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Probing the mutational interplay between primary and promiscuous protein functions: a computational-experimental approach.

Garcia-Seisdedos H, Ibarra-Molero B, Sanchez-Ruiz JM - PLoS Comput. Biol. (2012)

Bottom Line: Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities.Overall, the results reported may help clarify the mechanisms of the evolution of new functions.From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes.

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias, Departamento de Quimica Fisica, Universidad de Granada, Granada, Spain.

ABSTRACT
Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes.

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Full-library partial-least-squares reconstructions of the reductase/catalyis-of-oxidative-folding data based upon the expanded 40-variant set.The black squares represent the first-round 29-variant set and the circles represent the 11 variants added as a result of the experimental validation of the Pareto prediction shown in Figure 5C (green data points are used here for the Pareto set). Error bars have been omitted for clarity, but they are shown in Figures 5E and 5F. The reconstructed data (grey squares) are derived from 20 bootstrapping replicas of the 40-variant data. Note that the PLS reconstructions shown here are based on the expanded 40-variant set, while those of Figure 5B were based on the first-round 29-variant set. The full-library reconstructions shown here suggest only small enhancements over the 40-variant Pareto set would be obtained in additional screening rounds and, further, they support that the 40-variant Pareto set is already close to the full-library Pareto set.
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pcbi-1002558-g006: Full-library partial-least-squares reconstructions of the reductase/catalyis-of-oxidative-folding data based upon the expanded 40-variant set.The black squares represent the first-round 29-variant set and the circles represent the 11 variants added as a result of the experimental validation of the Pareto prediction shown in Figure 5C (green data points are used here for the Pareto set). Error bars have been omitted for clarity, but they are shown in Figures 5E and 5F. The reconstructed data (grey squares) are derived from 20 bootstrapping replicas of the 40-variant data. Note that the PLS reconstructions shown here are based on the expanded 40-variant set, while those of Figure 5B were based on the first-round 29-variant set. The full-library reconstructions shown here suggest only small enhancements over the 40-variant Pareto set would be obtained in additional screening rounds and, further, they support that the 40-variant Pareto set is already close to the full-library Pareto set.

Mentions: It is relevant to note at this point that the PLS-reconstruction/Pareto-prediction analysis leads to an expansion of our experimental variant set (from 29 variants to 40 variants) but in a manner that is not random. Actually, the 11 variants added to the experimental set allow us to move in the space of primary/promiscuous activities in the general direction of the simultaneous enhancement of both activities, as is visually apparent in Figures 5E and 5F. The Pareto set from the experimental data for the 29+11 = 40 variant set (Figures 5E and 5F) is still only an approximation to the Pareto set for the whole library, since additional cycles of PLS-reconstruction/Pareto-prediction could in principle lead to further enhancements in both activities. However, PLS-reconstruction starting with the 40-variant experimental data set suggests that additional improvements are expected to be small (see Figure 6), supporting that 40-variant Pareto set is likely close to the Pareto set of the full library. Furthermore, the main result of the analysis is already apparent with the 40-variant set: E. coli thioredoxin, as well as the background P34H variant for library construction, does not belong to the Pareto set and, therefore, simultaneous enhancement of the primary and promiscuous activities is feasible (and has been experimentally achieved: Figures 5E and 5F). Note that, in addition to the targeted simultaneous enhancement (implying enhanced levels for both activities), the experimental data set (as well as the PLS reconstructions of the full combinatorial library) indicates that the two activities can be modulated in an independent-like manner and includes “specialist” variants with a high level for one activity and low value for the other.


Probing the mutational interplay between primary and promiscuous protein functions: a computational-experimental approach.

Garcia-Seisdedos H, Ibarra-Molero B, Sanchez-Ruiz JM - PLoS Comput. Biol. (2012)

