Sequence Tolerance of a Highly Stable Single Domain Antibody: Comparison of Computational and Experimental Profiles
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The sequence fitness of a single-domain antibody with unusually high thermal stability is explored by a combined computational and experimental study. Starting with the crystallographic structure, RosettaBackrub simulations were applied to model sequence-structure tolerance profiles and identify key substitution sites. Experimental site-directed mutagenesis was used to produce a panel of mutants and their melting temperatures were determined by thermal denaturation. The results reveal an excess stability margin of approximately 12 deg.C, a value taken from a decrease in the melting temperature of an electrostatic charge reversal substitution in the CRD3 without a deleterious effect on the binding affinity to the antigen target. Tolerance for disruption of antigen recognition without loss in thermal stability was demonstrated by the introduction of a proline in place of a tyrosine in the CDR2, producing a mutant that eliminated binding. To reconcile the differences between the modeled energies and their relationship to the observed experimental changes in melting temperatures, an approximation was developed by combining a statistical potential with a linearly scaled implicit solvent model to calculate the net contribution from a two-state model of folded and unfolded conformations. The derived computational model improves prediction accuracy and should prove applicable to other designs of antibodies.