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

Grady, Connor; Schossau, Jory; Ashbaugh, Ryan C.; Pelled, Galit; Gilad, Assaf A.

doi:10.1101/2022.11.30.518522

Cited by 8 publications

(12 citation statements)

References 39 publications

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“…This indicated potential for EPG be used to remotely treat nervous system disorders 12 . EPG can potentially be used in synthetic biological technologies as a way of remotely controlling the activation of a system 13 . In order to utilize EPG effectively in these applications, it is essential to characterize its structure and function so it can be optimized for the purpose at hand.…”

Section: Introductionmentioning

confidence: 99%

Proposed three-phenylalanine motif involved in magnetoreception signaling of an Actinopterygii protein expressed in mammalian cells

Ricker

Mitra

Castellanos

et al. 2022

Preprint

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Studies at the cellular and molecular level of magnetoreception - sensing and responding to magnetic fields - is a relatively new research area. As it appears that different mechanisms of magnetoreception in animals evolved from different origins, many questions about the mechanisms remain left open. Here we present new information regarding the Electromagnetic Perceptive Gene (EPG) from Kryptopterus vitreolus that may serve as part of the foundation to understanding and applying magnetoreception. Using HaloTag coupled with fluorescent ligands and phosphatidylinositol specific phospholipase C (PI-PLC) we show that EPG is associated to the membrane via glycosylphosphatidylinositol (GPI) anchor. EPGs function of increasing intracellular calcium was also used to generate an assay using GCaMP6m to observe the function of EPG and to compare its function with homologous proteins. It was also revealed that EPG relies on a motif of three phenylalanine residues in order to function - stably swapping these residues using site directed mutagenesis resulted in a loss of function in EPG. This information not only expands upon our current understanding of magnetoreception but may provide a foundation and template to continue characterizing and discovering more within the field.

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Section: Introductionmentioning

confidence: 99%

Proposed three-phenylalanine motif involved in magnetoreception signaling of an Actinopterygii protein expressed in mammalian cells

Ricker

Mitra

Castellanos

et al. 2022

Preprint

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“…POET can be useful for optimizing short peptides and especially when only small data sets are available by reliance more on generational evolution. Such examples for short peptides or motifs could be peptides for drug, gene, or exosome targeting, metal binding domains, functional protein motifs, or peptide linkers for fusion proteins. , All the above applications rely on short peptides and could benefit from optimization by POET.…”

Section: Resultsmentioning

confidence: 99%

A Genetic Programming Approach to Engineering MRI Reporter Genes

Bricco

Miralavy

et al. 2023

ACS Synth. Biol.

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Here we develop a mechanism of protein optimization using a computational approach known as “genetic programming”. We developed an algorithm called Protein Optimization Engineering Tool (POET). Starting from a small library of literature values, the use of this tool allowed us to develop proteins that produce four times more MRI contrast than what was previously state-of-the-art. Interestingly, many of the peptides produced using POET were dramatically different with respect to their sequence and chemical environment than existing CEST producing peptides, and challenge prior understandings of how those peptides function. While existing algorithms for protein engineering rely on divergent evolution, POET relies on convergent evolution and consequently allows discovery of peptides with completely different sequences that perform the same function with as good or even better efficiency. Thus, this novel approach can be expanded beyond developing imaging agents and can be used widely in protein engineering.

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“…EPG is a small (∼9kD) 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, 15-17 ), 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.…”

Section: Introductionmentioning

confidence: 99%

“…EPG is a small (~9kD) 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.…”

Section: Introductionmentioning

confidence: 99%

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

Ricker,

Castellanos Franco,

de los Campos

et al. 2024

Preprint

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Magnetoreceptive biology as a field remains relatively obscure; compared to the breadth of species believed to sense magnetic fields, it remains under-studied. Here, we present grounds for the expansion of magnetoreception studies among Teleosts. We begin with the electromagnetic perceptive gene (EPG) from Kryptopterus vitreolus and expand to identify 72 Teleosts with homologous proteins containing a conserved three-phenylalanine (3F) motif. Phylogenetic analysis provides insight as to how EPG may have evolved over time, and indicates that certain clades may have experienced a loss of function driven by different fitness pressures. One potential factor is water type with freshwater fish significantly more likely to possess the functional motif version (FFF), and saltwater fish to have the non-functional variant (FXF). It was also revealed that when the 3F motif from the homolog of Brachyhypopomus gauderio (B.g.) is inserted into EPG - EPG(B.g.) - the response (as indicated by increased intracellular calcium) is faster. This indicates that EPG has the potential to be engineered to improve upon its response and increase its utility to be used as a controller for specific outcomes.

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

Cited by 8 publications

References 39 publications

Proposed three-phenylalanine motif involved in magnetoreception signaling of an Actinopterygii protein expressed in mammalian cells

Proposed three-phenylalanine motif involved in magnetoreception signaling of an Actinopterygii protein expressed in mammalian cells

A Genetic Programming Approach to Engineering MRI Reporter Genes

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

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