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.

Highlights

  • Cystine is hydrophobic, and most of naturally occurring disulfide bonds are buried in the protein (1– 4)

  • In a previous study (12), we screened the amino acid pairs substituting for the disulfide bonds of four different immunoglobulin fold domains by a method based on the cellular quality control system (13, 14)

  • In the three-dimensional structure of llama VHH raised against the ␣-subunit of human chorionic gonadotropin (17), Ala49 and Ile70 are buried in the domain, and the distance between ␤-carbons of these amino acids is within a normal distance of naturally occurring ␤-carbons of cystines

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Summary

Introduction

Most of naturally occurring disulfide bonds are buried in the protein (1– 4). The introduction of a disulfide bond into the buried hydrophobic core of human carbonic anhydrase (A23C/L203C) markedly stabilizes this enzyme; the midpoint temperature of thermal unfolding (Tm) of the mutant is 10 °C higher than that of the wild-type protein (6). We replaced these two amino acids with Cys in the wild-type and mutant domains, substituting the native disulfide bond with the Trp/Ala amino acid pair.

Results
Conclusion

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