High-Throughput Reconstruction of Ancestral Protein Sequence, Structure, and Molecular Function

  • Kelsey Aadland
  • Charles Pugh
  • Bryan Kolaczkowski
Part of the Methods in Molecular Biology book series (MIMB, volume 1851)


Ancestral protein sequence reconstruction is a powerful technique for explicitly testing hypotheses about the evolution of molecular function, allowing researchers to meticulously dissect how historical changes in protein sequence impacted functional repertoire by altering the protein’s 3D structure. These techniques have provided concrete, experimentally validated insights into ancient evolutionary processes and help illuminate the complex relationship between protein sequence, structure, and function. Inferring the protein family phylogenies on which ancestral sequence reconstruction depends and reconstructing the sequences, themselves, are amenable to high-throughput computational analysis. However, determining the structures of ancestral-reconstructed proteins and characterizing their functions typically rely on time-consuming and expensive laboratory analyses, limiting most current studies to examining a relatively small number of specific hypotheses. For this reason, we have little detailed, unbiased information about how molecular function evolves across large protein family phylogenies. Here we describe a generalized protocol that integrates ancestral sequence reconstruction with structural homology modeling and structure-based molecular affinity prediction to characterize historical changes in protein function across families with thousands of individual sequences. We highlight key steps in the analysis protocol requiring particularly careful attention to avoid introducing potential errors as well as steps for which computationally efficient subroutines can be substituted for more intensive approaches, allowing researchers to scale the analysis up or down, depending on available resources and requirements for reproducibility and scientific rigor. In our view, this approach provides a compelling compliment to more laboratory-intensive procedures, generating important contextual information that can help guide detailed experiments.

Key words

Ancestral sequence reconstruction Structural modeling Protein function prediction Affinity prediction Protein evolution Molecular evolution 

Supplementary material (10 kb)
Data 1 Python scripts and data required for the presented examples. (ZIP 10 KB)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Kelsey Aadland
    • 1
  • Charles Pugh
    • 1
  • Bryan Kolaczkowski
    • 1
    • 2
  1. 1.Department of Microbiology & Cell Science, Institute for Food and Agricultural SciencesUniversity of FloridaGainesvilleUSA
  2. 2.Genetics InstituteUniversity of FloridaGainesvilleUSA

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