| The maximum excitation and emission of Near infra-red fluorescent(NIR)proteins are all in a range of tissue-imaging 'optic window',which have been broadly used in living deep tissue imaging.Researchers can label some intriguing moleculars with these fluorescent proteins,which directly and dynamically track the moleculars probably how to play a role in livings.This kind of research method taking the advantage of fluorescent imaging,which will provide another powerfully technological support for rapid expansion of biology field.Recently,although there have been little near infra-red fluorescent proteins that still have the future of evolution,which must be optimized for their optical properties,such as,the red-shifted excitation and emission,pH stability,monomeric property,quantum yield increasing,maturation rate raising,and so on.These key natures that influence fluorescent protein applying in imaging in vivo,are always main indexes for researchers to develop the fluorescent protein evolution.In recent years,our lab had acquired a series of NIR proteins through the evolution in vitro.Among them,so far,mNeptune684 has the longest emission,which maximum excitation/emission is 604/684 nm.However,it is a little pity that the quantum yield of mNeptune684 is only 0.03.The structural reason of the high quantum yield is an environment ensuring the planarity of the chromophore and restricting the rotation.The more increasing the red shift emission of the RFP,the more difficult for maintaining the environment that mentioned above.At last,the longer emission wavelength,the lower quantum yield,the FP's application of imaging in vivo is limited.Our study is mainly through rational design and random mutation that are based on analyzing protein crystalline structure,and should ensure the emission of mNeptune684 that is at near infra-red spectrum,at the same time,increasing the quantum yield to the most.The experiment would have two aspect:(1)reading other researcher's paper for reference,in which they remold the seven strand of ?-sheet in ECFP,making the structure of chromophore stable and increasing the quantum yield,and introducing this similar concept in remolding the ?-sheet in mNeptune684.In that paper,after the amino residue of 148His is substituted by Asp in the ECFP,the hydrogen bond interaction between the seven strand and ten strand of p-sheet should be more stable,then to firm the external structure of the chromophore and at last to steady the chromophore.Returning to the mNeptune684,through analysis of crystalline structure,we find that there is a random coil in the seven strand of its ?-barrel structure.Due to this helix,the structure of this protein has been loosely organized,which refers to two amino residues at 141/142.Above all these finding,we take up to remold this protein.During the experiment,we use secondary structure predicting software to narrow the number of mutation amino acid down,but none of each mutation has good result,some is no fluorescence and some is blue-shifted emission;(2)return to the analysis about amino acid surrounding chromophore,through hydrogen bond interaction between amino acid side chain and the chromophore to develop the mutation that can stable chromophore conformation.We also use analysis of crystalline structure in this test.In the mNeptune684,the site of Asn143 and Asn158 play a key role in red-shifted spectrum,which expand the hydrogen bond net work around the chromophore,and simultaneously,the hydrogen bond interaction among Arg155,His157 and Pro159 make chromophore steady.As the amino acid mutations mentioned above are gathering around p-hydrroxybenzylidene group of chromophore,so in our test,we select mutative sites are surrounding acylimine group,which are located in another side of chromophore.Via screening,we finally obtain a mutation mNeptune684_M11V that the methionine at 11 site is substituted by valine.Its quantum yield is 0.07,almost 2.5 times compared to the parental mNeptune684,and can apply in cell imaging.In the future,we would try to use this protein in animal imaging in vivo. |
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