The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies.
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.
National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan.
Yoko
Y
Akazawa-Ogawa
National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan k-uegaki@aist.go.jp.
Koichi
K
Uegaki
National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan hagihara-kappael@aist.go.jp.
Yoshihisa
Y
Hagihara
D000595:Amino Acid Sequence; D000818:Animals; D001409:Bacillus cereus / Q000201:enzymology; D001426:Bacterial Proteins / Q000276:immunology; D002161:Camelids, New World; D006063:Chorionic Gonadotropin / Q000276:immunology; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D055550:Protein Stability; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics; D001618:beta-Lactamases / Q000276:immunology
D001426:Bacterial Proteins; D006063:Chorionic Gonadotropin; D061905:Single-Domain Antibodies; D001618:beta-Lactamases
antibody engineering; disulfide bonds; protein chemical modification; protein engineering; protein stability