Effect On The Spectroscopic Behavior Of Near-infrared Fluorescent Protein PAiRFP1 And Its Stability And Photoactivation

Bacteriophytochrome(abbreviated as Bph P)is a Biliverdin(desigated as BV)-containing protein widely existing in bacteria,which can make response to red or far-red light.Although BphP has natural biological function of light-dependent signal transduction,Bph P can be used as near infrared(NIR)fluorescence probe to achieve the deeper tissue imaging when they are modified by protein engineering strategy to emit fluorescence in the near infrared region.In this paper,we choose a NIR fluorescent protein PAiRFP1 engineerred from Bph P of Agrobacterium tumefaciens.Different from other BphP-based modified fluorescence proteins(BphP-FP),PAiRFP1 contains the PHY domain,therefore it exhibits the photoactivation behavior,i.e.the NIR fluorescence intensity is enhanced following the photo-conversion from Pfr to Pr state upon irradiation.This behavior helped to improve the signal-to-noise ratio in vivo imaging of tumors.Little useful information is available concerning what factors would affect the photoactivation behavior.In the present paper,we combined PCR site-directed mutagenesis method,protein engineering and spectroscopic methods to investigate the effect of both environmental factors(including pH,metal ions,redox conditions,temperature)and amino acid residues on the photoactivation behavior.It was found(1)As pH increased,PAiRFP1 displayed the higher photoactivation efficiency and lower photoactivation rate.(2)More complicated and heterogeneous photoactivation processes were observed upon increasing the temperature.(3)The displacement of some amino acid residuse surrounding BV(for example Cys-13,Asp-196 and His-248)gave rise to the disappearance of Pfr state.As a result,the mutants exhibited no photoactivation behavior.Although similar phenomena were observed for Gln-190 and Tyr-251 mutants,the spectral broadening was clearly detected,probably indicating that there might coexist the heterogeneous distribution of BV molecules.Additionally,it was also found that the variation of IIe-20,which is far away from BV,led to the instability of Pfr state.In this paper,some information was also obtained from the pH-induced spectroscopic changes,activation energy involved in the photoactivation,as well as guanidine hydrochloride-induced denaturation.These data will provide some guidance for deeper understanding the roles of amino acid residues in the photoactivation.