Full-library partial-least-squares reconstructions of the reductase/catalyis-of-oxidative-folding data based upon the expanded 40-variant set.The black squares represent the first-round 29-variant set and the circles represent the 11 variants added as a result of the experimental validation of the Pareto prediction shown in Figure 5C (green data points are used here for the Pareto set). Error bars have been omitted for clarity, but they are shown in Figures 5E and 5F. The reconstructed data (grey squares) are derived from 20 bootstrapping replicas of the 40-variant data. Note that the PLS reconstructions shown here are based on the expanded 40-variant set, while those of Figure 5B were based on the first-round 29-variant set. The full-library reconstructions shown here suggest only small enhancements over the 40-variant Pareto set would be obtained in additional screening rounds and, further, they support that the 40-variant Pareto set is already close to the full-library Pareto set.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375227&req=5

pcbi-1002558-g006: Full-library partial-least-squares reconstructions of the reductase/catalyis-of-oxidative-folding data based upon the expanded 40-variant set.The black squares represent the first-round 29-variant set and the circles represent the 11 variants added as a result of the experimental validation of the Pareto prediction shown in Figure 5C (green data points are used here for the Pareto set). Error bars have been omitted for clarity, but they are shown in Figures 5E and 5F. The reconstructed data (grey squares) are derived from 20 bootstrapping replicas of the 40-variant data. Note that the PLS reconstructions shown here are based on the expanded 40-variant set, while those of Figure 5B were based on the first-round 29-variant set. The full-library reconstructions shown here suggest only small enhancements over the 40-variant Pareto set would be obtained in additional screening rounds and, further, they support that the 40-variant Pareto set is already close to the full-library Pareto set.
Mentions: It is relevant to note at this point that the PLS-reconstruction/Pareto-prediction analysis leads to an expansion of our experimental variant set (from 29 variants to 40 variants) but in a manner that is not random. Actually, the 11 variants added to the experimental set allow us to move in the space of primary/promiscuous activities in the general direction of the simultaneous enhancement of both activities, as is visually apparent in Figures 5E and 5F. The Pareto set from the experimental data for the 29+11 = 40 variant set (Figures 5E and 5F) is still only an approximation to the Pareto set for the whole library, since additional cycles of PLS-reconstruction/Pareto-prediction could in principle lead to further enhancements in both activities. However, PLS-reconstruction starting with the 40-variant experimental data set suggests that additional improvements are expected to be small (see Figure 6), supporting that 40-variant Pareto set is likely close to the Pareto set of the full library. Furthermore, the main result of the analysis is already apparent with the 40-variant set: E. coli thioredoxin, as well as the background P34H variant for library construction, does not belong to the Pareto set and, therefore, simultaneous enhancement of the primary and promiscuous activities is feasible (and has been experimentally achieved: Figures 5E and 5F). Note that, in addition to the targeted simultaneous enhancement (implying enhanced levels for both activities), the experimental data set (as well as the PLS reconstructions of the full combinatorial library) indicates that the two activities can be modulated in an independent-like manner and includes “specialist” variants with a high level for one activity and low value for the other.

Bottom Line: Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities.Overall, the results reported may help clarify the mechanisms of the evolution of new functions.From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes.

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias, Departamento de Quimica Fisica, Universidad de Granada, Granada, Spain.

ABSTRACT
Protein promiscuity is of considerable interest due its role in adaptive metabolic plasticity, its fundamental connection with molecular evolution and also because of its biotechnological applications. Current views on the relation between primary and promiscuous protein activities stem largely from laboratory evolution experiments aimed at increasing promiscuous activity levels. Here, on the other hand, we attempt to assess the main features of the simultaneous modulation of the primary and promiscuous functions during the course of natural evolution. The computational/experimental approach we propose for this task involves the following steps: a function-targeted, statistical coupling analysis of evolutionary data is used to determine a set of positions likely linked to the recruitment of a promiscuous activity for a new function; a combinatorial library of mutations on this set of positions is prepared and screened for both, the primary and the promiscuous activities; a partial-least-squares reconstruction of the full combinatorial space is carried out; finally, an approximation to the Pareto set of variants with optimal primary/promiscuous activities is derived. Application of the approach to the emergence of folding catalysis in thioredoxin scaffolds reveals an unanticipated scenario: diverse patterns of primary/promiscuous activity modulation are possible, including a moderate (but likely significant in a biological context) simultaneous enhancement of both activities. We show that this scenario can be most simply explained on the basis of the conformational diversity hypothesis, although alternative interpretations cannot be ruled out. Overall, the results reported may help clarify the mechanisms of the evolution of new functions. From a different viewpoint, the partial-least-squares-reconstruction/Pareto-set-prediction approach we have introduced provides the computational basis for an efficient directed-evolution protocol aimed at the simultaneous enhancement of several protein features and should therefore open new possibilities in the engineering of multi-functional enzymes.

Show MeSH