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, Ala 49 and Ile 70 are buried in the structure. A mutant with an artificial disulfide bond at this position showed a 10°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. Ala 49 and Ile 70 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.One of the major objectives of antibody engineering is to stabilize the three-dimensional structure of the antibody. Disulfide bonds often significantly stabilize the structure of native proteins. Thus, the introduction of artificial disulfide bonds is recognized as a useful protein engineering technique to increase conformational stability. Although this technique has been applied to many proteins, there are no reports of engineered disulfide bonds in an immunoglobulin fold framework.Cystine is hydrophobic, and thus, most of naturally occurring disulfide bonds are buried in the protein (1-4). Therefore, the introduction of an engineered disulfide bond into the hydrophobic core better maintains the biophysical properties of the target protein. There are several examples of artificial disulfide bonds that can replace a pair of buried hydrophobic residues, the accessible surface areas of which were Ͻ20% (5-11). 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 (T m ) of the mutant is 10°C higher than that of the wild-type protein (6). The engineered disulfide bonds in alkaline protease AprP (G199C/F236C) (11), xylanase (V98C/ A152C) (7), and manganese peroxidase (A48C/A63C) (10) mutants increase their tolerance against heat inactivation. On the other hand, subtilisin BPNЈ mutants (V26C/A232C and A29C/M119C) exhibit similar or slightly lower stability to irreversible thermal inactivation (8). Tolerance against heat denaturation is not directly correlated with conformational stability. Only the mutational effect on human carbonic anhydrase II (6) was examined in a reversible system. Little information is available about the thermodynamic effe...

The role of intra-domain disulfide bonds in heat-induced irreversible denaturation of camelid single domain VHH antibodies

“…6 and Table I). The removal of the native disulfide bond from wild-type anti-hCG and anti-bLA VHHs significantly reduced T m (À15 to À25 C) (9,25). The changes in t 1/2 90 C of mutants lacking disulfide bonds were dependent on the amino acid pair used to replace the disulfide bond, and ranged from À9 to 27% (À15 to 59 min) as compared with wild-type VHH.…”

“…Anti-hCG VHH with an additional disulfide bond at the positions of Ala49 and Ile69 in the Kabat numbering scheme (2-SS anti-hCG VHH) exhibited a T m of 74 C, which is 10 C higher than that of wild-type anti-hCG VHH (9). The same artificial disulfide bond was introduced on the 0-SS (WA) anti-hCG VHH sequence, resulting in 1-SS (WA) anti-hCG VHH (9). We also prepared antibLA VHH mutants lacking disulfide bonds, by replacing the Cys residues at positions 22 and 92 by Ala and Ala (0-SS (AA) anti-bLA VHH), or Ala and Val (0-SS (AV) anti-bLA VHH).…”

“…The resulting mutants were cloned into pAED4 (26). VHHs were produced according to previously described methods (7,9,25). In brief, anti-hCG VHH mutants with double disulfide bonds were expressed in Pichia pastoris in a secreted form and purified by SP-Sepharose FF and Superdex 75 (GE Healthcare, Little Chalfont, UK).…”

“…Construction of the expression vectors of wild-type and disulfide mutants of anti-hCG VHHs and wild-type anti-bLA VHH were previously described (7,9,25). Cys residues at positions 22 and 92 in anti-bLA VHH were replaced by Ala and Ala, or by Ala and Val through PCR-based site-directed mutagenesis.…”

Camelid single-domain antibody fragments: Uses and prospects to investigate protein misfolding and aggregation, and to treat diseases associated with these phenomena

“…The same inhibition was observed for cAb-HuL6 and the I56T variant [82], and for cAb-HuL22 and both the I56T and D67H variants [44]. On the other hand, these yellow peaks are observed in the mass spectra of the D67H variant in the presence of cAb-HuL5; the binding of cAb-HuL5 does therefore not inhibit the was added between the two b-sheets thanks to the S54C and I78C mutations (IMGT numbering) [70,71]. The variant obtained (named cAb-HuL22-S54C/I78C) is significantly more thermostable (i.e.…”