A putative design for the electromagnetic activation of split proteins for molecular and cellular manipulation

Abstract

The ability to manipulate cellular function using an external stimulus is a powerful strategy for studying complex biological phenomena. One approach to modulate the function of the cellular environment is split proteins. In this method, a biologically active protein or an enzyme is fragmented so that it reassembles only upon a specific stimulus. Although many tools are available to induce these systems, nature has provided other mechanisms to expand the split protein toolbox. Here, we show a novel method for reconstituting split proteins using magnetic stimulation. We found that the electromagnetic perceptive gene (EPG) changes conformation due to magnetic field stimulation. By fusing split fragments of a certain protein to both termini of the EPG, the fragments can be reassembled into a functional protein under magnetic stimulation due to conformational change. We show this effect with three separate split proteins: NanoLuc, APEX2, and herpes simplex virus type-1 thymidine kinase. Our results show, for the first time, that reconstitution of split proteins can be achieved only with magnetic fields. We anticipate that this study will be a starting point for future magnetically inducible split protein designs for cellular perturbation and manipulation. With this technology, we can help expand the toolbox of the split protein platform and allow better elucidation of complex biological systems.

A conserved phenylalanine motif among teleost fish provides insight for improving electromagnetic perception

“…It is possible that this particular mutation affected the protein’s kinetics by altering the spacing and/or flexibility of the F residues in the 3F motif. This coincides with the theory that EPG undergoes a conformational change upon activation [ 18 ], where certain amino acids may produce more favourable conditions to allow this shift in confirmation.…”

“…EPG is a small (~9 kDa) protein that adopts a Ly6/UPAR three-finger structure and is membrane associated via glycosylphosphatidylinositol (GPI) anchor [15]. EPG has shown response to magnetic stimuli in mammalian cells in the form of increased intracellular calcium [12,[15][16][17], in activatable split protein systems [18] and in rat models with increased neuronal plasticity [19] and reduced seizure activity [20], but its mechanism of action remains elusive. Site-directed mutagenesis has indicated a region of interest-the three-phenylalanine (3F) motif-in the protein that is essential for its function [15], but it remains unclear how this region facilitates magnetoreception.…”

Theranostics - a sure cure for cancer after 100 years?

“…Magnetogenetics has been deployed to activate enzymes in E. coli and mammalian cells. Notable examples of this technology include the creation of a split NanoLuc, a split Peroxidase and a split version of the theranostic gene HSV1-TK [189]. Upon stimulation with an electromagnetic field, it was demonstrated that the EPG split HSV1-TK can kill 4T1-Luc2 cancer cells in culture in the presence of the prodrug ganciclovir.…”