Folding pathway of human alpha 1-antitrypsin: characterization of an intermediate that is active but prone to aggregation.
The folding-unfolding kinetics of human alpha 1-antitrypsin (alpha 1-AT) were examined by monitoring intrinsic Trp fluorescence and extrinsic ANS fluorescence. While the unfolding of alpha 1-AT followed a single exponential phase, refolding exhibited three exponential phases. The fast phase (tau 1r < 40 sec). which was independent of urea concentration, appears to be hydrophobic collapse that may be limited by cis-trans isomerization of prolyl residue. The medium phase (tau 2s = 200 sec) yielded an intermediate (IN), which is capable of elastase binding. The slowest (tau 3r = 1000 sec) phase completes refolding to the native protein, which intersects with the unfolding kinetics at the same urea concentration (1.9 M) as the equilibrium midpoint. Concentration-dependence of the amplitude of major refolding phases indicated that IN is prone to kinetic competition between the on-pathway to native protein and aggregation.
Kim
D
D
Division of Protein Engineering, Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea.
Yu
M H
MH
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D010196:Pancreatic Elastase / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; C027132:1-anilino-8-naphthalenesulfonate; D010196:Pancreatic Elastase