occupied by the transition state, the all-atom model offers a more detailed description of the structural and energetic properties of the conformations included in the TSE. The combination of low-resolution Ca results with all-atom mol-ecular dynamics simulations provides a powerful and general method for determining the nature of TSEs from protein engineering data
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Biological context
2014Co-Authors: Giuliana Fusco, Fabrizio Chiti, Francesco Bemporad, Michele Vendruscolo, Simone Shang-te, Danny Hsu, Christopher M. DobsonAbstract:Abstract Human muscle Acylphosphatase (mAcP) is an enzyme with a ferrodoxin-like topology whose primary role is to hydrolyze the carboxyl-phosphate bonds of acylphosphates. The protein has been widely used as a model system for elucidating the molecular determinants of protein folding and misfolding. We present here the full NMR assignments of the backbone and side chains reso-nances of mAcP complexed with phosphate, thus providing an important resource for future solution-state NMR spectroscopic studies of the structure and dynamics of this protein in the contexts of protein folding and misfolding
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amyloid fibril formation can proceed from different conformations of a partially unfolded protein
Biophysical Journal, 2005Co-Authors: Martino Calamai, Fabrizio Chiti, Christopher M. DobsonAbstract: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 β-sheet content from partially unfolded states with very different structural features. Moreover, the presence of α-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.
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Glycine residues appear to be evolutionarily conserved for their ability to inhibit aggregation.
Structure (London England : 1993), 2005Co-Authors: Claudia Parrini, Matteo Ramazzotti, Niccolo Taddei, Giampietro Ramponi, Christopher M. Dobson, Donatella Degl'innocenti, Fabrizio ChitiAbstract:Six glycine residues of human muscle Acylphosphatase (AcP) are evolutionarily conserved across the three domains of life. We have generated six variants of AcP, each having a glycine substituted by an alanine (G15A, G19A, G37A, G45A, G53A, and G69A). Three additional variants had Gly45 replaced by serine, glutamate, and arginine, respectively. The mutational variants do not, on average, have a lower conformational stability than other variants with substitutions of nonconserved residues. In addition, only the G15A variant is enzymatically inactive. However, all variants, with the exception of the G15A mutant, form amyloid aggregates more rapidly than the wild-type. Dynamic light-scattering experiments carried out under conditions close to physiological confirm that aggregate formation is generally more pronounced for the glycine-substituted variants. Apart from the glycine at position 15, all other conserved glycine residues in this protein could have been maintained during evolution because of their ability to inhibit aggregation.
Fabrizio Chiti - One of the best experts on this subject based on the ideXlab platform.
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direct conversion of an enzyme from native like to amyloid like aggregates within inclusion bodies
Biophysical Journal, 2017Co-Authors: Francesco Elia, Fabrizio Chiti, Christopher M. Dobson, Francesca Cantini, Francesco BemporadAbstract:Abstract The Acylphosphatase from Sulfolobus solfataricus (Sso AcP) is a globular protein able to aggregate in vitro from a native-like conformational ensemble without the need for a transition across the major unfolding energy barrier. This process leads to the formation of assemblies in which the protein retains its native-like structure, which subsequently convert into amyloid-like aggregates. Here, we investigate the mechanism by which Sso AcP aggregates in vivo to form bacterial inclusion bodies after expression in E. coli . Shortly after the initiation of expression, Sso AcP is incorporated into inclusion bodies as a native-like protein, still exhibiting small but significant enzymatic activity. Additional experiments revealed that this overall process of aggregation is enhanced by the presence of the unfolded N-terminal region of the sequence and by destabilization of the globular segment of the protein. At later times, the Sso AcP molecules in the inclusion bodies lose their native-like properties and convert into β -sheet-rich amyloid-like structures, as indicated by their ability to bind thioflavin T and Congo red. These results show that the aggregation behavior of this protein is similar in vivo to that observed in vitro, and that, at least for a predominant part of the protein population, the transition from a native to an amyloid-like structure occurs within the aggregate state.
