Design of stimulus-responsive two-state hinge proteins

Abstract

Proteins that switch between two structural states as a function of environmental stimuli are widespread in nature. These proteins structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Engineering challenges ranging from biological computing devices to molecular motors require such two-state switches, but designing these is an unsolved problem as it requires sculpting an energy landscape with two low-energy but structurally distinct conformations that can be modulated by external inputs. Here we describe a general design approach for creating 'hinge' proteins that populate one distinct state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, and double electron-electron resonance spectroscopy demonstrate that despite the significant structural differences, the two states are designed with atomic level accuracy. The kinetics and thermodynamics of effector binding can be finely tuned by modulating the free energy differences between the two states; when this difference becomes sufficiently small, we obtain bistable proteins that populate both states in the absence of effector, but collapse to a single state upon effector addition. Like the transistor, these switches now enable the design of a wide array of molecular information processing systems.

Heme-substituted protein assembly bridged by synthetic porphyrin: achieving controlled configuration while maintaining rotational freedom

“…The introduction of dened exibility into articial protein assemblies could allow control of their motion, thereby providing them with motion-based functions, leading to the development of dynamic articial protein assemblies, a eld that currently has only limited examples. 7,36,37…”

Scalable design of repeat protein structural dynamics via probabilistic coarse-grained models

“…Many displayed very wide distributions with no clearly separated modes. While these candidates may not exhibit strong multi-state behaviour, the large range of movement (60 Å for H5) would provide an ideal starting point for establishing multi-stable dynamics using external stimuli, such as an additional peptide chain 43 .…”

De Novo Design of Polyhedral Protein Assemblies: Before and After the AI Revolution

“…Next steps include the creation of modular scaffolds for off-the-shelf programmability of assemblies with tailored geometric constraints, dynamic behaviours in response to environmental cues, or built-in functional sites. Multistate design pipelines that account for several protein states and/or conformations, [152,153] in-painting methods, [68,154,155] and prediction methods that identify alternative biophysically relevant states [84,156] or flexible protein regions [157] will enable the design of functional protein materials capable of sensing and interacting with their surroundings. While most current methods for protein-protein interface design are primarily based on hydrophobic and metal-mediated interactions, improvements in the design of polar protein-protein interactions, including those involving water, [132,[158][159][160] would facilitate the design of dynamic protein materials with tunable conformational and assembly dynamics, self-assembly mechanisms coupled with other processes, or assemblies interfacing with inorganic materials.…”