1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Tracing a protein's folding pathway over evolutionary time using ancestral sequence reconstruction and hydrogen exchange

  1. Shion An Lim
  2. Eric Richard Bolin
  3. Susan Marqusee  Is a corresponding author
  1. University of California, Berkeley, United States
Research Article
  • Cited 6
  • Views 2,598
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Cite this article as: eLife 2018;7:e38369 doi: 10.7554/eLife.38369


The conformations populated during protein folding have been studied for decades; yet, their evolutionary importance remains largely unexplored. Ancestral sequence reconstruction allows access to proteins across evolutionary time, and new methods such as pulsed-labeling hydrogen exchange coupled with mass spectrometry allow determination of folding intermediate structures at near amino-acid resolution. Here, we combine these techniques to monitor the folding of the ribonuclease H family along the evolutionary lineages of T. thermophilus and E. coli RNase H. All homologs and ancestral proteins studied populate a similar folding intermediate despite being separated by billions of years of evolution. Even though this conformation is conserved, the pathway leading to it has diverged over evolutionary time, and rational mutations can alter this trajectory. Our results demonstrate that evolutionary processes can affect the energy landscape to preserve or alter specific features of a protein's folding pathway.

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All data generated or analyzed during this study are included in the manuscript main text, supporting files, and source data

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Author details

  1. Shion An Lim

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2136-2732
  2. Eric Richard Bolin

    Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Susan Marqusee

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7648-2163


National Institute of General Medical Sciences (GM050945)

  • Shion An Lim
  • Eric Richard Bolin
  • Susan Marqusee

National Science Foundation (Graduate Research Fellowship)

  • Shion An Lim

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Lewis E Kay, University of Toronto, Canada

Publication history

  1. Received: May 14, 2018
  2. Accepted: September 9, 2018
  3. Accepted Manuscript published: September 11, 2018 (version 1)
  4. Version of Record published: September 26, 2018 (version 2)


© 2018, Lim et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.


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