Manipulating the stability of fibronectin type III domains by protein engineering

We have previously shown that a 'weak' fibronectin type III (fnIII) domain can be engineered to have enhanced mechanical strength by replacing the hydrophobic core with the core of a homologous 'strong' fnIII domain. Here we show that engineering the core is a robust method for manipulating the mechanical strength of this class of proteins. We performed an experiment that is the reverse of one described earlier. The hydrophobic core of a 'weak' domain (FNfn10) was grafted into a 'strong' fnIII domain, TNfn3. This newly engineered protein, TNoFNc, is indeed much less mechanically resistant than TNfn3. Interestingly, TNoFNc is very unstable, approximately 10 kcal mol(-1) less stable than FNfn10, yet its mechanical stability is very similar-a clear reflection of the fact that thermodynamic and mechanical stability are unrelated properties, even where they are both assumed to reflect properties of the hydrophobic core.