# A Method to Insert Locked cis- and trans-Proline Analogues in Full-Length Proteins

> **NIH NIH R21** · VIRGINIA POLYTECHNIC INST AND ST UNIV · 2020 · $182,350

## Abstract

PROJECT SUMMARY
Proline isomerization is emerging as a significant mechanism of regulating cell signaling, transcription and the
cell cycle. Determination of the proline isomerization state in vivo is extremely challenging with existing methods.
We propose to incorporate unique molecular tools, cis-locked and trans-locked Pro analogues, into full-length
proteins to permit analysis of conformation-specific activity in living cells (Figure 1). We will insert our locked
alkene analogues into Protein Aurora Borealis (Bora) using in vitro transcription-translation as a proof-of-principle
for the method. We will validate locked protein tools against a known outcome, activation of Aurora A by Bora in
living cells. The effect of proline isomerization states of Bora, with and without phosphate, will be investigated in
vitro, and phenotypically in living cells. The overall goal of this work is to develop a robust method to insert
locked cis- and trans-Pro analogues into full-length proteins. Developing the tools to examine the effects of
proline isomerization state in living cells is critical to a broader understanding of cell signaling, transcription, and
cell cycle regulation. These tools will enable determination of the precise isomer of cis- or trans-Pro that is active
in a specific cellular process. To achieve these goals, we will pursue the following aims.
Aim 1. Incorporate cis- and trans-locked Ser-Pro analogues into full-length proteins. Native and
conformationally locked full-length Bora proteins will be synthesized in an E. coli expression system with tRNA
chemically misacylated with locked Ser-Pro dipeptide analogues. Proteins will be verified as substrates for the
proline-directed Ser/Thr protein kinases, GSK3 and Cdk1-cyclin B in vitro. The phosphorylation state at the Ser-
Pro analogue positions will be determined by LC-MSMS.
Aim 2. Incorporate phosphorylated cis- and trans-locked Ser-Pro analogues into full-length proteins.
Native and conformationally locked phosphorylated Bora proteins will be synthesized in an E. coli expression
system with tRNA chemically misacylated with phosphorylated and locked Ser-Pro dipeptide analogues. The
effect of locked phosphorylated Bora proteins on Aurora A kinase activity will be verified in vitro.
Aim 3. Demonstrate the feasibility of using locked full-length proteins as a tool in cell biology. Full-length
Bora proteins with locked Ser-Pro dipeptide analogues, in both unphosphorylated and phosphorylated states,
will be microinjected into cell cycle synchronized human cells depleted of the endogenous Bora protein, and the
cell phenotypes will be evaluated by live-cell microscopy.

## Key facts

- **NIH application ID:** 9937769
- **Project number:** 5R21GM132715-02
- **Recipient organization:** VIRGINIA POLYTECHNIC INST AND ST UNIV
- **Principal Investigator:** FELICIA A ETZKORN
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $182,350
- **Award type:** 5
- **Project period:** 2019-06-01 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9937769

## Citation

> US National Institutes of Health, RePORTER application 9937769, A Method to Insert Locked cis- and trans-Proline Analogues in Full-Length Proteins (5R21GM132715-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9937769. Licensed CC0.

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