title: Stabilization of an immunoglobulin fold domain by an engineered disulfide bond at the buried hydrophobic region.
abstract: We report for the first time the stabilization of an immunoglobulin fold domain by an engineered disulfide bond. In the llama single-domain antibody, which has human chorionic gonadotropin as its specific antigen, Ala49 and Ile70 are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10 degrees C higher midpoint temperature of thermal unfolding than that without the extra disulfide bond. The modified domains exhibited an antigen binding affinity comparable with that of the wild-type domain. Ala49 and Ile70 are conserved in camel and llama single-domain antibody frameworks. Therefore, domains against different antigens are expected to be stabilized by the engineered disulfide bond examined here. In addition to the effect of the loop constraints in the unfolded state, thermodynamic analysis indicated that internal interaction and hydration also control the stability of domains with disulfide bonds. The change in physical properties resulting from mutation often causes unpredictable and destabilizing effects on these interactions. The introduction of a hydrophobic cystine into the hydrophobic region maintains the hydrophobicity of the protein and is expected to minimize the unfavorable mutational effects.
authors.affiliation: Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Ikeda, Osaka 563-8577, Japan. hagihara-kappael@aist.go.jp
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Hagihara
authors.forename: Shouhei
authors.initials: S
authors.lastname: Mine
authors.forename: Koichi
authors.initials: K
authors.lastname: Uegaki
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000276:immunology; D004220:Disulfides / Q000737:chemistry* / Q000276:immunology; D006358:Hot Temperature; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics
chemical_list: D006063:Chorionic Gonadotropin; D004220:Disulfides; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region