Tracing a protein's folding pathway over evolutionary time using ancestral sequence reconstruction and hydrogen exchange

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Abstract

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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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.

"This ORCID iD identifies the author of this article:" 0000-0003-2136-2732
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.
Susan Marqusee

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

For correspondence
marqusee@berkeley.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-7648-2163

Funding

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.

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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.