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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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Stochastic simulations of primary/promiscuous activities based on different conformational diversity models.(A) Simulation based on a 3-conformation model identical to that used in the simulation of Figure 8E, except that a low (but different from zero) level of primary and promiscuous activities for the suboptimal conformation (a0) is assumed. Here, as well as in the other panels, the Pareto set is shown with green circles. (B) Simulation based on a 4-conformation model with two suboptimal conformations. (C) Same as in (B), except that each one of the optimal conformations (a2 and a3) has a low level of the alternative activity. (D) Same as in (C), but assuming that the optimal conformations show high level of both activities. The models used in (A), (B) and (C) include the existence of trade-offs between the optimal conformations and yield roughly linear Pareto sets with a significant number of data points below the Pareto set due to the presence of suboptimal conformations. This is actually the experimental pattern we have found for the catalysis-of-oxidative-folding/reductase activities (Figure 8A). The model used in (D) does not include trade-offs between the optimal conformations (i.e., a2 and a3 efficiently catalyze both, the primary and the promiscuous processes) and the pattern is completely different: a significant correlation between the two activities and a very small Pareto set are observed.
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pcbi-1002558-g009: Stochastic simulations of primary/promiscuous activities based on different conformational diversity models.(A) Simulation based on a 3-conformation model identical to that used in the simulation of Figure 8E, except that a low (but different from zero) level of primary and promiscuous activities for the suboptimal conformation (a0) is assumed. Here, as well as in the other panels, the Pareto set is shown with green circles. (B) Simulation based on a 4-conformation model with two suboptimal conformations. (C) Same as in (B), except that each one of the optimal conformations (a2 and a3) has a low level of the alternative activity. (D) Same as in (C), but assuming that the optimal conformations show high level of both activities. The models used in (A), (B) and (C) include the existence of trade-offs between the optimal conformations and yield roughly linear Pareto sets with a significant number of data points below the Pareto set due to the presence of suboptimal conformations. This is actually the experimental pattern we have found for the catalysis-of-oxidative-folding/reductase activities (Figure 8A). The model used in (D) does not include trade-offs between the optimal conformations (i.e., a2 and a3 efficiently catalyze both, the primary and the promiscuous processes) and the pattern is completely different: a significant correlation between the two activities and a very small Pareto set are observed.

Mentions: For simplicity and illustration, the simulations included in Figure 8 assume that there is only one sub-optimal conformation and that it has zero primary and promiscuous activity levels. It is important to note, however, that the general result of the simulations is robust and it is obtained with different conformation models (see Figures 9A–C) including several sub-optimal conformations with non-zero activity levels. Apparently, all that is required for a linear-like Pareto set to be obtained in these simulations is a model with two optimal conformations with quite different capabilities to catalyze the primary and promiscuous processes. Actually, if the two optimal conformations are efficient at catalyzing both activities (and, therefore, there are no trade-offs!), the pattern of a linear-like Pareto set with a triangular-like data points cloud is not obtained (see Figure 9D for a representative simulation).


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)

Stochastic simulations of primary/promiscuous activities based on different conformational diversity models.(A) Simulation based on a 3-conformation model identical to that used in the simulation of Figure 8E, except that a low (but different from zero) level of primary and promiscuous activities for the suboptimal conformation (a0) is assumed. Here, as well as in the other panels, the Pareto set is shown with green circles. (B) Simulation based on a 4-conformation model with two suboptimal conformations. (C) Same as in (B), except that each one of the optimal conformations (a2 and a3) has a low level of the alternative activity. (D) Same as in (C), but assuming that the optimal conformations show high level of both activities. The models used in (A), (B) and (C) include the existence of trade-offs between the optimal conformations and yield roughly linear Pareto sets with a significant number of data points below the Pareto set due to the presence of suboptimal conformations. This is actually the experimental pattern we have found for the catalysis-of-oxidative-folding/reductase activities (Figure 8A). The model used in (D) does not include trade-offs between the optimal conformations (i.e., a2 and a3 efficiently catalyze both, the primary and the promiscuous processes) and the pattern is completely different: a significant correlation between the two activities and a very small Pareto set are observed.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375227&req=5

pcbi-1002558-g009: Stochastic simulations of primary/promiscuous activities based on different conformational diversity models.(A) Simulation based on a 3-conformation model identical to that used in the simulation of Figure 8E, except that a low (but different from zero) level of primary and promiscuous activities for the suboptimal conformation (a0) is assumed. Here, as well as in the other panels, the Pareto set is shown with green circles. (B) Simulation based on a 4-conformation model with two suboptimal conformations. (C) Same as in (B), except that each one of the optimal conformations (a2 and a3) has a low level of the alternative activity. (D) Same as in (C), but assuming that the optimal conformations show high level of both activities. The models used in (A), (B) and (C) include the existence of trade-offs between the optimal conformations and yield roughly linear Pareto sets with a significant number of data points below the Pareto set due to the presence of suboptimal conformations. This is actually the experimental pattern we have found for the catalysis-of-oxidative-folding/reductase activities (Figure 8A). The model used in (D) does not include trade-offs between the optimal conformations (i.e., a2 and a3 efficiently catalyze both, the primary and the promiscuous processes) and the pattern is completely different: a significant correlation between the two activities and a very small Pareto set are observed.
Mentions: For simplicity and illustration, the simulations included in Figure 8 assume that there is only one sub-optimal conformation and that it has zero primary and promiscuous activity levels. It is important to note, however, that the general result of the simulations is robust and it is obtained with different conformation models (see Figures 9A–C) including several sub-optimal conformations with non-zero activity levels. Apparently, all that is required for a linear-like Pareto set to be obtained in these simulations is a model with two optimal conformations with quite different capabilities to catalyze the primary and promiscuous processes. Actually, if the two optimal conformations are efficient at catalyzing both activities (and, therefore, there are no trade-offs!), the pattern of a linear-like Pareto set with a triangular-like data points cloud is not obtained (see Figure 9D for a representative simulation).

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