Cold denaturation of ubiquitin.

Temperature induced unfolding of bovine ubiquitin in solutions with different concentrations of guanidinium hydrochloride (GdmCl) has been measured using differential scanning calorimetry. It has been shown that at high concentrations of GdmCl the ubiquitin molecule can undergo both heat and cold induced denaturation. Analysis of the enthalpy of unfolding of ubiquitin in the presence of GdmCl shows a good agreement with the thermodynamic denaturant binding model. The unfolding Gibbs energy is found to change linearly with guanidine concentration up to zero denaturant concentration.

Preformed secondary structure drives the association reaction of GCN4-p1, a model coiled-coil system.

The structure of the transition state for the rate-limiting step in the folding and association of the homodimeric coiled-coil peptide GCN4-p1, was probed by mutational analysis. A series of quadruple amino acid replacements that spanned the helix propensity scale were made at the four external f positions in the heptad repeat. Equilibrium and kinetic circular dichroism studies demonstrate that both the stability and the unfolding and refolding rate constants vary with helix propensity but also reflect interactions of the altered side-chains with their local environments. Pairwise replacements and fragment studies show that the two C-terminal heptads are the likely source of the nucleating helices. Helix-helix recognition between preformed elements of secondary structure plays an important role in this fundamental folding reaction.

The sarcosine effect on protein stability: a case of nonadditivity?

We have used differential scanning calorimetry to determine the effect of low concentrations (C = 0-2 M) of the osmolyte sarcosine on the Gibbs energy changes (deltaG) for the unfolding of hen-egg-white lysozyme, ribonuclease A, and ubiquitin, under the same buffer and pH conditions. We have also computed this effect on the basis of the additivity assumption and using published values of the transfer Gibbs energies for the amino acid side chains and the peptide backbone unit. The values thus predicted for the slope delta deltaG/deltaC agree with the experimental ones, but only if the unfolded state is assumed to be compact (that is, if the accessibility to solvent of the unfolded state is modeled using segments excised from native structures). The additivity-based calculations predict similar delta deltaG/deltaC values for the three proteins studied. We point out that, to the extent that this approximate constancy of delta deltaG/deltaC holds, osmolyte-induced increases in denaturation temperature will be larger for proteins with low unfolding enthalpy (small proteins that bury a large proportion of apolar surface). The experimental results reported here are consistent with this hypothesis.

Are there equilibrium intermediate states in the urea-induced unfolding of hen egg-white lysozyme?

Protein folding intermediates that are sometimes populated at equilibrium under mild denaturing conditions have attracted much attention as plausible models for the kinetic intermediates transiently populated in the refolding kinetic pathways. Hen egg-white lysozyme is often considered as a typical example of close adherence to the equilibrium, two-state unfolding mechanism. However, recent small-angle X-ray scattering studies suggest that an equilibrium intermediate state is significantly populated in the urea-induced unfolding of this protein at moderately acidic pH. In this work, we analyze the urea-induced unfolding of hen egg-white lysozyme on the basis of steady-state fluorescence measurements, characterization of the folding-unfolding kinetics, double-jump unfolding assays for the amount of native protein, and double-jump refolding assays for the amount of unfolded protein. Our results do not provide support for the presence of an intermediate state and, in particular, disfavor that the following two types of intermediates be significantly populated at equilibrium: (1) intermediates showing a substantial quenching of the tryptophan fluorescence (such as that observed in the transient intermediates found in the refolding kinetic pathway under strongly native conditions) and (2) associating intermediates. Also, the deconvolution of the radius of gyration unfolding profile by using the values for the amount of native state derived from our double-jump unfolding assays is consistent with a two-state unfolding equilibrium and suggests, furthermore, that, in this case, large alterations in the average structure of the unfolded ensemble do not take place in response to changes in urea concentration. This work points up possible pitfalls in the experimental detection of equilibrium folding intermediates and suggests procedures to circumvent them.

A model-independent, nonlinear extrapolation procedure for the characterization of protein folding energetics from solvent-denaturation data.

We have characterized the guanidine-induced denaturation of hen egg white lysozyme within the 30-75 degrees C temperature range on the basis of equilibrium fluorescence measurements, unfolding assays, kinetic fluorescence measurements, and differential scanning calorimetry. Analysis of the guanidine denaturation profiles according to the linear extrapolation method yields values for the denaturation Gibbs energy which are about 15 kJ/mol lower than those derived from differential scanning calorimetry. Our results strongly suggest that this discrepancy is not due to deviations from the two-state denaturation mechanism. We propose a new method for the determination of denaturation Gibbs energies from solvent-denaturation data (the constant-delta G extrapolation procedure). It employs several solvent-denaturation profiles (obtained at different temperatures) to generate the protein stability curve at zero denaturant concentration within the -8 to 8 kJ/mol delta G range. The method is model-independent and provides a practical, nonlinear alternative to the commonly employed linear extrapolation procedure. The application of the constant-delta G method to our data suggests that the guanidine-concentration dependence of the denaturation Gibbs energy is approximately linear over an extended concentration range but, also, that strong deviations from linearity may occur at low guanidine concentrations. We tentatively attribute these deviations to the abrupt change of the contribution to protein stability that arises from pairwise charge-charge electrostatic interactions. This contribution may be positive, negative, or close to zero, depending on the pH value and the charge distribution on the native protein surface [Yang, A.-S., & Honig, B. (1993) J. Mol. Biol. 231, 459-474], which may help to explain why disparate effects have been found when studying protein denaturation at low guanidine concentrations. Kinetic m values for lysozyme denaturation depend on temperature, in a manner which appears consistent with Hammond behavior.

Mapping the energy surface for the folding reaction of the coiled-coil peptide GCN4-p1.

The energy surface for the folding/unfolding reactions of the homodimeric coiled-coil peptide M2V GCN4-p1, a 33-residue segment comprising the leucine zipper domain of the transcriptional activator GCN4, was mapped by equilibrium and kinetic methods. Circular dichroism (CD) spectroscopy was used to monitor the urea-induced unfolding reaction at a series of temperatures and temperature-induced unfolding at a series of urea concentrations. A global analysis of the urea- and temperature-induced equilibrium unfolding data provides strong support for a two-state mechanism. The absence of a detectable population of intermediate states is also consistent with differential scanning calorimetry and thermal CD melts as a function of peptide concentration. Furthermore, a global analysis of stopped-flow CD kinetic data is consistent with a kinetic two-state mechanism as well. The urea dependence of the apparent folding and unfolding rate constants at a series of temperatures reveals that the activation enthalpy and entropy for unfolding in the absence of denaturant are both significantly greater than those for the refolding reaction. Although the unfolding barrier is dominated by the activation enthalpy, the activation entropy dominates the refolding barrier. The relative magnitudes of the urea dependence of the unfolding and refolding rate constants indicate that 55-65% of the surface area is buried in the transition state. The activation parameters imply a partially organized transition state and are consistent with a previous model in which the pair of C-terminal heptad repeats are docked in a coiled-coil-like motif [Zitzewitz et al. (2000) J. Mol. Biol. 296, 1105-1116].

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 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.

Thermal versus guanidine-induced unfolding of ubiquitin. An analysis in terms of the contributions from charge-charge interactions to protein stability.

We have characterized the guanidine-induced unfolding of both yeast and bovine ubiquitin at 25 degrees 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.

Engineering a thermostable protein via optimization of charge-charge interactions on the protein surface.

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 wild-type 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.