# Structural Dynamics and Mechanochemical Coupling in Nucleoprotein Machines

> **NIH NIH R01** · STANFORD UNIVERSITY · 2023 · $377,173

## Abstract

SUMMARY
 Central functions in DNA biology and biotechnology are carried out by nucleoprotein
machines. In these dynamic macromolecular assemblies, the DNA duplex is bound and
distorted in complex with protein and sometimes RNA. Biophysical measurements and
models are needed to understand the mechanisms of these machines, in which coordinated
conformational changes in protein and nucleic acid components are coupled with chemical
steps such as backbone cleavage or nucleotide hydrolysis. This is a renewal application for
a grant in which we previously developed high-resolution and multimodal single-molecule
approaches and applied them to elucidate mechanochemical coupling in the ATP-dependent
supercoiling motor DNA gyrase from E. coli. Here, we propose to leverage our methods and
insights to dissect the dynamics and mechanics of additional nucleoprotein machines,
focusing on the RNA-guided nucleases Cas9 and Cas12a and comparing DNA gyrase motors
across species. We will characterize substeps in DNA interrogation and DNA supercoiling,
molecular determinants of energy landscapes and kinetics, and the effects of mechanical
strains experienced in the genome. If successful, the project will determine the physical
mechanisms of DNA interrogation by RNA-guided nucleases in dynamic and mechanical
detail, providing a quantitative description of the target search process for enzymes that are
currently being exploited for gene editing and for a rapidly expanding set of other applications
involving specific targeting of activities to sites in the genome. New DNA gyrase
measurements will further elucidate biophysical specializations, structural properties, and
mechanical regulation of enzymes that are important targets for antibacterial drugs. Finally,
single-molecule methods development driven by these biophysical questions will have broad
applications in systems ranging from transcription to nucleosome remodeling.

## Key facts

- **NIH application ID:** 10617217
- **Project number:** 5R01GM106159-07
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Zev Bryant
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $377,173
- **Award type:** 5
- **Project period:** 2014-01-10 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10617217

## Citation

> US National Institutes of Health, RePORTER application 10617217, Structural Dynamics and Mechanochemical Coupling in Nucleoprotein Machines (5R01GM106159-07). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10617217. Licensed CC0.

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