Edge strand engineering prevents native‐like aggregation in <i><scp>S</scp>ulfolobus solfataricus</i> acylphosphatase

Rosa, Matteo de; Bemporad, Francesco; Pellegrino, Sara; Chiti, Fabrizio; Bolognesi, Martino; Ricagno, Stéfano

doi:10.1111/febs.12861

Cited by 14 publications

(19 citation statements)

References 48 publications

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“…All three mutations are located on the protein surface, and two of them are also naturally occurring in vertebrates other than human; hence they should cause little perturbation to the protein structure and stability. It is noteworthy that the effects of surface mutations introducing or removing surface charges, do not correlate simply with aggregation propensity: the V84D and Y86E mutations in acylphosphatase from S. solfataricus do not protect the protein from unfolding and aggregation 37 . Furthermore, a D to N systematic scanning in β 2m indicated that the removal of a negative charge triggers measurable effects in aggregation propensity only when the D to N mutation occurs at a specific site 38 .…”

Section: Resultsmentioning

confidence: 99%

Rational design of mutations that change the aggregation rate of a protein while maintaining its native structure and stability

Camilloni

Sala

Sormanni

et al. 2016

Sci Rep

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A wide range of human diseases is associated with mutations that, destabilizing proteins native state, promote their aggregation. However, the mechanisms leading from folded to aggregated states are still incompletely understood. To investigate these mechanisms, we used a combination of NMR spectroscopy and molecular dynamics simulations to compare the native state dynamics of Beta-2 microglobulin (β2m), whose aggregation is associated with dialysis-related amyloidosis, and its aggregation-resistant mutant W60G. Our results indicate that W60G low aggregation propensity can be explained, beyond its higher stability, by an increased average protection of the aggregation-prone residues at its surface. To validate these findings, we designed β2m variants that alter the aggregation-prone exposed surface of wild-type and W60G β2m modifying their aggregation propensity. These results allowed us to pinpoint the role of dynamics in β2m aggregation and to provide a new strategy to tune protein aggregation by modulating the exposure of aggregation-prone residues.

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Section: Resultsmentioning

confidence: 99%

Rational design of mutations that change the aggregation rate of a protein while maintaining its native structure and stability

Camilloni

Sala

Sormanni

et al. 2016

Sci Rep

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“…The elegant crystal structure of hexameric β2m reveals many interesting intermolecular interactions, however such hexameric form does not aggregate under standard conditions [ 29 ], leaving open the question on which are the interactions that set the hexamer off-pathway. Specific mutations may structurally protect the protein from the aggregation pathway undertaken by the wt protein, but in parallel open new paths to amyloid formation, as recently exemplified by protective mutations on Acylphosphatase from Sulfolobus solfataricus [ 30 , 31 ].…”

Section: Discussionmentioning

confidence: 99%

Wild Type Beta-2 Microglobulin and DE Loop Mutants Display a Common Fibrillar Architecture

et al. 2015

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Beta-2 microglobulin (β2m) is the protein responsible for a pathologic condition known as dialysis related amyloidosis. In recent years an important role has been assigned to the peptide loop linking strands D and E (DE loop) in determining β2m stability and amyloid propensity. Several mutants of the DE loop have been studied, showing a good correlation between DE loop geometrical strain, protein stability and aggregation propensity. However, it remains unclear whether the aggregates formed by wild type (wt) β2m and by the DE loop variants are of the same kind, or whether the mutations open new aggregation pathways. In order to address this question, fibrillar samples of wt and mutated β2m variants have been analysed by means of atomic force microscopy and infrared spectroscopy. The data here reported indicate that the DE loop mutants form aggregates with morphology and structural organisation very similar to the wt protein. Therefore, the main effect of β2m DE loop mutations is proposed to stem from the different stabilities of the native fold. Considerations on the structural role of the DE loop in the free monomeric β2m and as part of the Major Histocompatibility Complex are also presented.

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“…The unfolded N-terminus and destabilization of the folded unit promote native-like aggregation in vivo A crucial role in the aggregation process of Sso AcP is played in vitro by the N-terminus and b-strand 4, as shown by the fact that the establishment of an intermolecular interaction between this segment and the edge b-strand 4 initiates the process (21,(26)(27)(28)(29)(30). A similarly important role for the N-terminal segment is also observed in vivo by the reduced rate of incorporation into IBs of the mutated variants lacking the N-terminal segments.…”

Section: Native-like Aggregation Of Sso Acp Can Occur In Vivomentioning

confidence: 99%

“…The b-strand 4 of Sso AcP appears to be protected only poorly against edge-to-edge interactions compared to other acylphosphatases (29). In fact, three single-point mutants of Sso AcP in which single-residue mutations introduce additional protection of b-strand 4 have been found to resist native-like aggregation but to induce self-assembly through a different mechanism (29,30).…”

Section: Introductionmentioning

confidence: 99%

“…Although the process of native-like aggregation is well characterized in vitro for Sso AcP (21,22,(24)(25)(26)(27)(28)(29)(30) and for several other proteins (11,15,20), similar processes in vivo have not yet been described. In this study, we addressed this issue by expressing in Escherichia coli wild-type (WT) Sso AcP and three of its mutants, which are devoid of the N-terminal segment, contain a destabilizing mutation in the globular unit, or display both modifications.…”

Section: Introductionmentioning

confidence: 99%

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Direct Conversion of an Enzyme from Native-like to Amyloid-like Aggregates within Inclusion Bodies

Elia

Cantini

Chiti

et al. 2017

Biophysical Journal

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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 b-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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Edge strand engineering prevents native‐like aggregation in Sulfolobus solfataricus acylphosphatase

Cited by 14 publications

References 48 publications

Rational design of mutations that change the aggregation rate of a protein while maintaining its native structure and stability

Rational design of mutations that change the aggregation rate of a protein while maintaining its native structure and stability

Wild Type Beta-2 Microglobulin and DE Loop Mutants Display a Common Fibrillar Architecture

Direct Conversion of an Enzyme from Native-like to Amyloid-like Aggregates within Inclusion Bodies

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