# Dynamics and function of soluble and membrane proteins viewed by simulation and hydrogen exchange

> **NIH NIH R01** · UNIVERSITY OF CHICAGO · 2022 · $324,566

## Abstract

Summary
The Sosnick lab has studied the dynamics and function of soluble proteins using an integrated experimental and
computational approach for the past 25 years. This proposal expands our focus to include membrane proteins
with an emphasis on applying hydrogen-deuterium exchange (HDX) to study the relationship between dynamics
and function. In Aim 1, we will address a central challenge in protein biophysics – apply and rigorously test our
ability to accurately simulate the free energy surface including the generation of the Boltzmann ensemble of all
major species. The lab has made considerable progress in this area, developing Upside, a molecular dynamics
algorithm that can cooperatively fold proteins with an accuracy comparable to all-atom methods yet is 103-104
fold faster. The algorithm will be advanced, validated, and applied across the three specific aims. Testing will be
done largely using HDX/NMR as this powerful combination of methods can determine the free energies of
individual H-bonds throughout a protein and quantitatively describe the free energy surface with high precision.
Test systems include designed and naturally occurring proteins. In Aims 2 and 3, we will continue our studies of
the folding and dynamics of KcsA and other ion channels in liposomes, developing methods to measure HDX
on membrane proteins during folding and activation by voltage, pH and tension to explore the relationship
between their dynamics and function. Preliminary results on the folding of the KcsA find that the initial folding
event is a rapid association into a protein-dense phase, while the rate-limiting step in forming the native tetramer
is a unimolecular event. We will continue to investigate the protein-dense phase and the overall folding kinetics,
testing our prediction that the rate-limiting step involves the formation of a solvent channel followed by insertion
of the four selectivity filters and pore helices. To identify when specific H-bonds form during KcsA folding, we will
use HDX pulse-labeling with mass spectrometry and thus shed light on this final pathway to the tetramer. Finally,
it is often the case that structural biologists can only obtain the structure of a membrane protein in either its
resting or activated state, and even if both structures are known, the dynamics and pathways between the states
remain to be determined. We propose to develop robust protocols to obtain HDX patterns of both states for KcsA,
KvAP, and the mechano-sensing channel MscS using HDX/mass spectrometry to identify the structural
dynamics associated with the proteins’ functional processes. In doing so, we will further expand the repertoire of
applications in which the HDX method, so rich in information content, can be applied.
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## Key facts

- **NIH application ID:** 10488676
- **Project number:** 5R01GM055694-27
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **Principal Investigator:** Tobin R Sosnick
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $324,566
- **Award type:** 5
- **Project period:** 1996-08-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10488676

## Citation

> US National Institutes of Health, RePORTER application 10488676, Dynamics and function of soluble and membrane proteins viewed by simulation and hydrogen exchange (5R01GM055694-27). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10488676. Licensed CC0.

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