Lower kinetic limit to protein thermal stability: A proposal regarding protein stability in vivo and its relation with misfolding diseases

In vitro thermal denaturation experiments suggest that, because of the poss ibility of irreversible alterations, thermodynamic stability (i.e., a posit ive value for the unfolding Gibbs energy) does not guarantee that a protein will remain in the native state during a given timescale, Furthermore, irr eversible alterations are more likely to occur in vivo than in vitro becaus e (a) some irreversible processes (e.g., aggregation, "undesirable" interac tions with other macromolecular components, and proteolysis) are expected t o be fast in the "crowded" cellular environment and (b) in many cases, the relevant timescale in vivo (probably related to the bah-life for protein de gradation) is expected to be longer than the timescale of the usual in vitr o experiments (of the order of minutes), We propose, therefore, that many p roteins tin particular, thermophilic proteins and "complex" proteins system s) are designed (by evolution) to have significant kinetic stability when c onfronted with the destabilizing effect of irreversible alterations, We sho w that, as long as these alterations occur mainly from nonnative states (a Lumry-Eyring scenario), the required kinetic stability may be achieved thro ugh the design of a sufficiently high activation barrier for unfolding, whi ch we define as the Gibbs energy barrier that separates the native state fr om the non-native ensemble (unfolded, partially folded, and misfolded state s) in the following generalized Lumry-Eyring model: Native State <-> Non-Native Ensemble --> Irreversibly Denatured Protein Finally, using familial amyloid polyneuropathy (PAP) as an illustrative exa mple, we discuss the relation between stability and amyloid fibril formatio n in terms of the above viewpoint, which leads us to the two following tent ative suggestions: (a) the hot spot defined by the FAP-associated amyloidog enic mutations of transthyretin reflects the structure of the transition st ate for unfolding and (b) substances that decrease the in vitro rate of tra nsthyretin unfolding could also be inhibitors of amyloid fibril formation. (C) 2000 Wiley-Liss, Inc.