# Repeat and Consensus Proteins: Stability, Cooperativity, Function, & Design

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2021 · $348,224

## Abstract

Project Summary
The broad objectives are 1) to determine the mechanisms by which protein stability is achieved,
and in particular, how stabilities of distant segments of proteins influence each other, giving rise
to cooperativity, using a combination of natural and designed repeat proteins, and 2) to
understand and leverage phylogenic-based consensus approaches to design proteins for high
stability and high levels of activity. These objectives will be achieved through three specific
aims. 1) Apply our nearest-neighbor 1D Ising formalism that we developed for natural repeat
proteins to quantify local folding and nearest neighbor coupling energies to a series of de novo
designed helical repeat proteins from the Baker lab. In parallel, we will measure folding kinetics,
using the energy landscape framework that results from the Ising analysis as a framework for
interpretation. Comparison to natural repeat proteins will reveal differences in folding between
designed and natural proteins. 2) Apply consensus design methods that we have used to
stabilize linear repeat proteins to globular proteins of different folds, sizes, and functions, and 3)
determine the extent to which biological activity is maintained. In Aim 2, we have identified
sixteen targets, and have strong preliminary results for six. We will determine structures by
NMR and x-ray crystallography, and stabilities using solution thermodynamics and kinetics. We
will dissect the basis of increased stability using sequence and structure metrics, and compare
with the "ancestral reconstruction" approach. In Aim 3, we will measure binding affinities,
specificities, and enzyme activities, and will focus on whether high stabilities decrease activity,
and whether dynamics changes is a general correlate.
 All three of these aims will use large numbers of comparisons among different proteins
to build a statistically rigorous and general picture of design and consensus features, allowing
us to generalize, determining what works, what does not, and why. This is a significant
improvement over the one-off anecdotal studies that have been described to date.
 Achieving these objectives will advance our understanding of the constraints on naturally
occurring protein sequences and evolution, and will address several paradigms including the "
principle of minimal frustration" and the "stability-activity tradeoff", and will identify key
differences between de novo-designed and natural protein sequences. These studies will
provide a deeper and more complete understanding of protein folding, and will also improve our
ability to design proteins for biotechnology and medicine.

## Key facts

- **NIH application ID:** 10159263
- **Project number:** 5R01GM068462-16
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** DOUGLAS E. BARRICK
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $348,224
- **Award type:** 5
- **Project period:** 2005-03-01 → 2022-09-19

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10159263

## Citation

> US National Institutes of Health, RePORTER application 10159263, Repeat and Consensus Proteins: Stability, Cooperativity, Function, & Design (5R01GM068462-16). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10159263. Licensed CC0.

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