# Evolution of molecular complexes:  genetic, structural, and functional mechanisms for the evolution of oligomers and allostery

> **NIH NIH R01** · UNIVERSITY OF CHICAGO · 2021 · $299,091

## Abstract

We propose the first experimental studies of the historical origin and elaboration of molecular
complexes. Virtually all proteins assemble with specific molecular partners into precise geometric
arrangements, but we know little about the genetic and structural mechanisms by which these
complexes evolved or the evolutionary forces that explain their origin, elaboration, and long-term
persistence. We will combine ancestral protein reconstruction with biochemical, structural, and
functional experiments to reconstruct the evolution of molecular complexes in three model protein
families, enabling us to formulate and test general hypotheses about the evolutionary causes and
consequences of changes in stoichiometry, allostery, and specificity. All three protein families are
biologically essential, experimentally tractable, and exemplify distinct questions. The models are: 1)
hemoglobin, the major oxygen carrier in vertebrates, a heterotetramer that is biochemistry's iconic
case of an allosterically regulated molecular complex; 2) citrate synthase, an essential metabolic
enzyme that is a dimer in some lineages and an allosterically regulated hexamer in others, which
provides a rich case-study of the evolutionary relationship and long-term persistence of allostery and
complexity; and 3) steroid hormone receptors, a family of transcription factors that regulate
vertebrate reproduction and development and which evolved after gene duplication to specifically
assemble as homodimers, each with distinct regulatory functions. The project will address these
questions: 1) How and why do complexes originate and increase in stoichiometry from simpler forms?
2) What genetic and biophysical mechanisms mediate evolution of new and specific interfaces? 3) By
what mechanisms did allostery evolve? 4) Does selection for oligomer-associated functions account
entirely for the emergence and long-term persistence of molecular complexes? Or did substitutions
compatible only with the assembled form occur neutrally and entrench the complex, creating an
evolutionary ratchet towards greater complexity? And 5) After duplication, how did homomers evolve
to selectively assemble with copies of themselves (excluding their sister paralogs), and was evolution
of new functions constrained until this selectivity was achieved? By combining advanced techniques
from protein biochemistry and evolutionary biology, the project will articulate and test at
unprecedented resolution hypotheses about the evolutionary forces and biochemical mechanisms that
underlie the physical and functional properties of molecular complexes. It will also help to explain
why multimers are so widespread and, by revealing how evolution achieved specificity and allosteric
regulation, enhance engineering efforts to design complexes with these properties. The project will
also provide a methdological and conceptual template for future studies of other molecular complexes.

## Key facts

- **NIH application ID:** 10251124
- **Project number:** 5R01GM131128-03
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **Principal Investigator:** Joseph W Thornton
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $299,091
- **Award type:** 5
- **Project period:** 2019-09-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10251124

## Citation

> US National Institutes of Health, RePORTER application 10251124, Evolution of molecular complexes:  genetic, structural, and functional mechanisms for the evolution of oligomers and allostery (5R01GM131128-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10251124. Licensed CC0.

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