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The equilibrium unfolding at neutral pH of the third PDZ domain of PSD95, as followed by DSC, is characterized by the presence of an equilibrium intermediate with clear signs of oligomerization. DLS and SEC measurements indicate that at 60-70 degrees C small oligomers populate, showing a typical beta-sheet far-UV CD spectrum. These intermediate species lead to the formation of rodlike particulates of approximately 12 nm, which remain in solution after 2 weeks incubation and grow until they adopt annular/spherical shapes of approximately 50 nm and protofibrils, which are subsequently fully transformed into fibrils. The fibrils can also disaggregate after the addition of 1:1 buffer dilution followed by cooling to room temperature, thus returning to the initial monomeric state. Growth kinetics, as shown by ThT and ANS fluorescence, show that the organization of the different supramacromolecular structures comes from a common nucleation unit, the small oligomers, which organize themselves before reaching the incubation temperature of 60 degrees C. Our experiments point toward the existence of a well-defined reversible, stepwise, and downhill organization of the processes involved in the association-dissociation of the intermediate. We estimate the enthalpy change accompanying the association-dissociation equilibria to be 130 kJ x mol(-1). Furthermore, the coalescence under essentially reversible conditions of different kinds of supramacromolecular assemblies renders this protein system highly interesting for biophysical studies aimed at our further understanding of amyloid pathological conditions.

WW domains are the smallest naturally independent beta-sheet protein structures available to date and constitute attractive model systems for investigating the determinants of beta-sheet folding and stability. Nonetheless, their small size and low cooperativity pose a difficult challenge for a quantitative analysis of the folding equilibrium. We describe here a comprehensive thermodynamic characterization of the conformational equilibrium of the fourth WW domain from the human ubiquitin ligase Nedd4 (hNedd4-WW4) using a combination of calorimetric and spectroscopic techniques with several denaturing agents (temperature, pH, and chemical denaturants). Our results reveal that even though the experimental data can be described in terms of a two-state equilibrium, spectral data together with anomalous values for some thermodynamic parameters (a strikingly low temperature of maximum stability, a higher than expected native-state heat capacity, and a small specific enthalpy of unfolding) could be indicative of more complex types of equilibria, such as one-state downhill folding or alternative native conformations. Moreover, double-perturbation experiments reveal some features that, in spite of the apparent linear correlation between the thermodynamic parameters, seem to be indicative of a complex conformational equilibrium in the presence of urea. In summary, the data presented here point toward the existence of a low-energy barrier between the different macrostates of hNedd4-WW4, placing it at the frontier of cooperative folding.

YAP is a WW domain-containing effector of the Hippo tumor suppressor pathway, and the object of heightened interest as a potent oncogene and stemness factor. YAP has two major isoforms that differ in the number of WW domains they harbor. Elucidating the degree of co-operation between these WW domains is important for a full understanding of the molecular function of YAP. We present here a detailed biophysical study of the structural stability and binding properties of the two YAP WW domains aimed at investigating the relationship between both domains in terms of structural stability and partner recognition. We have carried out a calorimetric study of the structural stability of the two YAP WW domains, both isolated and in a tandem configuration, and their interaction with a set of functionally relevant ligands derived from PTCH1 and LATS kinases. We find that the two YAP WW domains behave as independent units with different binding preferences, suggesting that the presence of the second WW domain might contribute to modulate target recognition between the two YAP isoforms. Analysis of structural models and phage-display studies indicate that electrostatic interactions play a critical role in binding specificity. Together, these results are relevant to understand of YAP function and open the door to the design of highly specific ligands of interest to delineate the functional role of each WW domain in YAP signaling.

The unfolding thermodynamics of the circular enterocin protein AS-48, produced by Enterococcus faecalis, has been characterized by differential scanning calorimetry. The native structure of the 70-residue protein is extremely thermally stable. Thus, at pH 2.5 and low ionic strength thermal denaturation occurs under equilibrium at 102³C, while the unfolded state irreversibly aggregates at neutral and alkaline pH. Calorimetric data analysis shows that the specific enthalpy change upon unfolding is unusually small and the heat capacity change is quite normal for a protein of this size, whereas the Gibbs energy change at 25³C is relatively high. At least part of this high stability might be put down to entropic constraints induced by the circular organization of the polypeptide chain. ß

Significance The envelope subunit gp41 is an attractive target for therapeutic intervention against HIV-1. Interfering with the interaction between the heptad-repeat regions of gp41 is a promising approach to inhibit HIV-1 fusion to the host cell membrane. Here, we present an alternative rational design and protein-engineering approach to produce highly stable single-chain proteins that accurately mimic the trimeric coiled-coil surface of the gp41 N-terminal heptad repeat. This approach has a strong potential for development to HIV-1 drugs, vaccines, or microbicides and could be extendable to the design of proteins interfering with other types of coiled-coil interactions.

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