# Structural characterization of large eukaryotic proteins containing both folded and disordered domains

> **NIH NIH R35** · SCRIPPS RESEARCH INSTITUTE, THE · 2023 · $452,500

## Abstract

Intrinsically disordered proteins (IDPs) are highly abundant in eukaryotes and play a central role in key
cellular regulatory pathways and in the spatial organization of the cell. Approximately half of the proteins
in the human proteome are either fully disordered or contain long disordered regions (IDRs). The cellular
abundance of disordered proteins is tightly regulated and dysregulation or mutation of IDPs and IDRs is
associated with devastating diseases such as cancer, diabetes, cardiovascular disease, and
neurodegenerative disease. Disordered proteins are highly flexible and undergo transient and dynamic
intramolecular and intermolecular interactions that are central to their regulatory functions. Molecular
level characterization of the numerous human regulatory proteins that contain both structured and
disordered domains represents an enormous challenge to the traditional methods of structural biology.
Most studies to date have relied upon a reductionist, divide-and-conquer approach, in which the ordered
and disordered regions are expressed independently and studied in isolation. However, within the cell,
the folded and disordered domains of a given protein act synergistically to allow it to perform its biological
function and a full understanding of the underlying molecular mechanism can only be achieved through
a holistic, rather than reductionist, approach. An overarching goal of our research is to utilize a non-
reductionist approach, aided by intein-based segmental isotope labeling, to characterize the structural
ensemble, dynamics, and interactions of eukaryotic proteins containing both folded and disordered
domains. This strategy is broadly applicable to large, dynamic proteins with disordered domains since it
is relatively straightforward to identify or engineer optimal intein splice sites within disordered regions.
Importantly, traceless ligation, where no cysteine or other non-native residues are introduced at the splice
site, can be accomplished using the Nrdj1 intein, allowing retention of the native protein sequence and
cysteine-mediated coupling of spin labels or fluorophores at desired probe sites. Initial efforts will focus
on the full-length, 180 kDa tumor suppressor p53. Current structural information on p53 is largely limited
to isolated domains and fails to explain how the disordered and folded regions function synergistically to
control p53 activity. There is a large and growing body of evidence that the intrinsically disordered regions
of p53 regulate its activity through dynamic intramolecular and intermolecular interactions that are
modulated by constitutive and stress-induced post-translational modifications. This research will provide
new molecular-level insights into the mechanisms by which this important tumor suppressor is regulated,
as well as providing new tools for structural and dynamic characterization of large eukaryotic regulatory
proteins that contain disordered regions.

## Key facts

- **NIH application ID:** 10552345
- **Project number:** 1R35GM148226-01
- **Recipient organization:** SCRIPPS RESEARCH INSTITUTE, THE
- **Principal Investigator:** PETER Edwin WRIGHT
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $452,500
- **Award type:** 1
- **Project period:** 2023-09-21 → 2028-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10552345

## Citation

> US National Institutes of Health, RePORTER application 10552345, Structural characterization of large eukaryotic proteins containing both folded and disordered domains (1R35GM148226-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10552345. Licensed CC0.

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