Explore The Effect Of Cysteine Residues On The Spectroscopic Behaviour Of PAiRFP1 In Different Redox Environments

Bacteriophytochrome are a class of Biliverdin-protein complexes widely distributed in bacteria that respond to red or far-red light.The natural biological function of bacteriophytochrome is dependent on light signalling.Protein engineering methods allow these Biliverdin-protein complexes to fluoresce in the near infrared region and thus serve as genetically encoded near infrared fluorescent probes for in-vivo imaging.PAiRFP1 is a light-activated near-infrared fluorescent protein obtained by modifying bacteriophytochrome as a template.Compared with other photoactivated fluorescent proteins,its biggest advantage is that excitation and emission are in the near-infrared region.It has been successfully applied to the early detection of tumors,especially in the case of a small number of cells in the early stage of tumors.The near-infrared fluorescence signal does not differ much from the noise level,and it can achieve a high signal-to-noise ratio by differentially subtracting the fluorescence signal before and after photoactivation to eliminate the interference of surrounding noise.Considering the high oxidative and reducing properties of the tumor microenvironment in PAiRFP1,this thesis focuses on the eight cysteine residues contained in PAiRFP1.By using PCR targeted mutagenesis,combined with protein engineering,spectroscopic detection,and computational biology.Invested the effects of Cys on protein spectroscopic behavior,stability,and antioxidant reduction capacity.We found that(1)eight Cys mutations affected not only the photoactivation behavior but also the NIR fluorescence emission properties of the protein.The C29 S mutant was the superior mutant over the wild type,and the molecular docking results provided some preliminary explanations;(2)Combined guanidine hydrochloride denaturation experiments with folding-defolding model,the Gibbs free energy of the defolding process is obtained.We found differences in the stability of different mutants,and the experimental results were further compared with the molecular dynamics results.(3)In vitro simulations of the redox environment in tumor cells revealed that the wildtype protein and each mutant protein responded differently to redox stress.Among them,the molar extinction coefficient of Cys-47 was significantly increased,indicating that it could be used as a target for subsequent modification of the antioxidant reduction capacity of the protein.The above work not only explored the functions of the eight Cys residues in PAiRFP1 protein,but also provided some theoretical guidance for the subsequent modification to obtain a light-activated NIR fluorescent protein more suitable for in vivo tumor imaging.