Exploring The Function Of Amino Acids Of The Conserved Sequence PRXSF In The PHY Domain Of The Near-infrared Fluorescent Protein PAiRFP1

PAiRFP1(deviated as photoactivatable near-infrared fluorescent protein 1) is one photoactivative near-infrared(NIR) fluorescent protein engineered from Agrobacterium tumefaciens bacteriophytochrome Agp2,and it has been successfully applied to in vivo imaging of tumor tissues.PAiRFP1 has one remarkable advantage when compared with other BphP-based permenant NIR fluroesent proteins,i.e.the imaging signal-to-noise ratio can be significantly enhanced by substracting the fluorescence signals before and after the photoactivation because in this case the background noise is almost completely eliminated.The aftermentioned photoactivation is strongly dependent on the PHY domain of PAiRFP1.On one hand,PHY is essential for the photoconversion from Pfr to Pr and the resultanting photoactivation behavior.On the other hand,the dark recovery from Pr to Pfr needs to be inhibited so as to enhance the NIR quantum yield of Pr state,which will help to achieve the higher photoactivation fluorescence contrast.Nevertheless,it is unclear how the amino acid residues in PHY domain regulate the photoactivation.Although the X-ray crystallographic structure of PAiRFP1 has not been successfully resolved,the alignment of amino acids showed that PAiRFP1 shared the highly convserved PRSXF motif with other BphPs.The homology modelling on PAiRFP1 revealed that F187,F192,Y251 are involed in the hydrogen bonding network to stabilize Biliverdin(BV).In this thesis,we combined PCR-directed mutagenesis,protein engineering and spectroscopic methods to investigate the role of PRXSF and F187,F192,F251.It was found that(1)the substitution of P459?S462 and P463 in PRXSF resulted in the disappearance of the photoactivation,therefore indicating that these three sites was essential for the photoactivation behavior.Different from this situation,both R460 mutants and K461 mutants exhibited the photoactivation behavior similar to PAiRFP1.The properties of the introduced amino acids affected various photophysical parameters,including the extincition coefficient,fluorescence quantum yield,molecular brightness and fluoscence contrast.Among them,R460A?R460D?K461A are the promising candidate for the further work.(2)It was also found that the replacement of F187,F192 and Y251 gave rise to the diminished photactivation or the absence of photoactivation.In order to explain the mechanism why these mutations affect the photoactivation,we explored the BV-assembing dynamcis,the dark recovery,the protein structure,as well as the stability of protein and BV-binding pocket against GdmCl in this work.More systematic work or molecular dynamics needs to be done in the future to gain deeper understanding on the mechanism.