De novo design of allosterically switchable protein assemblies.

Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central part in the control of metabolism and cell signalling1-3. There has been considerable interest in designing allosteric systems, both to gain insight into the mechanisms underlying such 'action at a distance' modulation and to create synthetic proteins whose functions can be regulated by effectors4-7. However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge8,9. Here, inspired by the classic Monod-Wyman-Changeux model of cooperativity10, we investigate the de novo design of allostery through rigid-body coupling of peptide-switchable hinge modules11 to protein interfaces12 that direct the formation of alternative oligomeric states. We find that this approach can be used to generate a wide variety of allosterically switchable systems, including cyclic rings that incorporate or eject subunits in response to peptide binding and dihedral cages that undergo effector-induced disassembly. Size-exclusion chromatography, mass photometry13 and electron microscopy reveal that these designed allosteric protein assemblies closely resemble the design models in both the presence and absence of peptide effectors and can have ligand-binding cooperativity comparable to classic natural systems such as haemoglobin14. Our results indicate that allostery can arise from global coupling of the energetics of protein substructures without optimized side-chain-side-chain allosteric communication pathways and provide a roadmap for generating allosterically triggerable delivery systems, protein nanomachines and cellular feedback control circuitry.

De novo design of modular peptide-binding proteins by superhelical matching.

General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone1-3. Here, inspired by natural and re-engineered protein-peptide systems4-11, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone12. The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins.

Design of stimulus-responsive two-state hinge proteins.

In nature, proteins that switch between two conformations in response to environmental stimuli structurally transduce biochemical information in a manner analogous to how transistors control information flow in computing devices. Designing proteins with two distinct but fully structured conformations is a challenge for protein design as it requires sculpting an energy landscape with two distinct minima. Here we describe the design of "hinge" proteins that populate one designed state in the absence of ligand and a second designed state in the presence of ligand. X-ray crystallography, electron microscopy, double electron-electron resonance spectroscopy, and binding measurements demonstrate that despite the significant structural differences the two states are designed with atomic level accuracy and that the conformational and binding equilibria are closely coupled.

Charged Poly(N-isopropylacrylamide) Nanogels for the Stabilization of High Isoelectric Point Proteins.

Storage and transportation of protein therapeutics using refrigeration is a costly process; a reliable electrical supply is vital, expensive equipment is needed, and unique transportation is required. Reducing the reliance on the cold chain would enable low-cost transportation and storage of biologics, ultimately improving accessibility of this class of therapeutics to patients in remote locations. Herein, we report on the synthesis of charged poly(N-isopropylacrylamide) nanogels that efficiently adsorb a range of different proteins of varying isoelectric points and molecular weights (e.g., adsorption capacity (Q) = 4.7 ± 0.2 mg/mg at 6 mg/mL initial IgG concentration), provide protection from external environmental factors (i.e., temperature), and subsequently release the proteins in an efficient manner (e.g., 100 ± 1% at 2 mg/mL initial IgG concentration). Both cationic and anionic nanogels were synthesized and selectively chosen based on the ability to form electrostatic interactions with adsorbed proteins (e.g., cationic nanogels adsorb low isoelectric point proteins whereas anionic nanogels adsorb high isoelectric point proteins). The nanogel-protein complex formed upon adsorption increases the stabilization of the protein's tertiary structure, providing protection against denaturation at elevated temperatures (e.g., 84 ± 4% of the protected IgG was stabilized when exposed to 65 °C). The addition of a high molar salt solution (e.g., 40 mM CaCl2 solution) to protein-laden nanogels disrupts the electrostatic interactions and collapses the nanogel, ultimately releasing the protein. The versatile materials utilized, in addition to the protein loading and release mechanisms described, provide a simple and efficient strategy to protect fragile biologics for their transport to remote areas without necessitating costly storage equipment.