Directed Evolution of Protoglobin Optimizes the Enzyme Electric Field

Abstract

To unravel why computational design fails in creating viable enzymes, while directed evolution (DE) succeeds, our research delves into the laboratory evolution of protoglobin. DE has adapted this protein to efficiently catalyze carbene transfer reactions. We show that the previously proposed enhanced substrate access and binding alone cannot account for increased yields during DE. The 3D electric field in the entire active site is tracked through protein dynamics, clustered using the affinity propagation algorithm, and subjected to principal component analysis. This analysis reveals notable changes in the electric field with DE, where distinct field topologies influence transition state energetics and mechanism. A chemically meaningful field component emerges and takes the lead during DE and facilitates crossing the barrier to carbene transfer. Our findings underscore intrinsic electric field dynamic's influence on enzyme function, the ability of the field to switch mechanisms within the same protein, and the crucial role of the field in enzyme design.

Machine-learning prediction of protein function from the portrait of its intramolecular electric field

“…Our group has previously used this protocol to interpret the dynamic heterogeneous field differences along a directed evolution pathway of catalytic protoglobin complexes. 26 We used these compressed representations of the electric field along the entire MD trajectory to further study the effects of the 3-D electric field on electronic populations within the active site. With this, we demonstrate the relationship between induced fields at the active site and the overall protein activity.…”

“…The fields were then compared to each other along the trajectory and clustered by the topology similarity. 26…”

“…To enhance the interpretability of our MD based 3-D electric field analysis and reduce its complexity, we have recently developed a protocol that captures dynamic information regarding the electric fields experienced by the active site of a protein, 26 as illustrated in Figure 4B, followed by mapping these clusters onto the principal components identified as critical. By capturing the complex dynamic fluctuations within the enzyme's active site, we aim to elucidate how these variations complicate the model's ability to accurately predict enzyme activity.…”

“…16,22,23 In the realm of metal-containing enzymes, significant attention has been devoted to exploring electric fields within Fe-heme containing enzymes and their model systems. [24][25][26][27] Even with identical Fe-heme coordination, mere variation in axial ligands such as cysteine, histidine, and tyrosine, heme enzymes exhibit diverse reactivity. Our prior research unearthed a pivotal revelation: beyond the axial ligand, the electric field from the surrounding protein (excluding the heme and the axial ligand) strongly influences reactivity.…”

“…24 In a recent study of laboratory evolved protoglobin for the catalysis of carbene transfer reactions, we furthermore showed that it is the catalytic component of the electric field in the active site that the evolution develops in its course. 26 We infer the impact of fields within protein active sites on chemical reactivity, and thus offer another avenue in the pursuit of effective protein design. 28 Such insights could bridge the existing gap between computationally designed proteins and genuinely effective enzymes, whether naturally occurring or laboratory evolved.…”