Three-state thermal unfolding of onconase.

Onconase is a member of the ribonuclease A superfamily currently in phase IIIb clinical trials as a treatment for malign mesothelioma due to its cytotoxic activity selective against tumor-cells. In this work, we have studied the equilibrium thermal unfolding of onconase using a combination of several structural and biophysical techniques. Our results indicate that at least one significantly populated intermediate, which implies the exposure of hydrophobic surface and significant changes in the environment around Trp3, occurs during the equilibrium unfolding process of this protein.

Using multi-objective computational design to extend protein promiscuity.

Many enzymes possess, besides their native function, additional promiscuous activities. Proteins with several activities (multipurpose catalysts) may have a wide range of biotechnological and biomedical applications. Natural promiscuity, however, appears to be of limited scope in this context, because the latent (promiscuous) function is often related to the evolved one (sharing the active site and even the chemical mechanism) and its enhancement upon suitable mutations usually brings about a decrease in the native activity.

Engineering proteins with tunable thermodynamic and kinetic stabilities.

It is widely recognized that enhancement of protein stability is an important biotechnological goal. However, some applications at least, could actually benefit from stability being strongly dependent on a suitable environment variable, in such a way that enhanced stability or decreased stability could be realized as required. In therapeutic applications, for instance, a long shelf-life under storage conditions may be convenient, but a sufficiently fast degradation of the protein after it has performed the planned molecular task in vivo may avoid side effects and toxicity.

Key role of coulombic interactions for the folding transition state of the cold shock protein.

The cold shock protein CspB shows a five-stranded beta-sheet structure, and it folds rapidly via a native-like transition state. A previous Phi value analysis showed that most of the residues with Phi values close to one reside in strand beta1, and two of them, Lys5 and Lys7 are partially exposed charged residues. To elucidate how coulombic interactions of these two residues contribute to the energetic organisation of the folding transition state we performed comparative folding experiments in the presence of an ionic denaturant (guanidinium chloride) and a non-ionic denaturant (urea) and a double-mutant analysis.

Empirical parametrization of pK values for carboxylic acids in proteins using a genetic algorithm.

Considerable effort has been devoted to the development of theoretical electrostatic methods to predict the pK values of ionizable residues in proteins. However, predictions appear often to be still at the qualitative or semi-quantitative level. We believe that, with the increasing number experimentally available pK values for proteins of known structure, an alternative approach becomes feasible: the empirical parametrization of the experimental protein pK database.