# Comprehensive analysis of fitness effects and epistasis along a billion-year evolutionary trajectory

> **NIH NIH R01** · UNIVERSITY OF CHICAGO · 2024 · $549,823

## Abstract

Epistatic interactions within proteins can, in principle, make the paths and outcomes of evolution
contingent on chance events; they can also entrench proteins with residues that appear to be optimal but
are accidents of history. The extent to which epistasis actually affected the trajectory and outcomes of
molecular evolution depends on the fitness effects of substitutions when they occurred in history
compared to their potential effects earlier or later in time and on the temporal order in which interacting
substitutions occurred. Deep mutational scanning studies have revealed pervasive epistasis among the
huge number of possible mutations, but no studies have directly assessed how the fitness effects of
substitutions that happened during history changed over time as the protein evolved. We will perform
the first comprehensive experimental analysis of the fitness effects of all amino acid
states that evolved in a protein during a long-term phylogenetic trajectory, both at the
time they occurred and if they had occurred at other points in history. These data will be
analyzed in the ordered temporal context of the protein's phylogeny and supplemented with biochemical
experiments, enabling a deep characterization of the causes and consequences of epistasis,
contingency and entrenchment across the billion-year history of an essential protein.
 Our model system is ideal for this purpose. Hsp90, the essential molecular chaperone in all eukary-
otic cells, plays key roles in protein folding and maturation, cell signaling, and a wide range of diseases.
Strong phylogenetic signal allows confident reconstruction of the billion-year evolutionary history of
Hsp90's protein sequence from the last common ancestor of animals, fungi and related protists to
present-day Saccharomyces cerevisiae. We will generate targeted protein libraries containing every
ancestral and derived state that occurred during this phylogenetic trajectory, singly and in every possible
pair, in the background of all 30 reconstructed ancestral proteins along the trajectory. Using a high-
resolution bulk competition assay in yeast, we will precisely measure selection coefficients and epistatic
interactions and quantify how these properties changed over time. This will reveal the fitness effects and
interactions of every substitution at the approximate time it occurred, as well as the effects and
interactions it would have had if it happened (or reverted to the ancestral state) at any point earlier or
later during the trajectory. We will also apply biophysical and structural techniques to elucidate the
underlying biochemical mechanisms that drove these genetic and evolutionary phenomena. This work
will provide deep new insight into the ways in which proteins' genetic and physical architecture
influences, and is influenced by, the processes by which they evolve; it will also strengthen our
understanding of sequence-structure-function relationships in a biologically essential protein.

## Key facts

- **NIH application ID:** 10833151
- **Project number:** 5R01GM139007-04
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **Principal Investigator:** DANIEL N BOLON
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $549,823
- **Award type:** 5
- **Project period:** 2021-06-01 → 2026-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10833151

## Citation

> US National Institutes of Health, RePORTER application 10833151, Comprehensive analysis of fitness effects and epistasis along a billion-year evolutionary trajectory (5R01GM139007-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10833151. Licensed CC0.

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