# Protein folding in the cell: Challenges and coping mechanisms

> **NIH NIH R35** · UNIVERSITY OF MASSACHUSETTS AMHERST · 2022 · $544,294

## Abstract

Project Summary
Hsp70 molecular chaperones are central players in protein homeostasis and quality control. They
mediate a diverse set of cellular functions using a deceptively simple allosteric mechanism. In
their ATP-bound states, they bind substrates with rapid on/off rates and relatively low affinity, and
in their ADP-bound states, substrates bind more tightly with slow association/dissociation. Hsp70s
bind short sequences within unfolded regions of their substrates, preferring hydrophobic residues
with flanking positively charged residues. Their transition between a high substrate affinity, ADP-
bound state, and a low substrate affinity, ATP-bound state, involves major conformational
rearrangement of both the N-terminal nucleotide-binding domain, and the C-terminal substrate-
binding domain. While recent research has shed light on this allosteric conformational change,
many questions remain and are the focus of the proposed research. What are the features of their
substrate-binding sites that enable binding to many but not all sequences (they are “selectively
promiscuous”)? Hsp70s work with partner co-chaperones, the J-proteins that help with cellular
localization and delivery of specific substrates, and the nucleotide-exchange factors that facilitate
replacement of ADP by ATP in the allosteric cycle. What are the structural origins of these
partnerships, and how do they affect substrate binding and release? Preliminary results and
literature observations suggest that Hsp70s can bind nearly isoenergetically to their extended
polypeptide substrates in either an N- to C-orientation or a C- to N-orientation. This provocative
bimodal substrate binding may have functional implications. The proposed work will examine the
structural origins of this capability as well as the potential impact on the functions of the
chaperone. Past work shows that the allosteric properties of Hsp70 are tunable by amino acid
substitutions and by post-translational modifications. The proposed work will delve into the
structural origins of this tuning, and the consequences of changes in the allosteric energy
landscape for specific Hsp70 functions. Past work leaned heavily on peptide models to
understand how Hsp70s bind their substrates. We will characterize the binding of Hsp70s to
protein substrates to learn how the affinity for short sequence motifs translates into a hierarchy of
site binding: are flanking sequences involved in selection of binding sites? How pivotal is
accessibility? Lastly, the role of the Hsc70 chaperone in preparing SNAP-25 to participate in pre-
synaptic vesicle docking and fusion in neurons will be studied. Methods that will be deployed in
the proposed work include biochemical assays, nuclear magnetic resonance, fluorescence, mass
spectrometry, and computational modeling.

## Key facts

- **NIH application ID:** 10410352
- **Project number:** 5R35GM118161-07
- **Recipient organization:** UNIVERSITY OF MASSACHUSETTS AMHERST
- **Principal Investigator:** LILA M GIERASCH
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $544,294
- **Award type:** 5
- **Project period:** 2016-06-01 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10410352

## Citation

> US National Institutes of Health, RePORTER application 10410352, Protein folding in the cell: Challenges and coping mechanisms (5R35GM118161-07). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10410352. Licensed CC0.

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