Cellular quality control screening to identify amino acid pairs for substituting the disulfide bonds in immunoglobulin fold domains.
We are interested in determining which amino acid pairs can be substituted for the disulfide (S-S) bonds in proteins without disrupting their native structures under physiological conditions. In this study, we focused on the intradomain S-S bonds in Ig fold domains and aimed to determine a simple rule for replacement of their S-S bonds. The cysteines of four different Ig fold domains were mutated randomly, and the amino acid pairs substituted for the S-S bonds were screened by the method utilizing a cellular quality control system. Among the 36 selected mutants, 31 were natively folded without S-S bonds, as judged from the cooperativity of thermal unfolding. In addition, the selected mutant llama heavy chain antibodies retained antigen-binding affinity. At least two of the pairs Ala:Ala, Ala:Val, Val: Ala, and Val:Val were found in the selected mutants for all four different Ig fold domains, and they were stably folded at 30 degrees C. This suggests that examination of these four pairs could be enough to obtain natively folded Ig fold domains without S-S bonds.
Hagihara
Yoshihisa
Y
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan. hagihara-kappael@aist.go.jp
Matsuda
Tomoki
T
Yumoto
Noboru
N
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000941:Antigens / Q000737:chemistry; D055592:Biophysical Phenomena; D001703:Biophysics; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D015723:Gene Library; D006358:Hot Temperature; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry* / Q000235:genetics; D015233:Models, Statistical; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011786:Quality Control; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013053:Spectrophotometry; D013696:Temperature
D000596:Amino Acids; D000941:Antigens; D004220:Disulfides; D007136:Immunoglobulins; D003545:Cysteine