Manipulating the stability of fibronectin type III domains by protein engineering

Abstract

We have previously shown that a ‘weak’ fibronectin type III (fnIII) domain can beengineered to have enhanced mechanical strength by replacing the hydrophobic core withthe core of a homologous ‘strong’ fnIII domain. Here we show that engineering the core is arobust method for manipulating the mechanical strength of this class of proteins.We performed an experiment that is the reverse of one described earlier. Thehydrophobic core of a ‘weak’ domain (FNfn10) was grafted into a ‘strong’ fnIII domain,TNfn3. This newly engineered protein, TNoFNc, is indeed much less mechanicallyresistant than TNfn3. Interestingly, TNoFNc is very unstable, approximately10 kcal mol−1 less stable than FNfn10, yet its mechanical stability is very similar—a clear reflection ofthe fact that thermodynamic and mechanical stability are unrelated properties,even where they are both assumed to reflect properties of the hydrophobic core.