Speaker
Description
NTF2-like proteins are compact α+β fold domains with cone-shaped architectures and internal pockets that provide versatile scaffolds for the de novo design of ligand-binding proteins and enzymes. A persistent challenge, however, is that engineering functional binding pockets often destabilizes the protein, creating a trade-off between stability and function. Here, we show that this trade-off can be overcome through structural buttressing. By computationally designing α-helical subdomains or homodimer interfaces that reinforce the convex face of the NTF2 β-sheet, we expand the hydrophobic core while preserving access to the functional pocket on the concave face.
Biochemical, biophysical, and crystallographic characterization demonstrates that these buttressing elements stabilize the designed fold while increasing pocket preorganization, yielding ligand-binding sites with enhanced affinity without compromising accessibility. Importantly, the effects of buttressing extend beyond molecular recognition. When applied to the de novo luciferase LuxSit-i, the same structural stabilization fundamentally remodels the enzyme's catalytic behavior. Rather than exhibiting the rapid flash kinetics characteristic of most natural and engineered luciferases, buttressed variants display a progressive increase in light output that reaches a sustained steady-state plateau before gradually decaying. The duration and stability of this plateau scale with substrate concentration, revealing a Michaelis–Menten-like kinetic regime that supports continuous, predictable luminescence for real-time biosensing.
These findings identify structural buttressing as a general design principle that couples protein stability to functional optimization across multiple levels of protein behavior. By stabilizing the scaffold, preorganizing the ligand-binding pocket, and reshaping the catalytic energy landscape, buttressing enables the simultaneous programming of stability, affinity, and enzyme kinetics, providing a versatile framework for the next generation of de novo binders and biocatalysts.