# A New Platform for Studying Protein Folding in Live Cells

> **NIH NIH F31** · UNIVERSITY OF MARYLAND BALTIMORE · 2022 · $28,514

## Abstract

Project Summary
 Understanding protein folding is critical for understanding proper protein function. It is well known that
various diseases are associated with misfolding or folding deficiencies. Protein folding has been studied in vitro
with residue resolution. However, it is well established that folding in the cell can be significantly different from
folding studies in an isolated environment, notably due to the interactions of chaperones. Studying protein folding
in a cellular context is limited with few approaches being able to tease out high resolution structural detail from
a complex cellular environment. To overcome these limitations, we aim to develop a new method to study protein
folding. This method, pulse chase in-cell fast photochemical oxidation of proteins (pcIC-FPOP), couples pulse-
chase technology with mass spectrometry-based in cell footprinting which will allow for quick analysis of short-
lived protein folding intermediates. This method aims to fill gaps in knowledge of protein folding and its role in
various diseases. We designed a new platform for in cell hydroxyl radical protein footprinting. The system called
Platform Incubator with an XY movable stage (PIXY) includes a sterile incubation system, XY movable stage
with controllers, peristaltic pumps, a 248 nm KrF excimer laser, and optic mirrors. The system has been tested
and optimized as the new platform for cell growth and IC-FPOP studies, referred to as PIXY IC- FPOP. Transient
transfections were performed to assess and compare cell culture quality under standard incubator and stage top
incubator conditions. Furthermore, a luciferase assay was performed to quantitate transfection efficiency. PIXY
IC-FPOP experiments were also carried out to access comparability with the standard IC-FPOP flow system
technique and results. PIXY will be configured into an automated system through LABVIEW software. Once
optimized, PIXY will be further validated as a protein folding tool by observing the folding dynamics of well-
studied abundant serpin protein, alpha-1 antitrypsin as well as its chaperone interaction. The sensitivity of the
PIXY platform to decipher between wild type protein folding dynamics and subsequent mutants will allow for the
expansion of this method to other protein groups. pcIC-FPOP will be used to study transthyretin (TTR) misfolding
and aggregation known to be associated with the amyloid disease, hereditary transthyretin amyloidosis (hATTR).

## Key facts

- **NIH application ID:** 10337041
- **Project number:** 5F31GM140651-02
- **Recipient organization:** UNIVERSITY OF MARYLAND BALTIMORE
- **Principal Investigator:** Dante Tenielle Johnson
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $28,514
- **Award type:** 5
- **Project period:** 2021-02-01 → 2022-10-16

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10337041

## Citation

> US National Institutes of Health, RePORTER application 10337041, A New Platform for Studying Protein Folding in Live Cells (5F31GM140651-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10337041. Licensed CC0.

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