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. The intermediate begins to populate at about 30° below the global unfolding temperature, reaching a maximum population of nearly 60%, 10° below the global unfolding temperature.

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. Here we explore the use of computational protein design to overcome these limitations. The high-plasticity positions close to the original ("native") active-site are the most promising candidates for mutations that create a second active-site associated to a new function. To avoid compromising protein folding and native activity, we propose a minimal-perturbation approach based on the combinatorial optimization of, both the de novo catalytic activity and the folding free-energy: essentially, we construct the Pareto Set of optimal stability/promiscuous-function solutions. We validate our approach by introducing a promiscuous esterase activity in E. coli thioredoxin on the basis of mutations at positions close to the native-active-site disulfide-bridge. Native oxidoreductase activity is not compromised and it is, in fact, found to be 1.5-fold enhanced, as determined by an insulin-reduction assay. This work provides general guidelines as to how computational design can be used to expand the scope and applications of protein promiscuity. From a more general viewpoint, it illustrates the potential of multi-objective optimization as the computational analogue of multi-feature natural selection.

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. Undesirable effects associated to high stability are also likely to occur in food-industry applications. Clearly, one fundamental factor involved here is the kinetic stability of the protein, which relates to the time-scale of the irreversible denaturation processes and which is determined to some significant extent by the free-energy barrier for unfolding (the barrier that "separates" the native state from the highly-susceptible-to-irreversible-alterations nonnative states). With an appropriate experimental model, we show that strong environment-dependencies of the thermodynamic and kinetic stabilities can be achieved using robust protein engineering. We use sequence-alignment analysis and simple computational electrostatics to design stabilizing and destabilizing mutations, the latter introducing interactions between like charges which are screened out at high salt. Our design procedures lead naturally to mutating regions which are mostly unstructured in the transition state for unfolding. As a result, the large salt effect on the thermodynamic stability of our consensus plus charge-reversal variant translates into dramatic changes in the time-scale associated to the unfolding barrier: from the order of years at high salt to the order of days at low salt. Certainly, large changes in salt concentration are not expected to occur in biological systems in vivo. Hence, proteins with strong salt-dependencies of the thermodynamic and kinetic stabilities are more likely to be of use in those cases in which high-stability is required only under storage conditions. A plausible scenario is that inclusion of high salt in liquid formulations will contribute to a long protein shelf-life, while the lower salt concentration under the conditions of the application will help prevent the side effects associated with high-stability which may potentially arise in some therapeutic and food-industry applications. From a more general viewpoint, this work shows that consensus engineering and electrostatic engineering can be readily combined and clarifies relevant aspects of the relation between thermodynamic stability and kinetic stability in proteins.

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. Lys5 contributes 6.6 kJ mol(-1) to the stability of the transition state, and half of it originates from screenable coulombic interactions. Lys7 contributes 5.3 kJ mol(-1), and 3.4 kJ mol(-1) of it are screened by salt. In the folded protein Lys7 interacts with Asp25, and the screenable coulombic interaction between these two residues is fully formed in the transition state. This suggests that long-range coulombic interactions such as those originating from Lys5 and Lys7 of CspB can be important for organizing and stabilizing native-like structure early in protein folding.

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. Of course, in the long term, this empirical approach is no substitute for rigorous electrostatic analysis but, in the short term, it may prove to have useful predictive power and it may help to pinpoint the main structural determinants of pK values in proteins. Here we demonstrate the feasibility of the parametrization approach by fitting (using a genetic algorithm as fitting tool) the database for carboxylic acid pK values in proteins on the basis of an empirical equation that takes into account the two following kinds of effects: (1) long-range charge-charge interactions; (2) interactions of the given carboxylic acid group with its environment in the protein, which are described in terms of contributions from the different kind of atoms present in the protein (atomic contributions).

Do proteins always benefit from a stability increase? Relevant and residual stabilisation in a three-state protein by charge optimisation.

