Genetically Encoded Aminocoumarin Lysine for Optical Control of Protein–Nucleotide Interactions in Zebrafish Embryos

Abstract

The strategic placement of unnatural amino acids into the active site of kinases and phosphatases has allowed for the generation of photocaged signaling proteins that offer spatiotemporal control over activation of these pathways through precise light exposure. However, deploying this technology to study cell signaling in the context of embryo development has been limited. The promise of optical control is especially useful in the early stages of an embryo where development is driven by tightly orchestrated signaling events. Here, we demonstrate light-induced activation of Protein Kinase A and a RASopathy mutant of NRAS in the zebrafish embryo using a new light-activated amino acid. We applied this approach to gain insight into the roles of these proteins in gastrulation and heart development and forge a path for further investigation of RASopathy mutant proteins in animals.

Expanding the Genetic Code of Xenopus laevis Embryos

“…RASopathies make up a family of diseases characterized by congenital hyperactivating mutations of the RAS/MAPK pathway. These diseases disrupt a variety of developmental processes, from gastrulation to brain and heart development. ,− We identified lysine 16 as the nucleotide binding lysine in the pocket (Figure A) and mutated it to an amber stop codon (K16TAG), then synthesized the corresponding mRNA through in vitro transcription . We incorporated the photocaged lysine 2 into the HA-tagged NRAS and performed Western blots to confirm the expression of the construct (Figure B).…”

“… 74 , 76 − 78 We identified lysine 16 as the nucleotide binding lysine in the pocket ( Figure 6 A) 79 and mutated it to an amber stop codon (K16TAG), then synthesized the corresponding mRNA through in vitro transcription. 80 We incorporated the photocaged lysine 2 into the HA-tagged NRAS and performed Western blots to confirm the expression of the construct ( Figure 6 B). No background amber suppression was seen in the no UAA control, and a band at the same molecular weight as the non-TAG control was seen in the presence of 2 , confirming caged NRAS expression.…”