| Light-activated fluorescent protein is a type of protein that can shift its fluorescence or change its intensity under light of a specific wavelength.Because they can improve the imaging spatial resolution and signal-to-noise ratio during imaging,they have attracted much attention in the imaging field.In this thesis,a new type of light-activated fluorescent protein PAiRFP1,which was modified using the bacterial phytochrome Bph P as a template,was selected.Under light induction,the biliverdin molecule C15=C16 double bond in Bph P undergoes cis-trans isomerization,resulting in the conversion of the non-fluorescent Pfr state to the fluorescent Pr state,and finally the fluorescence intensity increases.Compared with other light-activated fluorescent proteins,although PAiRFP1 has a lower near-infrared fluorescence quantum yield,its biggest advantage is that both excitation and emission are in the near-infrared region,which can avoid tissue self-absorption and autofluorescence,and achieve deep tissue imaging.In view of the advantages of this type of protein,discussing how this type of special light-activated fluorescent protein evolved and which amino acid mutations change the photoactivation can help guide us to discover and modify more of this type of protein.In addition,during the assembly process of this type of pigment-protein complex,the protoporphyrin molecule in the body will compete with the pigment molecule biliverdin in Bph P to bind to the bacterial phytochrome.Therefore,studying competitive binding and the influence of binding on near-infrared fluorescence is also very important for improving the luminescence characteristics of PAiRFP1.For this kind of special light-activated near-infrared fluorescent protein,it needs to have light-activated behavior at least to meet the following four conditions:（1）Pr and Pfr states must coexist in the dark;（2）Pfr state can transfer to Pr state under light conditions.（3）Pr state can emit near-infrared fluorescence;（4）Pr state has certain stability.In this paper,we used PCR saturation mutation,protein engineering,and spectroscopy to explore the influence of the nature of the amino acid residues at positions 244 and 163 near the biliverdin binding pocket on the above links,and found that:（1）In evolution During the process,the mutation of amino acid at position 163 to Leu is very important for obtaining photoactivation activity;when this position is occupied by other amino acid residues,the photoactivation activity is severely weakened or almost lost;（2）the amino acid properties at position 244 affect The maximum absorption peak of the Pr state,the position of the maximum fluorescence emission peak,and the fluorescence quantum yield are shown.By comparison,we have obtained a dominant mutant F244L,its molar extinction coefficient is 50345 M-1.cm-1,the maximum fluorescence emission peak is red-shifted by 5 nm,and the molecular brightness is slightly increased than that of the wild type;（3）244 The site also participates in the competitive binding of protoporphyrin,affecting the fluorescence emission of protoporphyrin and the near-infrared fluorescence emission of biliverdin.In addition to discussing the effects of mutations on photoactivation characteristics,we also tried to apply two-dimensional correlation spectroscopy to the field of bacterial phytochromes.By changing the excitation wavelength,we obtained 3fluorescence emission spectra.After the Matlab matrix calculation,we obtained the two-dimensional correlation synchronous and asynchronous spectra.From the cross peaks,we found that the protoporphyrin may be bound to the bacterial phytochrome in a variety of ways.These results laid the foundation for our follow-up in-depth research. |
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