# Investigations into ubiquitin binding proteins using structure guided reactivity

> **NIH NIH F31** · PURDUE UNIVERSITY · 2022 · $46,752

## Abstract

PROJECT SUMMARY
Ubiquitin (Ub) is a principal post-translational modifier crucial to eukaryotic biology. The Ub system relays an
intricate control over cellular processes through its attachment and detachment on substrate protein, giving rise
to a “ubiquitin code”. Deregulation in this code has severe consequences, primarily in disease pathogenesis. In
contrast to other post-translational modifications being singular, ubiquitination comes in many different flavors.
Subsequent attachment of the C-terminus of one Ub moiety onto a substrate lysine residue or one of eight
amines (Met1, Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, Lys63) of another Ub moiety gives rise to distinct Ub
linkages that are recognized by the decoders or readers of the “ubiquitin code”. These Ub receptors facilitate
important functions such as inflammatory response and DNA damage repair. Research over the past few
decades has provided critical insight into the writers that establish and erasers that remove the “ubiquitin code”,
yet our understanding of these Ub-receptors and Ub-interacting proteins is limited in comparison. This proposal
describes the use of Ub-based probes for profiling UBDs and Ub-dependent protein-protein interactions
using expanded protein chemistry. In two aims, we leverage genetic code expansion and liquid
chromatography-tandem mass spectrometry to address the goal of this project: to develop a means to identify
and structurally characterize Ub-dependent protein-protein interactions. Aim 1. Identify transiently interacting
UBDs of eukaryotic origin using photoaffinity biotinylated Ub probes and structurally characterize promising
candidates using chemo-selective ligation and X-ray crystallography. Aim 2. Develop a means to perform
chemical (poly)ubiquitination using the traceless Staudinger ligation for access to homogenous (poly)Ub probes
and (poly)ubiquitinated substrate protein. This will be applied to the atypical Lys29 ubiquitination of the 26S
proteasome receptor Rpn13, a regulatory effect of the E3 ligase UBE3C. These approaches in Ub chemical
biology will allow us to study Ub-dependent protein-protein interactions and their biophysical counterpart by
reconstituting the elements needed to chemically trap noncovalent interactions, a feature currently limiting the
study of the Ub system. Taken together, we seek to describe how these Ub-based interactions biochemically
attenuate function and corroborate these findings using structural evidence.

## Key facts

- **NIH application ID:** 10538227
- **Project number:** 1F31CA275390-01
- **Recipient organization:** PURDUE UNIVERSITY
- **Principal Investigator:** Rishi Patel
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $46,752
- **Award type:** 1
- **Project period:** 2022-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10538227

## Citation

> US National Institutes of Health, RePORTER application 10538227, Investigations into ubiquitin binding proteins using structure guided reactivity (1F31CA275390-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10538227. Licensed CC0.

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