Reconstructing Ancient Proteins to Understand the Causes of Structure and Function

Abstract

A central goal in biochemistry is to explain the causes of protein sequence, structure, and function. Mainstream approaches seek to rationalize sequence and structure in terms of their effects on function and to identify function’s underlying determinants by comparing related proteins to each other. Although productive, both strategies suffer from intrinsic limitations that have left important aspects of many proteins unexplained. These limits can be overcome by reconstructing ancient proteins, experimentally characterizing their properties, and retracing their evolution through time. This approach has proven to be a powerful means for discovering how historical changes in sequence produced the functions, structures, and other physical/chemical characteristics of modern proteins. It has also illuminated whether protein features evolved because of functional optimization, historical constraint, or blind chance. Here we review recent studies employing ancestral protein reconstruction and show how they have produced new knowledge not only of molecular evolutionary processes but also of the underlying determinants of modern proteins’ physical, chemical, and biological properties.

Evolution of protein specificity: insights from ancestral protein reconstruction

“…Finally, comparative studies of homologous proteins are limited in their capacity to identify the minimal causes of functional differences, because long periods of sequence divergence and epistatic interactions among substitutions might obscure relatively simple echanisms by which proteins in the deep past diverged in function. [23, 24]. …”

Higher-order epistatic networks underlie the evolutionary fitness landscape of a xenobiotic-degrading enzyme

“…Previous studies using ASR examined functions that evolved millions or billions of years ago [2][3][4][31][32][33][34][35] . Our study demonstrates that this technique, combined with biochemical and mutational assays, can effectively uncover the molecular mechanisms underlying recently evolved functional novelty.…”

Engineering indel and substitution variants of diverse and ancient enzymes using Graphical Representation of Ancestral Sequence Predictions (GRASP)

“…Sequencing technology is driving the identification of the extant (modern) portion of the universe of biological sequences 1,2,3 . With this increased coverage of natural diversity we are now better placed than ever before to leverage ancestral sequence reconstruction (ASR) to recover the ancestral portion and trace the evolutionary events that determine biological function and structure 4 . This is especially useful for protein engineering; the evolutionary record reveals essential cues for the discovery of new enzymes and resurrection of ancestral enzymes often generates enzymes with novel properties that can be exploited in biocatalysis 5,6,7,8 .…”

“…In addition, we also evaluated a large-scale inference of, but did not resurrect, the following family. 4. The ketol-acid reductoisomerase (KARI) family includes enzymes in the branched-chain amino acid biosynthetic pathway (similar to DHAD) present in bacteria, fungi, and plants.…”