# Characterization of RfaH evolution and folding dynamics: pairing in vitro and in situ methods

> **NIH NIH FI2** · U.S. NATIONAL HEART LUNG AND BLOOD INST · 2024 · —

## Abstract

Project Summary/Abstract
Historically 3D protein structure has been determined by the amino acid sequence and thought to only provide information
for one protein fold. Recent work has not only identified that proteins can adopt two distinct folds with different functions
but that this occurs in nature frequently. The current limit in understanding and observing these two distinct folds is that
expressing these fold-switching proteins is often complicated with solubility issues and homology identity recognition software
has bias based on the assumption proteins only have one possible fold.
RfaH is a fold-switching protein in the NusG/Spt5 family that has an ⍺-helical hairpin fold capable of autoinhibition and a β-
roll fold that can directly interact with the S10 unit of the ribosome, which is like the single-fold of NusG. The N-terminus
domain (NTD) or NusG and RfaH are very similar, though their C-terminus domains (CTD) are not. This variation in the
CTD is hypothesized to have evolved through stepwise mutations but there are currently no findings about RfaH evolution as
a fold-switching protein. Characterizing the evolution of fold-switching protein has been challenging due to the bias in
homology recognition software, but a recent workflow using full length sequences for alignments and ancestral reconstruction
has yielded promising results to identify other evolution pathways of fold-switching proteins.
Currently, identified fold-switchers were mostly found accidently by circular dichroism spectra. Other techniques commonly
used for characterizing the two-fold states are NMR, x-ray crystallography, and cryo-EM. However, recent developments in
confocal microscopy have broken the 200-micron resolution limit and new assays have been developed. Some of these assays
include Försters resonance energy transfer (FRET) efficiency determined by fluorescent lifetime intensity microscopy (FLIM)
also termed FLIM-FRET. This assay relies on two fluorescent tags, usually on separate molecules, when the molecules are
close and interact there is a change in fluorescence for each tag that can be quantified. Due to the increase in resolution limits,
we can now tag one protein on both ends to identify FLIM-FRET efficiency based on end-to-end distances.
This proposal aims to identify the common ancestors between NusG and RfaH through ancestral reconstruction by full-length
sequence alignments, identify the critical residues likely mutated through evolution that are important for folding dynamics,
and develop a new fold-switching screening tool of FLIM-FRET frequency analysis. This will be accomplished by using
structural analysis techniques including CD spectra, x-ray crystallography, FLIM-FRET, and site directed mutagenesis paired
with bioinformatics and predicted structure folding. The cumulative work of this proposal will contribution to understanding
the evolution and folding properties of RfaH and the sequence properties that bias alternatively spliced proteins to switch...

## Key facts

- **NIH application ID:** 10940629
- **Project number:** 1FI2GM154712-01
- **Recipient organization:** U.S. NATIONAL HEART LUNG AND BLOOD INST
- **Principal Investigator:** Leslie Ronish
- **Activity code:** FI2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** —
- **Award type:** 1
- **Project period:** 2024-09-01 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10940629

## Citation

> US National Institutes of Health, RePORTER application 10940629, Characterization of RfaH evolution and folding dynamics: pairing in vitro and in situ methods (1FI2GM154712-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10940629. Licensed CC0.

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