# Integrating experiment and theory for predicting protein folding pathways and structure

> **NIH NIH R01** · UNIVERSITY OF CHICAGO · 2020 · $399,787

## Abstract

Project Summary/Abstract
 Protein folding and dynamics are integral to many biological activities, including chaperone action,
degradation, amyloid diseases and aging. Our goal is to combine experimental and computational studies to
produce a predictive understanding of protein dynamics through the development of methods capable of
simulating folding and dynamics just using physio-chemical principles. Our past studies have focused on single
domain proteins. Our efforts have expanded to more complicated systems including the snow flea anti-freeze
protein (sfAFP) and TonB-dependent transporters (TBDT) that are relevant to iron sequestration in pathogenic
bacteria. Tying these studies together is our new molecular dynamics (MD) package, Upside, which can
reversibly fold some proteins up to 97 AA to under 4 Å Cα-RMSD in cpu-days without the use of fragments,
homology or evolution. Upside utilizes a number of unique features, including rapid side chain packing that
enables simulations using only 3 backbone atoms while retaining considerable detail and avoiding side chain
“rattling”, which slows all-atom methods. We will improve Upside and implement enhanced sampling methods
to increase our accuracy and size range, and study protein dynamics as monitored by hydrogen exchange.
 sfAFP's unique structure challenges conventional wisdom regarding cooperative folding and stability.
Lacking a hydrophobic core to promote folding, other factors must contribute to sfAFP's stability. We will test
our quantum calculations that sfAFP's H-bonds are unusually stable by measuring amide H/D fractionation
factors and NMR J-couplings. We will evaluate whether intrinsic biases in backbone dihedral angles for the
PP2 basin in the unfolded state are another major stabilizing factor. This information will be used to improve
the Upside simulations. Finally, we will apply our standard folding tools to characterize the folding pathway and
compare it to the behavior we expect based on principles derived from proteins with hydrophobic cores.
 Many aspects of the transport cycle in TBDT remain unknown despite protracted study, including the
conformational rearrangement of the plug domain during transport. We will provide the first structure of the
plug domain outside the barrel, and so answer whether this structure matches the crystal structure in the barrel.
Additionally, the study of Nakamoto's V10C-S120C variant of the BtuB plug enables the investigation of a
possible folding or transport intermediate. We will characterize the plug's dynamics while it is in the barrel
using HX to observe possible transport-competent states. The mechanism of plug folding and insertion into the
barrel will be investigated, with comparative studies for FhuA, a TBDT whose plug domain is intrinsically
disordered in solution. These studies represent an exciting combination of protein folding and function, at the
interface between soluble and membrane folding, using experiments and complementary foldi...

## Key facts

- **NIH application ID:** 9938596
- **Project number:** 5R01GM055694-25
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **Principal Investigator:** KARL F FREED
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $399,787
- **Award type:** 5
- **Project period:** 1996-08-01 → 2021-09-14

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9938596

## Citation

> US National Institutes of Health, RePORTER application 9938596, Integrating experiment and theory for predicting protein folding pathways and structure (5R01GM055694-25). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9938596. Licensed CC0.

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