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Biological context
2014Co-Authors: Giuliana Fusco, Fabrizio Chiti, Francesco Bemporad, Michele Vendruscolo, Simone Shang-te, Danny Hsu, Christopher M. DobsonAbstract:Abstract Human muscle Acylphosphatase (mAcP) is an enzyme with a ferrodoxin-like topology whose primary role is to hydrolyze the carboxyl-phosphate bonds of acylphosphates. The protein has been widely used as a model system for elucidating the molecular determinants of protein folding and misfolding. We present here the full NMR assignments of the backbone and side chains reso-nances of mAcP complexed with phosphate, thus providing an important resource for future solution-state NMR spectroscopic studies of the structure and dynamics of this protein in the contexts of protein folding and misfolding
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native like aggregation of the Acylphosphatase from sulfolobus solfataricus and its biological implications
FEBS Letters, 2009Co-Authors: Francesco Bemporad, Fabrizio ChitiAbstract:Studies in vitro show that globular proteins can experience the formation of native-like conformational states able to self-assemble with no need of transitions across the energy barrier for unfolding, and that such processes can lead eventually to the formation of amyloid-like species. Circumstantial evidence collected in vivo suggests that aggregation of native-like states can be a concrete possibility for living organisms and thus more relevant than previously thought. In this review we summarize the key observations collected on the “native-like aggregation” of the Acylphosphatase from Sulfolobus solfataricus, a protein that has allowed the direct monitoring and analysis of native-like aggregates for its propensity to form rapidly native-like aggregates and their slow conversion into amyloid-like aggregates.
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amyloid fibril formation can proceed from different conformations of a partially unfolded protein
Biophysical Journal, 2005Co-Authors: Martino Calamai, Fabrizio Chiti, Christopher M. DobsonAbstract: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 β-sheet content from partially unfolded states with very different structural features. Moreover, the presence of α-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.
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structure conformational stability and enzymatic properties of Acylphosphatase from the hyperthermophile sulfolobus solfataricus
Proteins, 2005Co-Authors: Alessandra Corazza, Francesco Bemporad, Cristina Capanni, Massimo Stefani, Georgia Plakoutsi, C. Rosano, Katiuscia Pagano, Vera Alverdi, Gennaro Esposito, Fabrizio ChitiAbstract:The structure of AcP from the hyperthermophilic archaeon Sulfolobus solfataricus has been determined by (1)H-NMR spectroscopy and X-ray crystallography. Solution and crystal structures (1.27 A resolution, R-factor 13.7%) were obtained on the full-length protein and on an N-truncated form lacking the first 12 residues, respectively. The overall Sso AcP fold, starting at residue 13, displays the same betaalphabetabetaalphabeta topology previously described for other members of the AcP family from mesophilic sources. The unstructured N-terminal tail may be crucial for the unusual aggregation mechanism of Sso AcP previously reported. Sso AcP catalytic activity is reduced at room temperature but rises at its working temperature to values comparable to those displayed by its mesophilic counterparts at 25-37 degrees C. Such a reduced activity can result from protein rigidity and from the active site stiffening due the presence of a salt bridge between the C-terminal carboxylate and the active site arginine. Sso AcP is characterized by a melting temperature, Tm, of 100.8 degrees C and an unfolding free energy, DeltaG(U-F)H2O, at 28 degrees C and 81 degrees C of 48.7 and 20.6 kJ mol(-1), respectively. The kinetic and structural data indicate that mesophilic and hyperthermophilic AcP's display similar enzymatic activities and conformational stabilities at their working conditions. Structural analysis of the factor responsible for Sso AcP thermostability with respect to mesophilic AcP's revealed the importance of a ion pair network stabilizing particularly the beta-sheet and the loop connecting the fourth and fifth strands, together with increased density packing, loop shortening and a higher alpha-helical propensity.
