Studying the folding process of the acylphosphatase from Sulfolobus solfataricus. A comparative analysis with other proteins from the same superfamily.
The folding process of the acylphosphatase from Sulfolobus solfataricus (Sso AcP) has been followed, starting from the fully unfolded state, using a variety of spectroscopic probes, including intrinsic fluorescence, circular dichroism, and ANS binding. The results indicate that an ensemble of partially folded or misfolded species form rapidly on the submillisecond time scale after initiation of folding. This conformational ensemble produces a pronounced downward curvature in the Chevron plot, appears to possess a content of secondary structure similar to that of the native state, as revealed by far-UV circular dichroism, and appears to have surface-exposed hydrophobic clusters, as indicated by the ability of this ensemble to bind to 8-anilino-1-naphthalenesulfonic acid (ANS). Sso AcP folds from this conformational state with a rate constant of ca. 5 s(-1) at pH 5.5 and 37 degrees C. A minor slow exponential phase detected during folding (rate constant of 0.2 s(-1) under these conditions) is accelerated by cyclophilin A and is absent in a mutant of Sso AcP in which alanine replaces the proline residue at position 50. This indicates that for a lower fraction of Sso AcP molecules the folding process is rate-limited by the cis-trans isomerism of the peptide bond preceding Pro50. A comparative analysis with four other homologous proteins from the acylphosphatase superfamily shows that sequence hydrophobicity is an important determinant of the conformational stability of partially folded states that may accumulate during folding of a protein. A low net charge and a high propensity to form alpha-helical structure also emerge as possibly important determinants of the stability of partially folded states. A significant correlation is also observed between folding rate and hydrophobic content of the sequence within this superfamily, lending support to the idea that sequence hydrophobicity, in addition to relative contact order and conformational stability of the native state, is a key determinant of folding rate.
Bemporad
Francesco
F
Dipartimento di Scienze Biochimiche, Università di Firenze, Viale Morgagni 50, 50134 Firenze, Italy.
Capanni
Cristina
C
Calamai
Martino
M
Tutino
Maria Luisa
ML
Stefani
Massimo
M
Chiti
Fabrizio
F
D017766:Acid Anhydride Hydrolases / Q000737:chemistry*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D021984:Cyclophilin A / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013057:Spectrum Analysis; D016944:Sulfolobus / Q000201:enzymology*; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D021984:Cyclophilin A; D000097024:Acylphosphatase