# Manipulating the Unfolded State of a Folded Protein through Site-Specific Backbone Modification

## TL;DR

Scientists modified parts of a protein's backbone to change its unfolded state while keeping its folded structure, offering new ways to study protein folding.

## Contribution

A new method to rationally tune the unfolded state of a folded protein through minimal backbone modifications.

## Key findings

- Backbone modifications can alter conformational freedom while maintaining folded structure.
- Substitution type systematically affects the sensitivity of folding free energy to denaturants.
- Variants show complex stability changes but retain structural identity.

## Abstract

Protein unfolded states are heterogeneous but can manifest
local
and long-range order. Replacement of side chains through site-directed
mutagenesis is a common method to manipulate the unfolded state and
elucidate its role in the folding process. Modification of the protein
backbone represents a less explored complementary approach with the
potential to elicit dramatic changes in conformational preferences
from minimal chemical alteration. Prior work has shown backbone modification
can affect unfolded ensembles as well as intrinsically disordered
sequences. Here, we show that it can be used to rationally tune structural
characteristics of the unfolded state of a folded protein. Using the
GCN4 leucine zipper as a host, canonical α-residues throughout
the chain are individually replaced by β3 or Cα-Me-α analogues. The former modification enhances
conformational freedom, the latter restricts it, and both retain the
side chain at the substitution site. Characterization by circular
dichroism and X-ray crystallography shows that the variants adopt
folded structures identical to the prototype. Thermal and thermodynamic
stability vary in complex ways with the context and nature of backbone
modification; however, a uniform relationship is observed between
substitution type and the sensitivity of folding free energy to chemical
denaturant. This finding suggests systematic changes in solvent-accessible
surface area of the unfolded ensemble among isomeric proteins differing
only in the position of a single CH2 group. Collectively,
these results demonstrate a platform for predictably tuning the properties
of the unfolded state through minimal chemical modification, enabling
new avenues for fundamental research on folding behavior of proteins
as well as protein mimetics.