We have characterized the guanidine-induced unfolding of both yeast and bovine ubiquitin at 25°C and in the acidic pH range on the basis of fluorescence and circular dichroism measurements. Unfolding Gibbs energy changes calculated by linear extrapolation from high guanidine unfolding data are found to depend very weakly on pH. A simple explanation for this result involves the two following assumptions: (1) charged atoms of ionizable groups are exposed to the solvent in native ubiquitin (as supported by accessible surface area calculations), and Gibbs energy contributions associated with charge desolvation upon folding (a source of pK shifts) are small; (2) charge-charge interactions (another source of pK shifts upon folding) are screened out in concentrated guanidinium chloride solutions. We have also characterized the thermal unfolding of both proteins using differential scanning calorimetry. Unfolding Gibbs energy changes calculated from the calorimetric data do depend strongly on pH, a result that we attribute to the pH dependence of charge-charge interactions (not eliminated in the absence of guanidine). In fact, we find good agreement between the difference between the two series of experimental unfolding Gibbs energy changes (determined from high guanidine unfolding data by linear extrapolation and from thermal denaturation data in the absence of guanidine) and the theoretical estimates of the contribution from charge-charge interactions to the Gibbs energy change for ubiquitin unfolding obtained by using the solvent-accessibility-corrected Tanford-Kirkwood model, together with the Bashford-Karplus (reduced-set-of-sites) approximation. This contribution is found to be stabilizing at neutral pH, because most charged groups on the native protein interact mainly with groups of the opposite charge, a fact that, together with the absence of large charge-desolvation contributions, may explain the high stability of ubiquitin at neutral pH. In general, our analysis suggests the possibility of enhancing protein thermal stability by adequately redesigning the distribution of solvent-exposed, charged residues on the native protein surface.The thermodynamic stability of proteins is commonly described by the unfolding Gibbs energy versus temperature profile, the so-called protein stability curve (1,2). It is a well-known fact that Gibbs energy changes of unfolding are small numbers, resulting from the cancellation of large contributions arising from the major "forces" that drive or oppose folding (hydrophobic effect, hydrogen bonding, van der Waals interactions, conformational entropy; see refs 3-8). As an obvious consequence of this, even minor contributions to the unfolding Gibbs energy are important regarding protein stability. Thus, for instance, electrostatic contributions to unfolding Gibbs energies due to the interactions between the charges on ionizable groups are comparable to the total unfolding Gibbs energy (9, 10); therefore, these electrostatic interactions might play a key role in determining o...

A simple theoretical model for increasing the protein stability by adequately redesigning the distribution of charged residues on the surface of the native protein was tested experimentally. Using the molecule of ubiquitin as a model system, we predicted possible amino acid substitutions on the surface of this protein which would lead to an increase in its stability. Experimental validation for this prediction was achieved by measuring the stabilities of single-site-substituted ubiquitin variants using urea-induced unfolding monitored by far-UV CD spectroscopy. We show that the generated variants of ubiquitin are indeed more stable than the wild-type protein, in qualitative agreement with the theoretical prediction. As a positive control, theoretical predictions for destabilizing amino acid substitutions on the surface of the ubiquitin molecule were considered as well. These predictions were also tested experimentally using correspondingly designed variants of ubiquitin. We found that these variants are less stable than the wildtype protein, again in agreement with the theoretical prediction. These observations provide guidelines for rational design of more stable proteins and suggest a possible mechanism of structural stability of proteins from thermophilic organisms.How to stabilize protein structure? This question is not just of scholastic interest. The answer to this question has immediate importance for the biotechnological industry, which is interested in improving the thermostability of enzymes. The efforts in this direction have concentrated on repacking the hydrophobic cores, engineering disulfide bridges, adding extra hydrogen bonds or salt bridges, and improving secondary structure propensities or side chain helix dipole interactions (1-8). All these methods can be characterized as optimizing the local short-range interactions. Such an approach is well justified because it is becoming more and more clear that the protein folding is a hierarchical process and thus is mostly driven by local interactions (9, 10). However, this does not mean that the final native state of the protein is stabilized exclusively by the short-range interactions (11,12). Long-range interactions such as chargecharge interactions will also contribute to the stability of proteins. To address this issue, we developed a simple approach [the "TK-BK procedure" (13)] that allows us, nevertheless, to determine the regions of the protein surface where redesign of the charge-charge interactions is likely to lead to stability enhancement. In the TK-BK procedure (13), the interaction energies between unit positive charges placed in the protonation sites of ionizable groups are estimated using the solvent-accessibility-corrected Tanford-Kirkwood model (14), and subsequently, the pairwise charge-charge interaction energies are calculated as averages over the protonation states of the native protein on the basis of the BashfordKarplus reduced-set-of-sites approximation (15). This approach is indeed a simple one (charge-charge intera...

No 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.