# Non-canonical amino acid mutagenesis in the engineering of insulin biophysics

> **NIH NIH R01** · CALIFORNIA INSTITUTE OF TECHNOLOGY · 2021 · $301,223

## Abstract

PROJECT SUMMARY/ABSTRACT
We propose to explore the use of proline analogs to engineer and probe the biophysical behavior of
insulin. Insulin contains a single proline residue – at position 28 of the B-chain – which is known to play
critical roles in controlling the rates of onset of action and fibrillation of pharmaceutical preparations of
the protein. Replacement of proline through conventional mutagenesis has led to FDA-approved rapid-
acting insulins, but destabilizes the protein with respect to fibrillation. Conventional mutagenesis suffers
from a fundamental limitation when applied to proline; any amino acid change converts the
conformationally restricted cyclic proline residue to a more flexible acyclic one. We have recently found
that replacement of the proline residue at position 28 of the insulin B-chain by (4S)-hydroxyproline –
through non-canonical amino acid mutagenesis – yields an active form of insulin that dissociates more
rapidly, and fibrillates more slowly, than the wild-type protein. This approach allows one to alter critical
molecular interactions around position B28 without sacrificing the unique conformational properties of
proline. This proposal seeks to expand the known ‘proline chemical space’ that can be accessed in the
bacterial expression of recombinant insulin. We will accomplish this objective by assessing the
translational activity of a carefully chosen set of proline analogs in E. coli, by creating new prolyl-tRNA
synthetases to activate the analogs of interest, and by analyzing the biophysical behavior of the resulting
insulin variants by experimental and computational means. The proposed work will provide new forms of
insulin with altered biophysical properties, expand the toolkit for engineering protein structure and
function, and enhance our understanding of protein association and dynamics.

## Key facts

- **NIH application ID:** 10141263
- **Project number:** 5R01GM134013-03
- **Recipient organization:** CALIFORNIA INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** DAVID A TIRRELL
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $301,223
- **Award type:** 5
- **Project period:** 2019-07-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10141263

## Citation

> US National Institutes of Health, RePORTER application 10141263, Non-canonical amino acid mutagenesis in the engineering of insulin biophysics (5R01GM134013-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10141263. Licensed CC0.

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