Study On Photosensitive Orange Carotenoid Protein OCP And Photosensitive Tetrapyrrole Chromophore

Phytochrome is a kind of infrared and far-infrared receptor protein which covalently binding a linear tetrapyrrol with apo-protein exist widely in cyanobacteria and plants.It has important significance for life activities.The spectral redshift of plant phytochromes which combined with phytochromobilin(P?B)is about 10 nm compared with that cyanobacteriochromes(CBCRs)which combined with phycocyanobilin(PCB).This phenomenon of red shift has important biological significance in the development of fluorescent probes and deep tissue imaging.Although the expression cycle of phytochrome in vivo is long and difficult to purify,it is possible to obtain similar phytochrome expressed in Escherichia coli expression system.In this work,we design and optimize a simple method to isolate efficiently useful quantities of P?B.By co-expressing the optimized chromophore-binding domain of core-membrane linker protein encoded by Synechococcus sp.PCC7335 apcE2 with the plasmids for P?B in E.coli.In this way,not only the expression yield of pigments can be greatly increased,but also obtain non-covalent binding of the pigment to its apo-proteins.This hole-protein can release free P?B through a simple method.The isolated P?B was successfully incorporated into phytochrome-related assemblies,and furthermore,the noncovalently bound P?B could be transferred directly from the ApcE2 construct to the apo-proteins of phytochromes,CBCRS and phycobiliproteins,without loss of relevant biological activity.In the study of cyanobacteria found that not only CBCRs can responded to light changes under suitable light conditions,but also other light receptor proteins such as blue light receptor protein:orange carotenoid protein(OCP).Under irradiation of high light,dark stable orange form of OCP(OCP~O)is converted to a red active form(OCP~R),which triggers NPQ and quenches the fluorescence of PBS to protect the cyanobacteria cells from the stress of light.In order to further explore the dynamic conversion process of OCP protein,in this study,native and high resolution crystals were obtained then used dynamic crystallography at the molecular level to elucidate this change.For the functional study of activated OCP,current scholars have explored specific action sites through non-photochemical quenching(NPQ)of various types of phycobilisome(PBS).However,due to the complex assembly of PBS,the specific action sites are still uncertain.In this study,the subunits of PBS was expressed separately in E.coli for the first time and activated OCP was added to study the fluorescence quenching of each subunit in vitro.In vitro quenching experiments showed that activated OCP could strongly quench the fluorescence of all of these fluorescent proteins including the APC,CPC,PEC,CBCRs,green fluorescent protein(GFP)and mCherry.Thus,quenching fluorescence is an intrinsic feature of activated OCP and there is no specificity with the target proteins.The difference of fluorescence quenching capacity between different proteins should be affected by the overlapping properties of the protein spectrum.This is a further study of the function of OCP protein and NPQ,and has important guiding significance for the further study of the quenching sites and mechanism of NPQ.