Ramponi Giampietro - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a novel Drosophila melanogaster Acylphosphatase
Federation of European Biochemical Societies. Published by Elsevier B.V., 2003Co-Authors: Degl’innocenti Donatella, Raugei Giovanni, Marzocchini Riccardo, Chiti Fabrizio, Ramazzotti Matteo, Ramponi GiampietroAbstract:AbstractAnalysis of the Drosophila melanogaster EST database led to the characterization of a novel Acylphosphatase (AcPDro2). This is coded by the CG18505 (Acyp2) gene and is clearly distinct from a previously described AcPDro coded by the CG16870 (Acyp) gene from D. melanogaster. The two proteins show a 60% homology with both vertebrate isoenzymes. All the residues involved in the catalytic mechanism are conserved. AcPDro2 is a stable enzyme with a correct globular folded structure. Its activity on benzoylphosphate shows higher Kcat but lower Km with respect to AcPDro. It is possible that AcPDro and AcPDro2 genes are not the direct ancestor of MT and CT vertebrate isoenzymes
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The inhibitory effect of the 5′ untranslated region of muscle Acylphosphatase mRNA on protein expression is relieved during cell differentiation
Federation of European Biochemical Societies. Published by Elsevier B.V., 2000Co-Authors: Fiaschi Tania, Raugei Giovanni, Chiarugi Paola, Veggi Daniele, Ramponi GiampietroAbstract:AbstractPrevious experiments suggested that the upstream AUG triplet present in the 5′ untranslated region (UTR) of muscle Acylphosphatase mRNA is involved in the regulation of protein expression. In this paper, we study the involvement of the 5′UTR secondary structure and upstream peptide on mRNA stability and protein translation. Our data, obtained using deletion and frame-shift mutants, demonstrate that the 5′UTR controls protein expression regulating translation together with mRNA stability. Furthermore, we demonstrate that the inhibitory effect of the 5′UTR of muscle Acylphosphatase is relieved during the differentiation process in agreement with previous data reporting an increase of Acylphosphatase content during cell differentiation. Finally, UV cross-linking experiments show that specific mRNA-binding proteins are associated with the 5′UTR of the muscle Acylphosphatase mRNA
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Designing conditions for in vitro formation of amyloid protofilaments and fibrils
The National Academy of Sciences, 1999Co-Authors: Chiti Fabrizio, Taddei Niccolò, Ramponi Giampietro, Stefani Massimo, Webster Paul, Clark Anne, Dobson, Christopher M.Abstract:We have been able to convert a small α/β protein, Acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30–50 Å in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive β-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavine T and cause a red-shift of the Congo red absorption spectrum. All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions
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Drosophila melanogaster Acylphosphatase: A common ancestor for Acylphosphatase isoenzymes of vertebrate species
Published by Elsevier B.V., 1998Co-Authors: Pieri Alessandro, Magherini Francesca, Liguri Gianfranco, Raugei Giovanni, Taddei Niccolò, Bozzetti, Maria Pia, Cecchi Cristina, Ramponi GiampietroAbstract:AbstractAn open reading frame encoding a putative Acylphosphatase was found in Drosophila melanogaster. The corresponding gene product shows 40% identity and 22 additional amino acid residues at the C-terminus as compared to muscle- and common-type human Acylphosphatases. Moreover, all the residues involved in the catalytic mechanism of vertebrate enzymes are conserved in the D. melanogaster Acylphosphatase. The D. melanogaster protein and a deletion mutant, similar in length to vertebrate Acylphosphatases, were produced by cloning the corresponding cDNA in Escherichia coli. The wild-type enzyme is a protein with a well-established three-dimensional fold and a markedly reduced conformational stability as compared to vertebrate isoenzymes. The specific activity of the enzyme is significantly lower than that found in vertebrate enzymes though the substrate binding capability is basically unaltered. The deletion of 22 residues does not cause a significant change in kcat, while affecting the apparent binding parameters. This work suggests that the genes encoding the vertebrate enzymes originate from an ancestor gene by duplication and subsequent evolution
