# Use of the Noncanonical Amino Acid Mutagenesis Technique in Combination with Other Approaches to Study Functions of Posttranslational Lysine Modifications in Proteins

> **NIH NIH R35** · TEXAS A&M UNIVERSITY · 2022 · $310,360

## Abstract

PROJECT SUMMARY/ABSTRACT
 As a naturally existing amber suppression system, the pyrrolysine (Pyl) incorporation machinery has turned
into an enormous tool for undergoing amber suppression-based noncanonical amino acid (ncAA) mutagenesis
in both prokaryotic and eukaryotic cells. By ectopically expressing tRNAPyl and pyrrolysyl-tRNA synthetase
(PylRS) or a PylRS mutant that charges tRNAPyl with an ncAA, about 200 ncAAs have been genetically encoded
by the amber codon in various cells. As one of the original pioneers in the research field of engineering the Pyl
system for the genetic incorporation of ncAAs, the PI’s group has contributed more than one third of the total
encoded ncAAs. These ncAAs contain a large variety of functionalities, allowing a myriad of applications in both
academia and industry possible. After more than a decade of engineering the Pyl system for the ncAA
incorporation, the field is now able to use the Pyl system-based ncAA mutagenesis technique to conduct grand
explorations to address fundamentally important biological questions. In the past, the PI’s lab has devised a
variety of ncAA mutagenesis-based approaches for the synthesis of proteins with posttranslational lysine
modifications (lysine PTMs). A novel method that allows direct functionalization of ubiquitin and ubiquitin like
proteins for their conjugation with other proteins has also been developed in the PI’s lab. With all these methods
coming to fruition, the PI’s lab is shifting its research focus to use their developed techniques to study basic and
fundamentally important biological questions. Five specific directions will be pursued. The first direction is to use
the ncAA mutagenesis technique to produce designer nucleosomes (nucleosomes with defined lysine PTMs) for
probing histone lysine sites and PTM types targeted by epigenetic erasers including SIRT6, SIRT7, HDAC1, and
LSD1 (HDAC1 and LSD1 in their native complexes). The second direction is to use reconstituted designer
nucleosomes as probes to enrich their binding partners from cells whose identities can be determined using
mass spectrometry-based proteomic analysis. The third direction is to conduct cryo-EM analysis of designer
nucleosomes bound with SIRT6, SIRT7, HDAC1, and LSD1 (HDAC1 and LSD1 in their native complexes) to
elucidate the structural basis of the four enzymes in their recognition of targeted lysine sites and PTMs in
chromatin. The fourth direction is to synthesize different triubiquitin isoforms and use them as probes to enrich
binding partners from cells whose identities will be confirmed with mass spectrometry-based proteomic analysis.
The last but not least direction is to synthesize cyclic GMP-AMP synthase (cGAS), which is a frontline sensor in
human cells that detect double-stranded DNA from pathogens and triggers innate immune responses, with lysine
PTMs and to study the functional roles of lysine PTMs in regulating activity, cellular localization and cellular half-
life of cGAS. A l...

## Key facts

- **NIH application ID:** 10406602
- **Project number:** 1R35GM145351-01
- **Recipient organization:** TEXAS A&M UNIVERSITY
- **Principal Investigator:** Wenshe Ray Liu
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $310,360
- **Award type:** 1
- **Project period:** 2022-04-01 → 2027-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10406602

## Citation

> US National Institutes of Health, RePORTER application 10406602, Use of the Noncanonical Amino Acid Mutagenesis Technique in Combination with Other Approaches to Study Functions of Posttranslational Lysine Modifications in Proteins (1R35GM145351-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10406602. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*