# Theoretical and experimental investigation of multi-domain protein folding and conformational dynamics

> **NIH NIH R01** · STATE UNIVERSITY NEW YORK STONY BROOK · 2020 · $456,568

## Abstract

PROJECT SUMMARY
Proper folding is crucial to achieving a protein’s unique three dimensional structure while the conformational
dynamics of the protein play a major role in its biological function. Although significant progress has been
made in understanding the folding/unfolding and conformational dynamics for single-domain proteins, these
two fundamental processes remain largely unexplored for multi-domain proteins, which have been suggested
to account for up to 80% of all eukaryotic proteins. Therefore, a significant disparity exists in our understanding
of the underlying mechanisms of folding/unfolding and conformational dynamics for the majority of human
proteins and we seek to address these uncertainties through theoretical and experimental investigation. In this
proposal, we devise a comprehensive strategy to answer the above, in-depth mechanistic unknowns regarding
multi-domain proteins through an energy landscape approach with subsequent experimental validation. The
energy landscape approach significantly improves technical capabilities through the establishment of
theoretical models for uncovering underlying mechanisms. By establishing the microscopic energy landscape
and structure based models, we will elucidate the folding/unfolding mechanisms of DPO4, a multi-domain,
model Y-family DNA polymerase critical for bypassing unrepaired DNA lesions, in vitro and in vivo (here means
mimicking in vivo conditions), and predict possible intermediate states and critical residues under various
environments, including the presence of the ribosome (co-translational) and a crowding agent (in vivo), as well
as different thermal and chemical denaturant conditions. Through our microscopic energy landscape and
structure based models, we will reveal the underlying mechanisms of conformational changes between various
conformational states of DPO4 upon binding to DNA or a protein replication factor PCNA through quantifying
the stability, kinetics, and structural hot spots critical for function. The theoretical model predictions will be
tested and validated through stopped-flow, circular dichroism, fluorescence energy transfer, and other
spectroscopic experiments. The results generated from the proposal will advance the DNA polymerase field
while the methods developed here are general and can serve as a framework for studies of folding/unfolding
and conformational dynamics of other multi-domain proteins. Moreover, the intricacies of protein
folding/unfolding and conformational transitions revealed by our proposed studies will facilitate protein design
and drug discovery.

## Key facts

- **NIH application ID:** 9985931
- **Project number:** 5R01GM124177-03
- **Recipient organization:** STATE UNIVERSITY NEW YORK STONY BROOK
- **Principal Investigator:** JIN WANG
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $456,568
- **Award type:** 5
- **Project period:** 2018-09-01 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9985931

## Citation

> US National Institutes of Health, RePORTER application 9985931, Theoretical and experimental investigation of multi-domain protein folding and conformational dynamics (5R01GM124177-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9985931. Licensed CC0.

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