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The 5′-untranslated region of the human muscle Acylphosphatase mRNA has an inhibitory effect on protein expression
Federation of European Biochemical Societies. Published by Elsevier B.V., 1997Co-Authors: Fiaschi Tania, Raugei Giovanni, Chiarugi Paola, Veggi Daniele, Marzocchini Riccardo, Ramponi GiampietroAbstract:AbstractThe cDNA of the human muscle type Acylphosphatase was isolated and characterized. The mRNA presents a very long 5′-untranslated region, covering the first half of the molecule: 175 bases of this part were cloned and prediction of the possible secondary structure showed that a very stable stem-loop structure could be formed in that region. Moreover, an additional AUG triplet was found upstream of the start codon of the protein, defining an open reading frame of 60 codons which overlapped that of Acylphosphatase. The possible regulatory effect on translation of this part of the mRNA molecule was studied by means of transient transfection experiments: a 10-fold decrease in the expression of a reporter protein and a dramatic decrease in the corresponding mRNA was observed, due to the presence of the 5′-untranslated region of Acylphosphatase mRNA. Mutagenesis of the upstream AUG triplet eliminated mRNA instability, leading to the hypothesis that the product of the upstream open reading frame could play a role in this mechanism
Niccolo Taddei - One of the best experts on this subject based on the ideXlab platform.
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Glycine residues appear to be evolutionarily conserved for their ability to inhibit aggregation.
Structure (London England : 1993), 2005Co-Authors: Claudia Parrini, Matteo Ramazzotti, Niccolo Taddei, Giampietro Ramponi, Christopher M. Dobson, Donatella Degl'innocenti, Fabrizio ChitiAbstract:Six glycine residues of human muscle Acylphosphatase (AcP) are evolutionarily conserved across the three domains of life. We have generated six variants of AcP, each having a glycine substituted by an alanine (G15A, G19A, G37A, G45A, G53A, and G69A). Three additional variants had Gly45 replaced by serine, glutamate, and arginine, respectively. The mutational variants do not, on average, have a lower conformational stability than other variants with substitutions of nonconserved residues. In addition, only the G15A variant is enzymatically inactive. However, all variants, with the exception of the G15A mutant, form amyloid aggregates more rapidly than the wild-type. Dynamic light-scattering experiments carried out under conditions close to physiological confirm that aggregate formation is generally more pronounced for the glycine-substituted variants. Apart from the glycine at position 15, all other conserved glycine residues in this protein could have been maintained during evolution because of their ability to inhibit aggregation.
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aggregation of the Acylphosphatase from sulfolobus solfataricus the folded and partially unfolded states can both be precursors for amyloid formation
Journal of Biological Chemistry, 2004Co-Authors: Georgia Plakoutsi, Massimo Stefani, Niccolo Taddei, Fabrizio ChitiAbstract: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.
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kinetic partitioning of protein folding and aggregation
Nature Structural & Molecular Biology, 2002Co-Authors: Fabrizio Chiti, Niccolo Taddei, Giampietro Ramponi, Christopher M. Dobson, Cristina Capanni, Massimo Stefani, Fabiana BaroniAbstract:We have systematically studied the effects of 40 single point mutations on the conversion of the denatured form of the α/β protein Acylphosphatase (AcP) into insoluble aggregates. All the mutations that significantly perturb the rate of aggregation are located in two regions of the protein sequence, residues 16–31 and 87–98, each of which has a relatively high hydrophobicity and propensity to form β-sheet structure. The measured changes in aggregation rate upon mutation correlate with changes in the hydrophobicity and β-sheet propensity of the regions of the protein in which the mutations are located. The two regions of the protein sequence that determine the aggregation rate are distinct from those parts of the sequence that determine the rate of protein folding. Dissection of the protein into six peptides corresponding to different regions of the sequence indicates that the kinetic partitioning between aggregation and folding can be attributed to the intrinsic conformational preferences of the denatured polypeptide chain.
