High resolution protein in-cell NMR in zebrafish embryos

In-cell NMR spectroscopy has emerged as a powerful tool to evaluate protein conformations and dynamics in the native environment of live cells. Here we extend these studies to a multicellular developing vertebrate. Zebrafish (Danio rerio) embryos are complex organisms with dynamic tissue organization and may be well suited for the high resolution NMR analysis of microinjected, isotopically enriched proteins. We used the intrinsically disordered protein Alpha-synuclein (aSyn) as a test model. aSyn has been thoroughly evaluated inside bacterial and mammalian cells, providing good reference points for NMR comparisons and the critical analysis of the advantages and disadvantages of the zebrafish system. High resolution 2D ¹H-¹⁵N NMR showed that aSyn in zebrafish embryos had the same conformational and biological features previously observed in mammalian cells, including conserved interactions with cellular biomolecules and the establishment of physiological protein post-translational modifications. A direct comparative analysis of gamma-synuclein (gSyn), a naturally occurring homolog of aSyn, in bacteria, mammalian cells and zebrafish embryos confirmed these observations. Our results showed that high resolution in-cell NMR is attainable in embryonic cells within the native environment of a live animal. This system provides more physiological cellular environments for high resolution, in situ protein biophysical studies.