Optimizing Stability in Dynamic Small-Molecule Binding Proteins

TL;DR

This paper introduces a method to stabilize dynamic proteins by designing mutations compatible with both open and closed conformations, improving thermal stability without harming ligand binding.

Contribution

A new method for stabilizing dynamic proteins by incorporating conformational compatibility and structural constraints into mutation design.

Findings

Designing mutations compatible with both conformations reliably enhances thermal stability.

Using evolutionary constraints alone is insufficient to maintain wild-type-like binding affinity.

16 stabilized variants of periplasmic binding proteins were successfully designed with 7–28 mutations each.

Abstract

The function of dynamic
proteins is determined by the stability
of distinct conformational states and the energy barriers that separate
these states. For most dynamic proteins, the molecular details of
the energy barriers are not known, implying a fundamental limit to
the ability of protein design methods to engineer beneficial mutations
without disrupting activity. We hypothesized that designing mutations
that are compatible with structurally distinct equilibrium conformations
may enable a reliable stability design. We focus on periplasmic binding
proteins (PBPs), a superfamily of dynamic proteins that change conformation
from open to closed states in response to binding their small-molecule
ligands. We find that the evolutionary constrained space of allowed
mutations computed for one conformation is incompatible with that
for the other. Therefore, putative conformational hinge points and
interface residues were additionally constrained, and incompatible
mutations were filtered out. Starting from four different PBPs, we
designed a total of 16 stabilized variants with 7–28 mutations
each. Our results show that design based on a single conformation
with evolutionary constraints is not sufficient to maintain a wild-type-like
binding affinity. Conversely, using a subset of mutations compatible
with both conformations and structural constraints reliably enhances
thermal stability while mitigating trade-offs in ligand binding. Our
work demonstrates a straightforward method for the one-shot stabilization
of dynamic proteins, which is critically required to generate robust
starting points for thermostable and responsive biosensors.

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