Contributions of a highly conserved VH/VL hydrogen bonding interaction to scFv folding stability and refolding efficiency.
The assembly of single-chain Fv (scFv) antibody fragments, consisting of an interconnected variable heavy chain (VH) and variable light chain (VL), is a cooperative process that requires coupled folding and domain association. We report here an initial investigation of VH/VL domain-domain assembly with a site-directed mutagenesis study that probes a highly conserved VH/VL hydrogen bonding interaction. Gln168 of the S5 scFv (Kabat VH 39) is absolutely conserved in 95% of all VH, and Gln44 (Kabat VL 38) is found in 94% of all kappa VL (Glx in 95% of all lambda VL). These side chains form two hydrogen bonds in head-to-tail alignment across the VH/VL interface. Double mutant cycles at Gln168 and Gln44 were constructed to first investigate their contribution to thermodynamic folding stability, second to investigate whether stability can be improved, and third to determine whether refolding efficiencies are affected by mutations at these positions. The results demonstrate that the Gln168-Gln44 interaction is not a key determinant of S5 scFv folding stability, as sequential modification to alanine has no significant effect on the free energy of folding. Several mutations that alter the glutamines to methionine or charged amino acids significantly increase the thermodynamic stability by increasing the m(g) associated with the unfolding isotherm. These effects are hypothesized to arise largely from an increase in the VH/VL association free energy that leads to tighter coupling between domain-domain association and folding. All of the mutants also display a reduced antigen binding affinity. Single and double methionine mutants also displayed significant increases in refolding efficiency of 2.4- to 3-fold over the native scFv, whereas the double alanine/methionine mutants displayed moderate 1.9- to 2.4-fold enhancement. The results suggest that reengineering the VH/VL interface could be useful in improving the stability of single-chain antibodies, as Ala/Met mutations at these conserved positions increase the free energy of folding by 46% while minimally perturbing binding affinity. They also could be useful in improving scFv recovery from inclusion bodies as the mutations increase the refolding efficiency by more than twofold.
Tan
P H
PH
Department of Bioengineering, University of Washington, Seattle 98195, USA.
Sandmaier
B M
BM
Stayton
P S
PS
D000818:Animals; D001483:Base Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004355:Drug Stability; D006860:Hydrogen Bonding; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics
D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region