# Metastable Crystallins: Structure and Stabilization

> **NIH NIH R01** · UNIVERSITY OF MISSOURI-COLUMBIA · 2021 · $375,875

## Abstract

ABSTRACT
-Crystallin is a complex macromolecule that accounts for nearly 40% of the adult lens proteins. The
chaperone-like activity of -crystallin, is implicated as a key component in the maintenance of lens
transparency by suppression of crystallin aggregation. In vitro studies of α-crystallin have shown that
chaperone activity is increased when α-crystallin is subjected to heat (48-60oC) and then brought back to 25-
37oC. Similarly, α-crystallin subjected to urea-induced unfolding and refolding also displays increased
chaperone activity. Understanding the molecular organization and properties of crystallin subunits in activated
chaperones would help answer questions on how -crystallin chaperone-like activity might be harnassed and
manipulated for the development of protein-based therapeutics. In our studies of the role of subunit interactions
in chaperone activity, a recombinant αB-crystallin expressed after deleting the 54-61 sequence (resulting in a
protein designated as αB∆54-61) was found to form ~ 40 % smaller oligomer than the wild-type protein
oligomer but to show a 10-fold increase in chaperone activity. The Specific Aims of this project will uncover the
molecular changes that account for the increased chaperone activity in heat- and urea-treated α-crystallin and
deletion mutant of αB-crystallin. Specific Aim 1: a) Determine the mechanism of αB-crystallin chaperone
activation after deletion of the 54-61 sequence and b) determine the biological implications of enhanced
chaperone activity in cell culture system. Specific Aim 2: a) Determine the functional units and mechanism of
activation in α-crystallin chaperone after thermal stress and urea-induced unfolding and refolding and b)
investigate the cytoprotective effect of heat- and urea-activated crystallins. Novel cross-linker(s) will be used to
gain fresh insights into the “cryptic” chaperone sites getting exposed in the activated crystallins. The studies
will also make use of site-directed mutagenesis, mass spectrometric analysis and biophysical techniques to
uncover the molecular changes at the secondary and tertiary structure levels and to delineate the quaternary
organization of the subunits in the oligomers showing increased chaperone activity. To see whether the
activated α-crystallins can be exploited to protect cells from oxidative injury, the effects of stress-inducing
agents such as H2O2, staurosporine or etoposide will be investigated in Cos-7, HeLa, HEK293 and ARPE-19
cells. Further, the ability of activated chaperones to suppress aggregation of mutant proteins (αAG98R) as well
as fibril-forming β-amyloid will be investigated both in vitro and ex-vivo. The long-term goals of the studies are
to understand the structure–function relationship of activated α-crystallin and develop crystallin proteins that
have therapeutic value in protein conformational diseases.

## Key facts

- **NIH application ID:** 10200048
- **Project number:** 5R01EY023219-09
- **Recipient organization:** UNIVERSITY OF MISSOURI-COLUMBIA
- **Principal Investigator:** KRISHNA K SHARMA
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $375,875
- **Award type:** 5
- **Project period:** 2013-05-01 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10200048

## Citation

> US National Institutes of Health, RePORTER application 10200048, Metastable Crystallins: Structure and Stabilization (5R01EY023219-09). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10200048. Licensed CC0.

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