# Structural Studies of Large Dynamic Complexes

> **NIH NIH R35** · SCRIPPS RESEARCH INSTITUTE, THE · 2020 · $514,710

## Abstract

The major overall aim of the research described in this MIRA proposal is to elucidate the structural and
dynamic characteristics of large macromolecules and their complexes, which generally contain both well-
structured and less-structured dynamic or unfolded regions, using a combination of solid-state methods such as
crystallography or cryo-EM and solution methods such as NMR, SAXS and fluorescence. Two major systems
have been under study in our lab for several years. Members of the NFκB family of transcription factors are held
in the cytoplasm in an inactive state by their bound inhibitors (IκBs) until the cell receives an external signal.
NFκB is activated by phosphorylation and ubiquitination of the IκBα, which is then targeted for proteasomal
degradation, releasing the NFκB to translocate into the nucleus. In a classical negative feedback mechanism,
NFκB upregulates transcription of IκBα in addition to signal-specific stress-response genes: newly-synthesized
IκBα kinetically enhances NFκB dissociation from the DNA in a process we have termed molecular stripping. A
transient ternary complex intermediate is formed during the stripping process and in an exciting new observation,
it was recently shown that the stability of the resting NFκB-IκBα complex in the cytoplasm is enhanced by
interaction with a specific long non-coding RNA (lncRNA), which appears to form a stable ternary complex
analogous to the transient NFκB-IκBα-DNA complex formed in the nucleus during molecular stripping. We
propose the structural characterization of this NFκB-IκBα-RNA complex using a variety of biophysical techniques,
including NMR, cryo-electron microscopy, in collaboration with Dr. Gabriel Lander, and small-angle X-ray
scattering, in collaboration with Dr. John Tainer, and will probe the structural and dynamic differences between
the binary and ternary complexes of NFκB, IκBα and DNA, and the ternary NFκB-IκBα-RNA complex using
specifically methyl-labeled proteins. Although a great deal is known about chaperone and co-chaperone
structure, the structural basis for the interaction between Hsp90 and its clients remains unknown. The
fundamental problem is that we still do not understand the physical state of the client protein when it is bound to
the Hsp90 chaperone, and we have only a rough idea of where on the Hsp90 molecule the client protein makes
contact. We propose an innovative method of preparation of a client protein-Hsp90 complex, by reconstituting
the chaperone cascade of the eukaryotic cell, but in the context of a cell-free expression system employing
bacterial cell extracts. We will prepare the complex of the estrogen receptor ligand-binding domain and Hsp90,
adapting methods that have been used in the literature to demonstrate the presence of this interaction in
mammalian cell extracts. Our cell-free protocol will include the use of a range of separate bacterial cell extracts
containing the over-expressed, folded co-chaperones required according to lite...

## Key facts

- **NIH application ID:** 9936446
- **Project number:** 5R35GM131693-02
- **Recipient organization:** SCRIPPS RESEARCH INSTITUTE, THE
- **Principal Investigator:** HELEN JANE DYSON
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $514,710
- **Award type:** 5
- **Project period:** 2019-06-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9936446

## Citation

> US National Institutes of Health, RePORTER application 9936446, Structural Studies of Large Dynamic Complexes (5R35GM131693-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9936446. Licensed CC0.

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