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- Title
The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies.
- Authors
Yoko Akazawa-Ogawa; Koichi Uegaki; Yoshihisa Hagihara
- Abstract
Camelid-derived single domain VHH antibodies are highly heat resistant, and the mechanism of heat-induced VHH denaturation predominantly relies on the chemical modification of amino acids. Although chemical modification of disulfide bonds has been recognized as a cause for heat-induced denaturation of many proteins, there have been no mutagenesis studies, in which the number of disulfide bonds was controlled. In this article, we examined a series of mutants of two different VHHs with single, double or no disulfide bonds, and scrutinized the effects of these disulfide bond modifications on VHH denaturation. With the exception of one mutant, the heat resistance of VHHs decreased when the number of disulfide bonds increased. The effect of disulfide bonds on heat denaturation was more striking if the VHH had a second disulfide bond, suggesting that the contribution of disulfide shuffling is significant in proteins with multiple disulfide bonds. Furthermore, our results directly indicate that removal of a disulfide bond can indeed increase the heat resistance of a protein, irrespective of the negative impact on equilibrium thermodynamic stability.
- Subjects
IMMUNOTECHNOLOGY; DISULFIDES; CHEMICAL modification of proteins; PROTEIN stability; PROTEIN engineering
- Publication
Journal of Biochemistry, 2016, Vol 159, Issue 1, p111
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/jb/mvv082