Novel Vibrational Proteins

• Published in: Analytical chemistry (Washington) 2024-10, Vol.96 (42), p.16481-16486
• Main Authors: Chen, Yage, Huang, Zhiliang, Cai, Erli, Zhong, Shuchen, Li, Haozheng, Ju, Wei, Yang, Jie, Chen, Wei, Tang, Chun, Wang, Ping
• Format: Article
• Language: English
• Subjects: Alkynes; Alkynes - chemistry; Amino acids; analytical chemistry; Chromophores; Emission spectra; Fluorescence; Genetic code; Green fluorescent protein; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Molecular biology; Protein Engineering; Proteins; Spectral emittance; Spectrum Analysis, Raman; Stop codon; Vibration; Vibrational spectra
• ISSN: 0003-2700; 1520-6882; 1520-6882
• DOI: 10.1021/acs.analchem.4c01569

Genetically encoded green fluorescent protein (GFP) and its brighter and redder variants have tremendously revolutionized modern molecular biology and life science by enabling direct visualization of gene regulated protein functions on microscopic and nanoscopic scales. However, the current fluorescent proteins (FPs) only emit a few colors with an emission width of about 30–50 nm. Here, we engineer novel vibrational proteins (VPs) that undergo much finer vibrational transitions and emit rather narrow vibrational spectra (0.1–0.3 nm, roughly 3–10 cm–1). In response to an amber stop codon (UAG), a terminal alkyne bearing an unnatural amino acid (UAA, pEtF) is directly incorporated in place of Tyr64 in the chromophore of pr-Kaede by genetic code expansion. Essentially, the UAA64 further conjugates into a large π system with the contiguous two editable amino acid residues (His63 and Gly65), resulting in a programmable Raman resonance shift of the embedded alkyne. In the proof-of-concept experiment, we constructed a series of novel pEtF-VP mutants and observed fine Raman shifts of the alkynyl group in different chromophores. The genetically encoded novel VPs, could potentially label tens of proteins in the future.