Do Proteins Always Benefit from a Stability Increase? Relevant and Residual Stabilisation in a Three-state Protein by Charge Optimisation

摘要: The vast majority of our knowledge on protein stability arises from the study simple two-state models. However, proteins displaying equilibrium intermediates under certain conditions abound and it is unclear whether energetics native/intermediate equilibria well represented in current knowledge. We consider here that overall conformational three-state made a "relevant" term "residual" one, corresponding to free energy differences native intermediate (N-to-I) denatured (I-to-D) equilibria, respectively. N-to-I difference considered be relevant because protein-unfolding are likely devoid biological activity. use surface charge optimisation first increase (N-to-D) model (apoflavodoxin) then investigate stabilisation obtained realised into or residual stability. Most mutations designed electrostatic calculations sequence conservation analysis produce large increases protein. most cases, this simply leads similarly Two mutations, nevertheless, show different trend substantially. When all mapped onto structure apoflavodoxin thermal-unfolding (obtained independently by phi-analysis NMR) they cluster perfectly so increasing appear small unstructured region others native-like region. This illustrates need for specific investigation intermediates, indicates possible rationally stabilise against partial unfolding once conformation known, even if at low resolution.