# Acetyllysine Structure-Function Analysis by 13C Direct-Detect NMR Spectroscopy

> **NIH NIH R21** · PENNSYLVANIA STATE UNIVERSITY, THE · 2020 · $221,903

## Abstract

PROJECT SUMMARY
Protein post-translational modifications are ubiquitous events that regulate the core biological functions of the
cell. Lysine acetylation was first characterized in the context of chromatin structure and transcription, where
abundant acetylation of histone proteins is known to regulate gene expression. Recent proteomic data have
expanded this paradigm and suggest that lysine acetylation is ubiquitous among both nuclear and cytosolic
proteins. Mechanistic studies to document how these marks are placed, utilized to transduce signals, and
eliminated when signals need to be turned off have not kept pace with proteomic discoveries. A major bottleneck
persists due to the paucity of transferrable chemical biology probes of acetyllysine that do not perturb structure,
which is pre-requisite for downstream structure-mechanism studies. The PI’s laboratory is uniquely positioned
to advance the acetylation signaling field through this proposal, which will seek to determine the technological
feasibility of direct acetyllysine readout through NMR spectroscopy applied to 13C-enriched acetyl
groups. The PI has a proven track record of technology development in 13C direct-detect biomolecular NMR,
which is used to probe the biophysics of disordered proteins and the complexes they form. The current project
proposes to generate acetyllysine with uniform 13C and 15N enrichment in order to leverage the similarity between
the peptide bond and the side chain acetamide functional group. In this context, the first specific aim of this
project is to develop a chemical biology route to site-specific isotopic-enrichment of acetyllysine. The initial
strategy involves synthetic coupling of 13C-acetate to Coenzyme-A, which will be used as a cofactor for enzymatic
transfer of the acetyl group to recombinant protein, with or without isotope labelling of the protein. To validate
this procedure with a thoroughly described enzyme-substrate pair, the preliminary work will employ acetylation
of Histone 3, Lysine 14 by the Ada2/Gcn5 complex. Because all known lysine acetyltransferases use acetyl-CoA
as a cofactor for substrate acetylation, the developed approach will be general and readily adapted to a broad
range of enzyme-substrate pairs. The second specific aim is to design broadly applicable NMR experiments to
target acetyllysine with high specificity. This project will lead to 13C direct-detect (N-Acetyl)-COCme and (N-
Acetyl)-CON experiments that will allow direct NMR readout of the acetyl group in a broader range of targets.
Further, 3D spectroscopy built up from these new 2D detection platforms will be developed to unambiguously
connect acetyl resonances with the aliphatic side chain. The new technology proposed here will enable
applications including site-specific assignment of newly-identified modifications, investigation of novel binding
modes as new reader proteins are discovered, and determination of solution NMR structures based on isotope
filtered NOE mea...

## Key facts

- **NIH application ID:** 9953232
- **Project number:** 1R21GM137129-01
- **Recipient organization:** PENNSYLVANIA STATE UNIVERSITY, THE
- **Principal Investigator:** Scott A Showalter
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $221,903
- **Award type:** 1
- **Project period:** 2020-05-01 → 2022-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9953232

## Citation

> US National Institutes of Health, RePORTER application 9953232, Acetyllysine Structure-Function Analysis by 13C Direct-Detect NMR Spectroscopy (1R21GM137129-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9953232. Licensed CC0.

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