Genetic Engineering And Spectral Characterization Of Molecular Vibrational Proteins

Fluorescent labeling has been the main tool for visualization of biomolecules,protein,cell structure,tissues and organs in life sciences.Genetically encoded green fluorescent proteins(GFP)and the derived fluorescent proteins(FPs)series with brighter colors have become the preferred strategies for protein-specific labeling and imaging.Although GFP is engineered from 20 natural amino acids,its three amino acids(Ser65-Tyr66-Gly67)on theα-helix form a π-electron conjugated chromophore via natural cyclization,further dehydration and oxidation reactions,and the new born chromophore can absorb external visible light and emit bright fluorescence.However,until today,the imaging channels of fluorescent proteins that can actually be used are still limited to 3～5.For more than 20 000 proteins in cells,the color channels of the gene targeted fluorescent proteins are not enough.Its essence lies in physical and chemical levels that the chromophores of FPs undergo electronic transition under excitation.However,the linewidths of all fluorescence spectra are 30～50 nm wide in full width at half maximum,depending on the lifetimes of the electronic excited states.Even the visible color range about 500～700 nm can only accommodate 3～5 fluorescent channels for simultaneous imaging.To overcome the limitations of fluorescent proteins in multicolor imaging applications,this work intends to discard the fluorescent property of FPs and develop molecular vibrational proteins(VPs)based on molecular vibration of chromophores and their Raman spectra,and thus VPs inherit both gene targeting specificity and narrow spectra linewidth for multiplexing.The linewidth of Raman spectrum is less than 1 nm.The concept of VPs provides new strategy for the exploration of the labeling technology that can accommodate labeling of 100 proteins.The details are as follows:(1)To overcome the color barrier of FPs and genetic specificity of Raman probes,this work proposes and establishes genetically encoded molecular vibrational proteins.The tyrosine 66 of EGFP was replaced by an unnatural amino acid p-Ethynylphenylalanine(p Et F)with a single phenyl ring and an alkynyl side group through the genetic code expansion.The side group of the unnatural amino acid(UAA)can be cyclized with threonine residue 65 and glycine residue 67 to form a larger π conjugated structure.Further,by verification of the protein expression,fluorescence spectra and protein mass spectrometry data,the chromophore maturing of VPs-p Et F was confirmed.(2)The UAA-p Et F was genetic incorporated into fluorescent protein chromophores with different conjugated amino acids residue at the position 65.The genetic engineering,protein expression,purification,fluorescence spectra,mass spectrometry and Raman spectra analysis of GFP-like,Ds Red-like and photoconversion Kaede-like fluorescent proteins chromophores were performed,respectively.Six VPs with different Raman resonance frequencies were produced successfully.This provides a new solution for the development of genetically encoded multicolor VP technology.(3)To verify the universality of VPs,para-cyano-L-phenylalanine(p CNF)with a single phenyl ring and a cyano side group was genetically incorporated into residue 66 of the chromophore of yellow,far-red and photoconversion fluorescent proteins,respectively.Two VPs with different Raman resonance frequencies were produced successfully.Meanwhile,this method was also explored in eukaryotic cells,and the fused expression with target protein was verified.In summary,this thesis establishes a method for the genetic engineering of molecular vibrational proteins,which inherit both gene targeting specificity and narrow spectra width for multiplexing.The construction,expression,purification and Raman spectra analysis of GFP-like,Ds Red-like and photoconversion Kaede-like VPs were performed.Eight VPs with different Raman resonance frequencies were produced successfully.The results pave the way for engineering of 50～100 VPs with different Raman resonance frequencies and overcoming the ‘color barrier’ in fluorescent proteins and protein-specific Raman microspectroscopy.