Initial denaturing conditions influence the slow folding phase of acylphosphatase associated with proline isomerization.
The folding kinetics of human common-type acylphosphatase (cAcP) from its urea- and TFE-denatured states have been determined by stopped-flow fluorescence techniques. The refolding reaction from the highly unfolded state formed in urea is characterized by double exponential behavior that includes a slow phase associated with isomerism of the Gly53-Pro54 peptide bond. However, this slow phase is absent when refolding is initiated by dilution of the highly a-helical denatured state formed in the presence of 40% trifluoroethanol (TFE). NMR studies of a peptide fragment corresponding to residues Gly53-Gly69 of cAcP indicate that only the native-like trans isomer of the Gly-Pro peptide bond is significantly populated in the presence of TFE, whereas both the cis and trans isomers are found in an approximately 1:9 ratio for the peptide bond in aqueous solution. Molecular modeling studies in conjunction with NMR experiments suggest that the trans isomer of the Gly53-Pro54 peptide bond is stabilized in TFE by the formation of a nonnative-like hydrogen bond between the CO group of Gly53 and the NH group of Lys57. These results therefore reveal that a specific nonnative interaction in the denatured state can increase significantly the overall efficiency of refolding.
Pertinhez
T A
TA
Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford, United Kingdom.
Hamada
D
D
Smith
L J
LJ
Chiti
F
F
Taddei
N
N
Stefani
M
M
Dobson
C M
CM
D017766:Acid Anhydride Hydrolases / Q000737:chemistry*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000737:chemistry; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D008956:Models, Chemical; D009132:Muscles / Q000201:enzymology; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000378:metabolism; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014270:Trifluoroethanol / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D005998:Glycine; D010446:Peptide Fragments; D011392:Proline; D014270:Trifluoroethanol; D014508:Urea; D000097024:Acylphosphatase