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

Abstract

In vitro thermal denaturation experiments suggest that, because of the possibility of irreversible alterations, thermodynamic stability (i.e., a positive value for the unfolding Gibbs energy) does not guarantee that a protein will remain in the native state during a given timescale. Furthermore, irreversible alterations are more likely to occur in vivo than in vitro because (a) some irreversible processes (e.g., aggregation, "undesirable" interactions with other macromolecular components, and proteolysis) are expected to be fast in the "crowded" cellular environment and (b) in many cases, the relevant timescale in vivo (probably related to the half-life for protein degradation) is expected to be longer than the timescale of the usual in vitro experiments (of the order of minutes). We propose, therefore, that many proteins (in particular, thermophilic proteins and "complex" proteins systems) are designed (by evolution) to have significant kinetic stability when confronted with the destabilizing effect of irreversible alterations. We show that, as long as these alterations occur mainly from non-native states (a Lumry-Eyring scenario), the required kinetic stability may be achieved through the design of a sufficiently high activation barrier for unfolding, which we define as the Gibbs energy barrier that separates the native state from the non-native ensemble (unfolded, partially folded, and misfolded states) in the following generalized Lumry-Eyring model: Native State <--> Non-Native Ensemble --> Irreversibly Denatured Protein. Finally, using familial amyloid polyneuropathy (FAP) as an illustrative example, we discuss the relation between stability and amyloid fibril formation in terms of the above viewpoint, which leads us to the two following tentative suggestions: (a) the hot spot defined by the FAP-associated amyloidogenic mutations of transthyretin reflects the structure of the transition state for unfolding and (b) substances that decrease the in vitro rate of transthyretin unfolding could also be inhibitors of amyloid fibril formation.

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

“…In our study, the CH2 domain is the least stable domain and the overall irreversible aggregation process is most likely determined by the activation barrier for unfolding of this particular domain. 51 We find it intriguing that the activation energy of the initial reaction (117 kcal/mol) is similar to the activation energy of the unfolding of the CH2 domain (98.4 kcal/mol). Thus, a structural change in the CH2 domain is likely to prime the molecule for aggregation.…”

Investigating protein unfolding kinetics by pulse proteolysis

“…The determined k u values were inversely proportional to the total maltose concentration as predicted in Eq. (2). By fitting the plot to Eq.…”

“…The kinetic stability of a protein, which is commonly defined by its unfolding kinetics, has a critical role in providing the protein with longevity and robustness. 1,2 Spectroscopic monitoring of conformational changes upon unfolding is the most preferred way to study unfolding kinetics of proteins. Typically, protein unfolding is initiated by chemical denaturants, and the change in conformations is observed by circular dichroism (CD) or fluorescence.…”

Thermally induced conformational changes in horseradish peroxidase

“…Nevertheless, it is well known that for different reasons many proteins cannot refold in vitro after denaturation such as proteolytic digestion [16], aggregation, loss of prosthetic group, the cis/trans izomerization of certain proline residues [17,18] or chemical modifications [19]. Generally, the thermal denaturation of proteins is often discussed in terms of the Lumry± Eyring model [20], in which a reversible unfolding step is followed by an irreversible denaturation step:where N, U and D are the native, unfolded or partially unfolded, and denatured states of the protein, respectively [21,22]. However, in many cases irreversible thermal denaturation of proteins can be phenomenologically described on the basis of simple irreversible models that are approximations to the Lumry±Eyring model [21,23±26].…”

“…where N, U and D are the native, unfolded or partially unfolded, and denatured states of the protein, respectively [21,22]. However, in many cases irreversible thermal denaturation of proteins can be phenomenologically described on the basis of simple irreversible models that are approximations to the Lumry±Eyring model [21,23±26].…”