Explore The Specific Role Of Amino Acid Substitution In The Evolution Of AtBphp2 Into Near-infrared Fluorescent Protein PAiRFP1

Bacteriophytochromes?designated as BphPs?are a class of biliverdin?BV?-protein complexes widely found in bacteria.They have a strong red/far-red light absorption capacity,and their natural biological function is light-dependent signaling.In the past 10 years,following the introduction of the protein engineering methods,the modified BphPs can emit fluorescence in the near infrared region.Based on this strategy,many BphPs have successfully evolved into near-infrared fluorescent proteins and have played an important role in vivo imaging.The research object of this thesis is the light-activated near-infrared fluorescent protein PAiRFP1,which is obtained through protein engineering methods based on the bacteriophytochromes AtBphP2from Agrobacterium tumefaciens as the template.In practical applications,compared to other near-infrared fluorescent proteins,the light-activated near-infrared fluorescent protein PAiRFP1 can achieve a higher imaging signal-to-noise ratio,and has an irreplaceable advantage in the vivo imaging.But it still has the following deficiencies,such as the low fluorescence quantum yield and molecular brightness.Given these things,the PAiRFP1 protein needs to be further modified.Since the spatial structure of the near-infrared fluorescent protein PAiRFP1 is unknown,the further protein modification will inevitably be accompanied by great blindness.Therefore,in this paper,we focus on the 15 amino acid replaced during the evolution of the PAiRFP1 protein.We used site-directed mutagenesis to reverse mutate the 15 amino acid positions of the PAiRFP1 protein to obtain 15 different reverse mutants.Subsequently,we analyzed the photochemical properties such as molar extinction coefficient,fluorescence quantum yield?QY?,molecular brightness,and maximum fluorescence emission peak position(F_max)of these 15 reversion mutants by spectroscopy method.The specific role of these 15 amino acid substitutions in regulating protein spectroscopy behavior was analyzed.We found that:1)Val-276 and Phe-244 amino acid residues have a greater influence on the position of the near infrared maximum fluorescence emission peak of PAiRFP1.When Val-276 is mutated to Ala,or Phe-244 to Val,Fmax is red-shifted by 3 nm and 8 nm,respectively;2)At the same time,Phe-244,Val-480,Tyr-498 have an important effect on the fluorescent quantum yield of PAiRFP1.When Phe-244 was mutated to Val,Val-480 to Ala,or Tyr-498 to His,QY decreased to²9%,²8%,and²2%,respectively.In addition,we preliminarily analyzed the structure-activity relationship of the 5amino acids of the protein active pocket that affects its spectroscopic properties.Finally,based on the above research,we tried to randomly mutate the key amino acid sites Val-276 and Cys-280 that regulate the spectral behavior of the PAiRFP1 protein.We obtained several dominant mutants such as PAiRFP1/V276G with the maximum fluorescence emission peak redshift of 8 nm,and PAiRFP1/C280V which dark recovery process was greatly inhibited.In summary,our study will provide valuable guidance for the subsequent directed evolution of the near-infrared fluorescent protein PAiRFP1.