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stabilisation of alpha helices by site directed mutagenesis reveals the importance of secondary structure in the transition state for Acylphosphatase folding
Journal of Molecular Biology, 2000Co-Authors: Niccolo Taddei, Fabrizio Chiti, Monica Bucciantini, Christopher M. Dobson, Tania Fiaschi, Cristina Capanni, Massimo Stefani, Luis Serrano, Giampietro RamponiAbstract:Abstract 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 α-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 α-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 α-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 α-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.
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mutational analysis of the propensity for amyloid formation by a globular protein
The EMBO Journal, 2000Co-Authors: Fabrizio Chiti, Niccolo Taddei, Monica Bucciantini, Giampietro Ramponi, Paul White, Christopher M. DobsonAbstract:Acylphosphatase can be converted in vitro, by addition of trifluoroethanol (TFE), into amyloid fibrils of the type observed in a range of human diseases. The propensity to form fibrils has been investigated for a series of mutants of Acylphosphatase by monitoring the range of TFE concentrations that result in aggregation. We have found that the tendency to aggregate correlates inversely with the conformational stability of the native state of the protein in the different mutants. In accord with this, the most strongly destabilized Acylphosphatase variant forms amyloid fibrils in aqueous solution in the absence of TFE. These results show that the aggregation process that leads to amyloid deposition takes place from an ensemble of denatured conformations under conditions in which non-covalent interactions are still favoured. These results support the hypothesis that the stability of the native state of globular proteins is a major factor preventing the in vivo conversion of natural proteins into amyloid fibrils under non-pathological conditions. They also suggest that stabilizing the native states of amyloidogenic proteins could aid prevention of amyloidotic diseases.
Giampietro Ramponi - One of the best experts on this subject based on the ideXlab platform.
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Glycine residues appear to be evolutionarily conserved for their ability to inhibit aggregation.
Structure (London England : 1993), 2005Co-Authors: Claudia Parrini, Matteo Ramazzotti, Niccolo Taddei, Giampietro Ramponi, Christopher M. Dobson, Donatella Degl'innocenti, Fabrizio ChitiAbstract:Six glycine residues of human muscle Acylphosphatase (AcP) are evolutionarily conserved across the three domains of life. We have generated six variants of AcP, each having a glycine substituted by an alanine (G15A, G19A, G37A, G45A, G53A, and G69A). Three additional variants had Gly45 replaced by serine, glutamate, and arginine, respectively. The mutational variants do not, on average, have a lower conformational stability than other variants with substitutions of nonconserved residues. In addition, only the G15A variant is enzymatically inactive. However, all variants, with the exception of the G15A mutant, form amyloid aggregates more rapidly than the wild-type. Dynamic light-scattering experiments carried out under conditions close to physiological confirm that aggregate formation is generally more pronounced for the glycine-substituted variants. Apart from the glycine at position 15, all other conserved glycine residues in this protein could have been maintained during evolution because of their ability to inhibit aggregation.
