Folding and stability of the N-terminus of human carbonic anhydrase II.
Truncations and mutations in the N-terminus of human carbonic anhydrase II were constructed in order to establish what role this part of the protein plays in the folding and stability of the protein. When incubated in various concentrations of guanidine hydrochloride (GuHCl), HCAII unfolds in two transitions, with an intermediate state at about 1.3 M GuHCl. N-Terminal truncations of 5, 17, or 24 amino acid residues destabilize the native state by 4-5 kcal/mol, relative to the intermediate state, but these amino acid residues have virtually no effect on the stability of the intermediate state relative to the unfolded state. These truncated variants of HCAII still have a high enzymatic activity. Deletion of 28 or more amino acid residues, however, results in inactive enzyme variants. The rates at which the active site is formed are practically unaffected by the removal of the 24-amino acid segment, i.e., the active site forms independently of the N-terminus. By using the tryptophans in positions 5 and 16 as intrinsic probes, we conclude that the structure of the N-terminal region is formed very late in folding. The results strongly indicate that this process is dependent on the prior formation of an enzymatically active native-like structure of the rest of the protein.
Department of Biochemistry, Umeå University, Sweden.
G
G
Aronsson
L G
LG
Mårtensson
U
U
Carlsson
B H
BH
Jonsson
D000595:Amino Acid Sequence; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry
D011994:Recombinant Proteins; D014364:Tryptophan; D002256:Carbonic Anhydrases