Paleoenzymology at the Single-Molecule Level: Probing the Chemistry of Resurrected Enzymes with Force-Clamp Spectroscopy

Abstract

A journey back in time is possible at the molecular level by resurrecting proteins from extinct organisms. In the last two decades several methods based on statistical theory have been developed to computationally reconstruct ancestral protein sequences. Laboratory resurrection of these ancestral proteins provides an excellent opportunity to explore aspects of ancient life that cannot be inferred from fossil records. Here we report the resurrection of ancestral thioredoxin enzymes (Trx) from the Precambrian era, dating back between 1.5 to 4 billion years (Gyr). Using single molecule force-clamp spectroscopy we demonstrate that all ancestral enzymes efficiently reduce disulfide bonds. From the force-dependency of the rate of reduction of an engineered substrate, we conclude that the Precambrian enzymes have similar chemical mechanisms of reduction that the extant enzymes. By contrast, the resurrected enzymes show thermal stabilities 20 to 30 °C higher than those of modern E. coli and human Trx as revealed by Differential Scanning Calorimetry (DSC). This high thermostability illustrate that ancient organisms lived in hot environments that have progressively cooled. Trx enzymes adapted to these environmental temperatures with similar chemical mechanisms than those observed in extant Trxs. Our work demonstrates that the combination of single molecule force spectroscopy together with the resurrection of ancestral proteins is a powerful new approach to study molecular evolution and the sequence-chemistry relationship in enzymes.