Ancestral Sequence Reconstruction Reveals the Evolutionary History of the Folding Pathway and Landscape of Ribonucleases H

Sequence changes that arise over evolution can alter various features of a protein's energy landscape, including its global stability and folding pathway. A comprehensive understanding of how the amino acid sequence dictates the energy landscape is critical to our understanding of biophysical properties of proteins, particularly for protein engineering applications. Here, we use ancestral sequence reconstruction (ASR) to examine the evolutionary history of homologous proteins by inferring and generating ancestral states and characterizing their energy landscapes. A systematic analysis of a protein family's evolutionary history should reveal how thermodynamic and kinetic features of the landscape are derived and maintained. This allows us to identify the sequence determinants of stability and folding pathways and reveal how different selective pressures alter the energy landscape and determine trajectories in sequence space.To access a protein family's evolutionary history, we utilized ASR to generate ancestral states along the lineages of mesophilic and thermophilic ribonucleases H. We observe pronounced trends that reveal the divergence in thermostability along the mesophilic and thermophilic lineages. Despite a clear selection for stability, the underlying mechanism was found to be variable, which we attribute to thermodyanmic system drift. Characterization of the ancestral folding pathway by kinetics, fragment models, and hydrogen-exchange mass spectrometry (HX-MS) reveals a subtly but distinctly changing landscape for the ribonuclease H family, revealing the role of critical mutations that alter the structure of partially folded intermediates and modulate barrier heights along the folding pathway.