Novel Vibrational\nProteins

Genetically encoded green fluorescent protein (GFP) and\nits brighter\nand redder variants have tremendously revolutionized modern molecular\nbiology and life science by enabling direct visualization of gene\nregulated protein functions on microscopic and nanoscopic scales.\nHowever, the current fluorescent proteins (FPs) only emit a few colors\nwith an emission width of about 30–50 nm. Here, we engineer\nnovel vibrational proteins (VPs) that undergo much finer vibrational\ntransitions and emit rather narrow vibrational spectra (0.1–0.3\nnm, roughly 3–10 cm<sup>–1</sup>). In response to an\namber stop codon (UAG), a terminal alkyne bearing an unnatural amino\nacid (UAA, <i>p</i>EtF) is directly incorporated in place\nof Tyr64 in the chromophore of pr-Kaede by genetic code expansion.\nEssentially, the UAA64 further conjugates into a large π system\nwith the contiguous two editable amino acid residues (His63 and Gly65),\nresulting in a programmable Raman resonance shift of the embedded\nalkyne. In the proof-of-concept experiment, we constructed a series\nof novel <i>p</i>EtF-VP mutants and observed fine Raman\nshifts of the alkynyl group in different chromophores. The genetically\nencoded novel VPs, could potentially label tens of proteins in the\nfuture.