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A Nucleophilic Catalysis Step is Involved in the Hydrolysis of Aryl Phosphate Monoesters by Human CT Acylphosphatase
Journal of Biological Chemistry, 2002Co-Authors: Paolo Paoli, L Pazzagli, G Manao, G Camici, Elisa Giannoni, Anna Caselli, Giampietro RamponiAbstract:Abstract Acylphosphatase, one of the smallest enzymes, is expressed in all organisms. It displays hydrolytic activity on acyl phosphates, nucleoside di- and triphosphates, aryl phosphate monoesters, and polynucleotides, with acyl phosphates being the most specific substrates in vitro. The mechanism of catalysis for human Acylphosphatase (the organ-common type isoenzyme) was investigated using both aryl phosphate monoesters and acyl phosphates as substrates. The enzyme is able to catalyze phosphotransfer from p-nitrophenyl phosphate to glycerol (but not from benzoyl phosphate to glycerol), as well as the inorganic phosphate-H2 18O oxygen exchange reaction in the absence of carboxylic acids or phenols. In short, our findings point to two different catalytic pathways for aryl phosphate monoesters and acyl phosphates. In particular, in the aryl phosphate monoester hydrolysis pathway, an enzyme-phosphate covalent intermediate is formed, whereas the hydrolysis of acyl phosphates seems a more simple process in which the Michaelis complex is attacked directly by a water molecule generating the reaction products. The formation of an enzyme-phosphate covalent complex is consistent with the experiments of isotope exchange and transphosphorylation from substrates to glycerol, as well as with the measurements of the Bronsted free energy relationships using a panel of aryl phosphates with different structures. His-25 involvement in the formation of the enzyme-phosphate covalent complex during the hydrolysis of aryl phosphate monoesters finds significant confirmation in experiments performed with the H25Q mutated enzyme.
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kinetic partitioning of protein folding and aggregation
Nature Structural & Molecular Biology, 2002Co-Authors: Fabrizio Chiti, Niccolo Taddei, Giampietro Ramponi, Christopher M. Dobson, Cristina Capanni, Massimo Stefani, Fabiana BaroniAbstract:We have systematically studied the effects of 40 single point mutations on the conversion of the denatured form of the α/β protein Acylphosphatase (AcP) into insoluble aggregates. All the mutations that significantly perturb the rate of aggregation are located in two regions of the protein sequence, residues 16–31 and 87–98, each of which has a relatively high hydrophobicity and propensity to form β-sheet structure. The measured changes in aggregation rate upon mutation correlate with changes in the hydrophobicity and β-sheet propensity of the regions of the protein in which the mutations are located. The two regions of the protein sequence that determine the aggregation rate are distinct from those parts of the sequence that determine the rate of protein folding. Dissection of the protein into six peptides corresponding to different regions of the sequence indicates that the kinetic partitioning between aggregation and folding can be attributed to the intrinsic conformational preferences of the denatured polypeptide chain.
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stabilisation of alpha helices by site directed mutagenesis reveals the importance of secondary structure in the transition state for Acylphosphatase folding
Journal of Molecular Biology, 2000Co-Authors: Niccolo Taddei, Fabrizio Chiti, Monica Bucciantini, Christopher M. Dobson, Tania Fiaschi, Cristina Capanni, Massimo Stefani, Luis Serrano, Giampietro RamponiAbstract:Abstract 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 α-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 α-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 α-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 α-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.
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mutational analysis of the propensity for amyloid formation by a globular protein
The EMBO Journal, 2000Co-Authors: Fabrizio Chiti, Niccolo Taddei, Monica Bucciantini, Giampietro Ramponi, Paul White, Christopher M. DobsonAbstract:Acylphosphatase can be converted in vitro, by addition of trifluoroethanol (TFE), into amyloid fibrils of the type observed in a range of human diseases. The propensity to form fibrils has been investigated for a series of mutants of Acylphosphatase by monitoring the range of TFE concentrations that result in aggregation. We have found that the tendency to aggregate correlates inversely with the conformational stability of the native state of the protein in the different mutants. In accord with this, the most strongly destabilized Acylphosphatase variant forms amyloid fibrils in aqueous solution in the absence of TFE. These results show that the aggregation process that leads to amyloid deposition takes place from an ensemble of denatured conformations under conditions in which non-covalent interactions are still favoured. These results support the hypothesis that the stability of the native state of globular proteins is a major factor preventing the in vivo conversion of natural proteins into amyloid fibrils under non-pathological conditions. They also suggest that stabilizing the native states of amyloidogenic proteins could aid prevention of amyloidotic diseases.