Optimizing Stability in Dynamic Small-Molecule Binding Proteins

Why this research matters

Many proteins must change shape to function. For proteins that bind small molecules, this motion is essential for activity but often makes them difficult to stabilize. Efforts to increase stability can unintentionally disrupt binding by shifting how the protein moves. This study examines how explicitly accounting for protein motion during design affects stability and binding outcomes.

Background: a challenge for protein design

The authors focus on periplasmic binding proteins (PBPs), a large family of bacterial proteins that switch between open and closed conformations when binding ligands. PBPs are widely used as scaffolds for biosensors, but engineering them often reduces expression yield or thermal stability. Previous attempts to stabilize PBPs using single-structure design methods have produced mixed results, including reduced activity.

The approach: design for both conformations

The study examines four PBPs with known open and closed crystal structures:

• PotF, a putrescine-binding protein
• TphC, which binds terephthalic acid
• MBP, the maltose-binding protein
• LAO, which binds L-lysine

For each protein, the authors first generated standard stability designs based on either the open or closed structure alone. They then compared the predicted effects of individual mutations across both conformations. Many mutations that were stabilizing in one state were destabilizing in the other.

To address this, the authors filtered out mutations that were not favorable in both conformations, producing a “two-state” design set. In a further refinement, they excluded mutations in hinge regions and lobe interfaces, which show the largest structural changes during opening and closing. This final strategy aimed to improve stability while minimizing disruption to the protein’s natural motion.

Across all four proteins, the authors selected 16 total designs, with 7 to 28 mutations per variant, for experimental testing.

Key results: higher stability, fewer binding losses

All designed variants showed increased thermal stability compared to their wild-type proteins. Apparent melting temperatures increased by 4 to 17 °C. For reference, wild-type melting temperatures were 63 °C for PotF, 61 °C for TphC, 63 °C for MBP, and 47 °C for LAO.

Binding outcomes depended strongly on the design strategy. Among single-state designs, three out of eight showed at least a fivefold reduction in binding affinity, with two of these originating from open-state designs. In contrast, two-state designs without hinge constraints showed reduced affinity in only one case.

Notably, all two-state designs that also excluded hinge and interface mutations retained wild-type-like binding affinity, while still gaining stability. In some cases, binding affinity was modestly improved, but the authors emphasize preservation rather than enhancement.

Validation: how performance was measured

The authors confirmed correct folding using circular dichroism spectroscopy. Thermal stability was measured by temperature-dependent unfolding experiments, both with and without ligand present. Ligand binding affinities were quantified using isothermal titration calorimetry, allowing direct comparison between wild-type proteins and each design.

Broader significance

The authors present this workflow as a practical strategy for generating stable starting points for PBP-based biosensors, where maintaining both responsiveness and stability is essential. More broadly, the study demonstrates that incorporating multiple functional conformations into design calculations can improve outcomes when engineering dynamic proteins.

This work was recently published by Hoecker, Fleishman, and colleagues in JACS.