The vast majority of our knowledge on protein stability arises from the study of simple two-state models. However, proteins displaying equilibrium intermediates under certain conditions abound and it is unclear whether the energetics of native/intermediate equilibria is well represented in current knowledge. We consider here that the overall conformational stability of three-state proteins is made of a "relevant" term and a "residual" one, corresponding to the free energy differences of the native to intermediate (N-to-I) and intermediate to denatured (I-to-D) equilibria, respectively. The N-to-I free energy difference is considered to be the relevant stability because protein-unfolding intermediates are likely devoid of biological activity. We use surface charge optimisation to first increase the overall (N-to-D) stability of a model three-state protein (apoflavodoxin) and then investigate whether the stabilisation obtained is realised into relevant or into residual stability. Most of the mutations designed from electrostatic calculations or from simple sequence conservation analysis produce large increases in the overall stability of the protein. However, in most cases, this simply leads to similarly large increases of the residual stability. Two mutations, nevertheless, show a different trend and increase the relevant stability of the protein substantially. When all the mutations are mapped onto the structure of the apoflavodoxin thermal-unfolding intermediate (obtained independently by equilibrium phi-analysis and NMR) they cluster perfectly so that the mutations increasing the relevant stability appear in the small unstructured region of the intermediate and the others in the native-like region. This illustrates the need for specific investigation of N-to-I equilibria and the structure of protein intermediates, and indicates that it is possible to rationally stabilise a protein against partial unfolding once the structure of the intermediate conformation is known, even if at low resolution.

The folding transition state of the cold shock protein is strongly polarized.

The cold shock protein CspB from Bacillus subtilis consists of a three-stranded (beta1-beta3) and a two stranded (beta4-beta5) sheet, which form a closed beta barrel structure. CspB folds and unfolds rapidly in a two-state reaction, and the unfolded and the folded molecules interconvert with a time constant of 30 ms at the midpoint of the urea-induced transition (at 25 degrees C). The transition state of folding is native-like, as judged by the Tanford betaT value of > or =0.9. By using a mutational approach and Phi value analysis, we find that the folding transition state of CspB is energetically polarized. Despite the high betaT value, most Phi values are low. Values close to 1 were found for only a few residues, particularly in strand beta1 (Lys5, Val6, Lys7, Asn10). The interactions of the Asn10 side-chain with the backbone at positions 12 and 13 define the turn that connects the strands beta1 and beta2. Lys5 and Val6 in beta1 interact with residues in beta4, and their high Phi values indicate that an energetic linkage between beta1 and beta4 and thus between the two sheets exists already in the transition state. We compared our experimental Phi values with theoretical predictions of the folding pathway of cold shock proteins. Several of them suggest that the entire first sheet is formed in the transition state, and some identify the beta1-beta4 pairing as a crucial step in folding. Alternative paths that involve formation of the second sheet and beta3-beta5 pairing reactions were, however, suggested as well. The calculations gave coarse-grained pictures that are limited in resolution to the two sheets of CspB or to the elements of secondary structure. They did not identify the key residues with the high Phi values within these structural elements.

Experimental identification of downhill protein folding.

Theory predicts the existence of barrierless protein folding. Without barriers, folding should be noncooperative and the degree of native structure should be coupled to overall protein stability. We investigated the thermal unfolding of the peripheral subunit binding domain from Escherichia coli's 2-oxoglutarate dehydrogenase multienzyme complex (termed BBL) with a combination of spectroscopic techniques and calorimetry. Each technique probed a different feature of protein structure. BBL has a defined three-dimensional structure at low temperatures. However, each technique showed a distinct unfolding transition. Global analysis with a statistical mechanical model identified BBL as a downhill-folding protein. Because of BBL's biological function, we propose that downhill folders may be molecular rheostats, in which effects could be modulated by altering the distribution of an ensemble of structures.

Energetics of heparin binding to human acidic fibroblast growth factor.

The binding of low-molecular-weight heparin to an amino-terminal-truncated, 132-amino-acid, human acidic fibroblast growth factor form has been studied by isothermal titration calorimetry. This technique yields values for the enthalpy change and equilibrium constant, from which the Gibbs energy and entropy change are also calculated. Experiments in different buffers and pH values show that the protonic balance during the reaction is negligible. Experiments made at pH 7.0 with NaCl concentrations ranging from 0.20 to 0.60 M revealed changes in enthalpy and Gibbs energy in the range of -30- -17 and -27- -24 kJ x mol(-1), respectively. Isothermal titration calorimetry was also performed at different temperatures to obtain a value for the heat-capacity change at pH 7.0 and 0.4 M NaCl concentration of -96 J K- x mol(-1). A change in the length of heparin brought about no change in the thermodynamic parameters at 25 degrees C under the same experimental conditions. Changes upon ligand binding in the range of -50- -200 A2 in both polar and non-polar solvent-accessible surface areas were calculated from thermodynamic data by using different parametric equations taken from the literature. These values suggest a negligible overall conformational change in the protein when it binds to heparin and no formation of any protein-protein interface.