Designing heterotropically activated allosteric conformational switches using supercharging

Abstract

Heterotropic allosteric activation of protein function, in which binding of one ligand thermodynamically activates the binding of another, different ligand or substrate, is a fundamental control mechanism in metabolism and as such has been a long-aspired capability in protein design. Here we show that greatly increasing the magnitude of a protein’s net charge using surface supercharging transforms that protein into an allosteric ligand- and counterion-gated conformational molecular switch. To demonstrate this we first modified the designed helical bundle hemoprotein H4, creating a highly charged protein which both unfolds reversibly at low ionic strength and undergoes the ligand-induced folding transition commonly observed in signal transduction by intrinsically disordered proteins in biology. As a result of the high surface-charge density, ligand binding to this protein is allosterically activated up to 1,300-fold by low concentrations of divalent cations and the polyamine spermine. To extend this process further using a natural protein, we similarly modified Escherichia coli cytochrome b562 and the resulting protein behaves in a like manner. These simple model systems not only establish a set of general engineering principles which can be used to convert natural and designed soluble proteins into allosteric molecular switches useful in biodesign, sensing, and synthetic biology, the behavior we have demonstrated––functional activation of supercharged intrinsically disordered proteins by low concentrations of multivalent ions––may be a control mechanism utilized by Nature which has yet to be appreciated.

Computational design of a sensitive, selective phase-changing sensor protein for the VX nerve agent

“…At pH 3.5, reduced charge repulsion from protonation of surface glutamates enabled a subset of designs to form helices at high α NaCl (fig. S3), possibly a result of condensed molten globule formation in the absence of ligand ( 19 ).…”

“…Strong intrachain charge repulsion produces an unfolded protein, but structure can be restored by screening electrostatic forces at high ionic strengths. By poising the solution ionic strength, a phase transition from the unfolded apoprotein to a folded ligand-bound protein can be achieved ( 19 ). Using this approach, we introduced a VX-binding site into the core of a supercharged protein scaffold using a distributed evolutionary algorithm implemented in the protein design platform, protCAD (protein Computer Aided Design) ( 20 ).…”

Non-Covalent Coatings on Carbon Nanotubes Mediate Photosensitizer Interactions

“…To assess the suitability of SWCNTs as a probe of direct and indirect porphyrin binding, we screened a library of SWCNT coatings for those capable of promoting a SWCNT emission red-shift to both free and protein-bound ferric hemin. A designed hemin binding protein, H4­(-28), with a net charge count (acidic minus basic residues) of −28, was used to sequester the hemin from direct contact with the SWCNT. The coating panel consisted of (A)- or (G)-rich ssDNA, surfactants (SDC or PEG-cera), cellulose (CMC), and in-house synthesized polycarbodiimide (PCD) polymers (Figure S1).…”

“…Binding Protein Expression and Purification. The negatively supercharged artificial hemin binding protein H4(-28) 26 was expressed in BL21(DE3) Escherichia coli expression strains using genes subcloned into pET32 expression plasmids (Novagen 69015) and purified using methods published previously. 27 Porphyrin complex formation was performed using six equimolar additions with 10 min incubation periods between additions, 28 and complexes were isolated using PD-10 desalting columns (GE Life Sciences 17085101).…”

Recent Progress Using De Novo Design to Study Protein Structure, Design and Binding Interactions

“…Linking binding reactions to other signaling events is a significant step. This has been shown with the LOCKR system, using other proteins, but the work of Schnatz, Koder and their colleagues apply this to small molecules [ 76 ]. Taking a cue from biologically common intrinsically disordered proteins, the authors supercharged the sequence of a de novo designed protein named H4 and were able to show that it was unstable in weakly ionic conditions but regained its structure upon addition of salts or spermine—A polycationic molecule.…”