Learning About Protein Stability and Functional Activity From Ancestral Reconstruction

Understanding how proteins have evolved to adapt their stability and function to changing temperatures remains a central question in molecular biology. While structural analyses, site-directed mutagenesis, and directed evolution have yielded valuable insights, ancestral sequence reconstruction (ASR) has recently emerged as a powerful tool for addressing the drivers behind protein evolution. Specifically, by enabling the inference and experimental characterization of reconstructed ancient proteins, ASR provides unique perspectives on the molecular mechanisms underlying both thermostability and low-temperature-adaptation. This review outlines the historical development of research on protein temperature adaptation and highlights the role of ASR in advancing the field. Selected case studies illustrate how ASR has uncovered structural and dynamic features associated with extreme thermostability or enhanced activity at low temperatures. Common sources of uncertainty in ASR and how they can be addressed are also discussed. Finally, the broader potential of ASR is described, both for elucidating early evolutionary processes and for guiding the design of enzymes useful for industrial applications.