Folding around the C‐terminus of human carbonic anhydrase II Kinetic characterization by use of a chemically reactive SH‐group introduced by protein engineering

FEBS Letters

Freskgård, Per-Ola¹  Mårtensson, Lars-Göran²  Carlsson, Uno¹  Jonsson, Bengt-Harald² 

[1] IFM-Department of Chemistry, Linköping University, S-581 83 Linköping, Sweden;Department of Biochemistry, Umeå University, S-901 87 Umeå, Sweden

关键词: Protein folding;    Site-directed mutagenesis;    Side chain accessibility;    Carbonic anhydrase;    Protein engineering;    HCAII;    human carbonic anhydrase II;   

DOI  :  10.1016/0014-5793(91)80922-P

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We are characterizing the process of refolding of the enzyme human carbonic anhydrase II from the denatured state in guanidine hydrochloride. To describe the folding in defined parts of the protein we use protein engineering to introduce cysteine residues as unique chemically reactive probes. The accessibility of the cysteine SH-group to the alkylating reagent iodoacetate, at different stages during refolding, is used to give a kinetic description of the folding process. The structuration of the C-terminal part of the polypeptide chain, which is involved in a unique ‘knot’ topology, was investigated. Our results show that the structure around the C-terminal, composed of the outermost β-strands in a dominating β-structure that extends through the entire protein, is formed relatively late during refolding. In contrast, it was found that β-strands located in the interior of the protein were structured very rapidly. The final native structure is formed in a process that is slower than those observed for formation of β-structure.

【 授权许可】

Unknown