# Mutational Pleiotropy, Epistasis, and the Adaptive Evolution of Hemoglobin Function

> **NIH NIH R01** · UNIVERSITY OF NEBRASKA LINCOLN · 2020 · $369,835

## Abstract

The step-by-step evolution of novel phenotypes is central to several fundamental questions in biology. In
studies of novel protein functions, the problem becomes experimentally tractable if it is possible to identify and
functionally characterize the complete set of causative mutations. With such a system, it is possible to address
key questions: Do novel functions evolve via the successive fixation of beneficial mutations that each produce
an adaptive change in phenotype when they first arise? Alternatively, are evolutionary transitions in protein
function facilitated by neutral mutations that produce no adaptive benefit when they first arise, but which
potentiate the function-altering effects of subsequent mutations? By reconstructing all possible mutational
pathways that connect ancestral and descendant proteins it is also possible to address fundamental questions
about the roles of contingency and determinism in protein evolution. For example: Can novel functions evolve
from any possible ancestral starting point, or are specific evolutionary outcomes contingent on prior history?
We will address these questions by experimentally dissecting the molecular basis of a key physiological
innovation during vertebrate evolution. Specifically, we will examine the evolution of a unique allosteric
mechanism for regulating hemoglobin (Hb) function in the red blood cells of crocodilians. This unique mode of
allosteric regulatory control contributes to crocodilians’ extraordinary capacities for breath-hold diving. Using
ancestral protein resurrection in conjunction with a combinatorial protein engineering approach based on site-
directed mutagenesis, we will examine the effects of sequential mutational steps in the evolution of the novel
allosteric mechanism of crocodilian Hb. We will also obtain insights into the structural basis of the change in Hb
function, as X-ray crystallography experiments will reveal biophysical mechanisms at atomic resolution. The
specific aims of the project are as follows: (1) Identify the specific mutations that are responsible for the
evolution of the novel protein function, and quantify their additive and nonadditive effects; and (2) Identify and
characterize the biophysical mechanisms responsible for the functional transition (gain of novel function, loss
of ancestral function). In combination, accomplishing Specific Aims 1 and 2 will reveal the molecular basis of a
key physiological innovation and will provide general insights into the pathways by which such innovations
evolve.

## Key facts

- **NIH application ID:** 9999011
- **Project number:** 5R01HL087216-12
- **Recipient organization:** UNIVERSITY OF NEBRASKA LINCOLN
- **Principal Investigator:** Jay Storz
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $369,835
- **Award type:** 5
- **Project period:** 2008-09-22 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9999011

## Citation

> US National Institutes of Health, RePORTER application 9999011, Mutational Pleiotropy, Epistasis, and the Adaptive Evolution of Hemoglobin Function (5R01HL087216-12). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9999011. Licensed CC0.

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