Characterization of folding intermediates of human carbonic anhydrase II: probing substructure by chemical labeling of SH groups introduced by site-directed mutagenesis.
By measurement of UV absorbance, CD spectra, and enzyme activity, we have shown that human carbonic anhydrase II forms a stable and compact folding intermediate at a moderate concentration of guanidine hydrochloride. The major aim of this study was to map the intermediate structure. For that reason, site-directed mutagenesis was used to introduce cysteine residues in various parts of the central beta-structure to give in each case a single cysteine residue. Thereafter, the accessibility of the introduced SH group to specific chemical labeling was used to probe the stability and compactness of the area surrounding each cysteine residue. Our results indicate that the folding intermediate has an ordered native-like secondary structure in the central part of the beta-sheet, whereas the peripheral part of the beta-sheet seems to be less ordered. A large hydrophobic cluster situated between the central beta-sheet core and secondary structure elements on the surface appears to be intact in the intermediate and is remarkably stable even at high GuHCl concentrations (> 5 M). This unusually stable substructure might function as a "seed" during the initiation of the folding process.
Mårtensson
L G
LG
Department of Biochemistry, Umeå University, Sweden.
Jonsson
B H
BH
Freskgård
P O
PO
Kihlgren
A
A
Svensson
M
M
Carlsson
U
U
D000478:Alkylation; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013816:Thermodynamics
D006146:Guanidines; D013438:Sulfhydryl Compounds; D002256:Carbonic Anhydrases; D019791:Guanidine; D003545:Cysteine