# Development of a novel pulse-chase in-cell footprinting method for protein folding analysis

> **NIH NIH R01** · UNIVERSITY OF MARYLAND BALTIMORE · 2021 · $294,437

## Abstract

Project Summary
An understanding of protein folding pathways is important in understanding protein
function. Protein folding can be studied in vitro using full-length proteins in dilute
solutions under conditions optimized for successful folding. However, in the complex
environment of the cell, co- and post-translational folding of proteins has been
observed. The vectorial nature of co-translational folding as well as interactions
between the nascent chain, modifying enzymes, and molecular chaperones presumably
inhibit unfavorable interactions such as aggregation and smooth the energy landscape,
thereby making folding in the cell quite different from folding in the test-tube. To date,
there is a dearth of information about conformations available to a nascent chain in cells
and how its interacting partners affect these conformations. This gap in knowledge is
mainly due to the experimental difficulty of observing the folding reaction in cells. To
overcome this limitation, we aim to develop a new method for study protein folding. This
method, entitled pulse-chase in-cell fast photochemical oxidation of proteins (pcIC-
FPOP) couples traditional pulse-chase technology with a mass spectrometry-based in-
cell footprinting method. pcIC-FPOP will provide higher resolution information than gel
electrophoresis, which is the current analytical technique for analysis of pulse-chase
data, as tandem mass spectrometry can provide information on the amino acid residue-
level. The development of this method requires a redesign of the footprinting platform.
We have designed a new platform for in-cell footprinting that includes a stage-top
incubator and nanopositioning system. We will assemble and optimize the new platform
to demonstrate its efficacy for pcIC-FPOP (specific aim 1). We will use alpha 1
antitrypsin (A1AT) as a model system to test the ability of the method to probe short-
lived folding intermediates (specific aim 2). We will also study two mutants of A1AT, S
and Z to determine whether pcIC-FPOP can detect protein misfolding (specific aim 3).
The S mutant has a mild folding defect while the Z mutant has a more severe defect.
The study of both proteins will determine the sensitivity of the method in detecting
protein misfolding. The developed method would provide a new, higher resolution tool
for studying protein folding in the native cellular environment.

## Key facts

- **NIH application ID:** 10145031
- **Project number:** 5R01GM128985-04
- **Recipient organization:** UNIVERSITY OF MARYLAND BALTIMORE
- **Principal Investigator:** Lisa M Jones
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $294,437
- **Award type:** 5
- **Project period:** 2018-08-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10145031

## Citation

> US National Institutes of Health, RePORTER application 10145031, Development of a novel pulse-chase in-cell footprinting method for protein folding analysis (5R01GM128985-04). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10145031. Licensed CC0.

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