# Protein stabilizers from tardigrades

> **NIH NIH R01** · UNIV OF NORTH CAROLINA CHAPEL HILL · 2020 · $379,270

## Abstract

Program Director/Principal Investigator (Last, First, Middle): Pielak, Gary J.
Project Summary.
Tardigrades are microscopic animals that survive extreme stresses, including desiccation, freezing, boiling,
large doses of ionizing radiation and even the vacuum of outer space. We have identified the family of proteins
that allows tardigrades to survive desiccation. These Cytosolic Abundant Heat Soluble (CAHS) proteins are
unique to tardigrades. The goal of the project is to define the protective mechanism used by CAHS proteins to
shield client proteins from desiccation-induced damage. This knowledge will facilitate the development of new
molecules to increase the stability and shelf life of protein-based drugs and industrial enzymes.
We hypothesize that CAHS proteins protect client proteins via encapsulation in a reversible matrix. We
propose a model where fully hydrated CAHS proteins possess partially-collapsed termini separated by a
flexible linker. Upon water removal, the termini interact intermolecularly, forming a protective gel matrix around
the client proteins. The matrix persists and maintains client protein protection even upon complete desiccation.
The model is supported by preliminary published and new data from studies of enzyme activity, NMR-detected
amide proton exchange, 19F NMR, circular dichroism spectropolarimetry, Fourier-transform infrared
spectroscopy and rheometry. The model will be further tested in three independent and complementary aims.
Aim 1: Determine sequence/function relationships in the solid state. We predict that protection is modular; linkers
can be swapped, and ends can be swapped as long as each molecule possesses two ends separated by a
linker. We will test this idea using fragments comprising the modules alone and combinations of modules. We
will determine the degree to which CAHS proteins and their fragments protect enzymes against desiccation-
induced inactivation and assess their ability to preserve the structure of a model protein in the solid state by
using NMR-detected amide proton/deuterium exchange.
Aim 2: Determine sequence/function relationships in gels and solution. We will quantify the effects of CAHS
proteins and the fragments on their modified standard-state free energy of unfolding of a client protein.
Aim 3: Define sequence/structure relationships in the solid state, gels and solution. We will quantify the ability
of CAHS proteins and their fragments to form gels using rheometry, a method to quantify gel strength. The
secondary structure of intact CAHS proteins and their fragments will be quantified using synchrotron-radiation
circular-dichroism spectropolarimetry and Fourier-transform infrared spectroscopy.
OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page

## Key facts

- **NIH application ID:** 9833528
- **Project number:** 5R01GM127291-02
- **Recipient organization:** UNIV OF NORTH CAROLINA CHAPEL HILL
- **Principal Investigator:** GARY JOSEPH PIELAK
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $379,270
- **Award type:** 5
- **Project period:** 2018-12-15 → 2021-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9833528

## Citation

> US National Institutes of Health, RePORTER application 9833528, Protein stabilizers from tardigrades (5R01GM127291-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9833528. Licensed CC0.

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