# Time-Resolved X-ray Crystallography of Dynamics in Cysteine-Dependent Enzymes

> **NIH NIH R01** · UNIVERSITY OF NEBRASKA LINCOLN · 2021 · $295,994

## Abstract

ABSTRACT
Catalysis by cysteine-dependent enzymes is required for many essential biochemical pathways, including
central metabolism, redox homeostasis, and cellular signaling. Derangements in these pathways occur in
many disease states and targeting reactive cysteine residues is an emerging approach for developing potent
new drugs. All cysteine-dependent enzymes are transiently modified during catalysis, however little is known
about how cysteine modifications alter the structure and functional dynamics of proteins. We will use newly
developed time-resolved serial crystallography methods to characterize functionally important non-equilibrium
motions in the cysteine-dependent enzyme isocyanide hydratase (ICH) during catalysis. ICH is the principal
enzyme that detoxifies isocyanide natural products that possess antibiotic, antiviral, and anticancer properties.
Our preliminary data show that transient cysteine modification during ICH catalysis activates a non-equilibrium
protein dynamics that can be mapped in atomic detail by mix-and-inject serial X-ray crystallography. The
objective of this proposal is to develop and apply new models of catalysis-activated non-equilibrium motions in
ICH by analyzing the unprecedentedly information-rich datasets now available from mix-and-inject serial
crystallography experiments. We will use serial crystallography and computational approaches to determine
how transient modification of the active site cysteine thiolate activates protein motions that involve the whole
protein, are asymmetric in the ICH dimer, and are responsible for kinetic heterogeneity in two active sites of the
ICH dimer. We have created mutations that alter the equilibrium ICH conformational ensemble, impair
catalysis, and diminish the ability of ICH to protect bacteria from isocyanides. Using serial crystallography and
enzyme kinetics, we will characterize how these mutations alter allosteric motions during ICH catalysis and
prevent efficient intermediate hydrolysis. Finally, we generalize a model of enzyme motions facilitated by
conformational strain by determining the role of unusual side-chain and backbone conformational strain in
catalysis by a distant ICH homolog that diffracts X-rays to ultrahigh resolution. Combining computation, serial
crystallography, and enzyme kinetics, we will determine how conformational strain evolves during ICH catalysis
in unprecedented detail. In total, our work will elucidate how catalytic cysteine modification alters
conformational ensembles and non-equilibrium motions in enzymes. This work will also drive urgently needed
advances in synchrotron serial crystallography methodology in order to dramatically expand the accessibility of
these new structural biological techniques.

## Key facts

- **NIH application ID:** 10259757
- **Project number:** 5R01GM139978-02
- **Recipient organization:** UNIVERSITY OF NEBRASKA LINCOLN
- **Principal Investigator:** Mark A. Wilson
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $295,994
- **Award type:** 5
- **Project period:** 2020-09-20 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10259757

## Citation

> US National Institutes of Health, RePORTER application 10259757, Time-Resolved X-ray Crystallography of Dynamics in Cysteine-Dependent Enzymes (5R01GM139978-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10259757. Licensed CC0.

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