Development of enzymatic activity during protein folding. Detection of a spectroscopically silent native-like intermediate of muscle acylphosphatase.

TitleDevelopment of enzymatic activity during protein folding. Detection of a spectroscopically silent native-like intermediate of muscle acylphosphatase.
Publication TypeJournal Article
Year of Publication1999
AuthorsChiti, F., N. Taddei, E. Giannoni, N. A. J. van Nuland, G. Ramponi, and C. M. Dobson
JournalJ Biol Chem
Volume274
Issue29
Pagination20151-8
Date Published1999 Jul 16
ISSN0021-9258
KeywordsAcid Anhydride Hydrolases, Humans, Magnetic Resonance Spectroscopy, Models, Molecular, Muscles, Mutagenesis, Site-Directed, Proline, Protein Conformation, Protein Denaturation, Protein Folding, Spectrometry, Fluorescence
Abstract

The recovery of enzymatic activity during the folding of muscle acylphosphatase and two single residue mutants (proline 54 to alanine and proline 71 to alanine) from 7 M urea has been monitored and compared with the development of intrinsic fluorescence emission. Fluorescence measurements reveal the presence in the wild-type protein of a major rapid refolding phase followed by a second low amplitude slow phase. The slow phase is absent in the fluorescence trace acquired with the proline 54 to alanine mutant, suggesting the involvement of this proline residue in the fluorescence-detected slow phase of the wild-type protein. The major kinetic phase is associated with a considerable recovery of enzymatic activity, indicating that a large fraction of molecules refolds with effective two-state behavior. The use of time-resolved enzymatic activity as a probe to follow the folding process reveals, however, the presence of another exponential slow phase arising from proline 71. This slow phase is not observable by utilizing optical probes, indicating that, unlike proline 54, the cis to trans isomerization of proline 71 can take place in an intermediate possessing a native-like fold. We suggest that, although spectroscopically silent and structurally insignificant, the cis-trans interconversion of proline residues in native-like intermediates may be crucial for the generation of enzymatic activity of functional enzymes.

Alternate JournalJ. Biol. Chem.
PubMed ID10400629