title: The folding process of acylphosphatase from Escherichia coli is remarkably accelerated by the presence of a disulfide bond.
abstract: 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.
authors.affiliation: Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Viale Morgagni 50, 50134 Firenze, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Parrini
authors.forename: Francesco
authors.initials: F
authors.lastname: Bemporad
authors.forename: Alessio
authors.initials: A
authors.lastname: Baroncelli
authors.forename: Stefano
authors.initials: S
authors.lastname: Gianni
authors.forename: Carlo
authors.initials: C
authors.lastname: Travaglini-Allocatelli
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Kohn
authors.forename: Matteo
authors.initials: M
authors.lastname: Ramazzotti
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Chiti
authors.forename: Niccolò
authors.initials: N
authors.lastname: Taddei
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D000097024:Acylphosphatase
chemical_list: D017766:Acid Anhydride Hydrolases; D003545:Cysteine; D004220:Disulfides; D000097024:Acylphosphatase