# The Curious Case of A31P, a Topology-Switching Mutant of the Repressor of Primer Protein: A Molecular Dynamics Study of Its Folding and Misfolding

## TL;DR

A single mutation in the Rop protein dramatically changes its structure, causing instability and challenging computational predictions.

## Contribution

A single mutation (A31P) in Rop protein causes a dramatic structural change and instability, contradicting computational predictions.

## Key findings

- The A31P mutation in Rop protein leads to a new topology with a 'bisecting U' structure.
- Molecular dynamics simulations show the A31P mutant structure is unstable and unfolds under mild conditions.
- The mutation's effect contradicts energy minimization predictions and supports a double-funneled energy landscape model.

## Abstract

The
effect of mutations on protein structures is usually rather
localized and minor. Finding a mutation that can single-handedly change
the fold and/or topology of a protein structure is a rare exception.
The A31P mutant of the homodimeric Repressor of primer (Rop) protein
is one such exception: This single mutation —and as demonstrated
by two independent crystal structure determinations— can convert
the canonical (left-handed/all-antiparallel) 4-α-helical bundle
of Rop to a new form (right-handed/mixed parallel and antiparallel
bundle) displaying a previously unobserved “bisecting U”
topology. The main problem with understanding the dramatic effect
of this mutation on the folding of Rop is to understand its very existence:
Most computational methods appear to agree that the mutation should
have had no appreciable effect, with the majority of energy minimization
methods and protein structure prediction protocols indicating that
this mutation is fully consistent with the native Rop structure, requiring
only a local and minor change at the mutation site. Here we use two
long (10 μs each) molecular dynamics simulations to compare
the stability and dynamics of the native Rop versus a hypothetical
structure that is identical with the native Rop but is carrying this
single Alanine31 to Proline mutation. Comparative analysis
of the two trajectories convincingly shows that, in contrast to the
indications from energy minimization —but in agreement with
the experimental data—, this hypothetical native-like A31P
structure is unstable, with its turn regions almost completely unfolding,
even under the relatively mild 320 K NpT simulations
that we have used for this study. We discuss the implication of these
findings for the folding of the A31P mutant, especially with respect
to the proposed model of a double-funneled energy landscape.