Molecular Design And Properties Of A New Far-red Fluorescent Protein With Large Stokes Shift

Fluorescent proteins have been widely used in life sciences,and simplified the experimental operation of intracellular components imaging with high precision.The fluorescent probes at the range of 650 nm~700 nm have some advantages,such as the higher transmittance,longer time of imaging,weak spontaneous fluorescence background and low light scattering rate in vivo.Far-red fluorescent proteins with a large Stokes shift also become the research focus in this field.Phycobiliproteins can be organized as the phycobilisomes,which can harvest orange,red and/or far-red lights for photosynthesis in cyanobacteria and red algae,phycobiliproteins in the phycobilisome cores such as allophycocyanins absorb far-red light for funneling the energy to the reaction centers.Thus,the allophycocyanin subunits have been engineered as far-red fluorescent proteins such as BDFP1.2/1.6.Nevertheless,most fluorescent proteins have small Stokes shifts at present,which limits their applications in multicolor bioimaging.mCherry is an excellent fluorescent protein that has maximal emittance in the red spectral range and high fluorescence quantum yield,thus can be used as a donor for energy transfer to the far-red acceptor such as BDFP1.6 via F?rster resonance energy transfer.In this study,mCherry was fused with BDFP1.6 in different ways,which results in a new fluorescent protein with a large Stokes shift,BDFP2.0.The excitation energy was absorbed maximally at 580 nm by mCherry and transferred to BDFP1.6 efficiently,thus the effective brightness of BDFP 2.0 in mammalian cells has been effectively enhanced at the far-red region.Meanwhile,it has the extraordinary stability of pH and guanidine hydrochloride.We fused BDFP 2.0 with different target proteins and transfected HeLa cells and the images were clear and bright,then BDFP2.0 transfected HeLa cells with BDFP 1.1 simultaneously,and high resolution dual-color imaging photographs can be clearly distinguished.Therefore,BDFP2.0 is not only a well bio-label marker of mammalian cells components,but also performances well in dual-color imaging.At the same time,the strategies of BDFP2.0 also provide some new ideas for researchers to develop new fluorescent proteins and FRET quantitative analysis.In the process of molecular evolution of subunit ApcF2,ApcF2-12,a mutant with special spectrum features was obtained.The mutant ApcF2-12 has low yield but two excitation peaks.By means of random mutagenesis and screen of ApcF2-12,not only did we screen the mutants with high yield,but also some mutants with special fluorescence spectrum features,such as large Stokes shift and blue shift spectrum.And blast by sequencing,attempt to making some analysis of the fluorescence spectrum changes.In molecular evolution of ApcF2-12,the random mutagenesis methods with a stable mutation rate and high accuracy of transformed rate were determined,which enrich the mutagenesis library for screening excellent fluorescent probes and provid some benefits for studying the fluorescence changes of BV-binding phycobiliproteins.The N-terminal domain(24-245)of core-membrane linker(ApcE2)from Synechococcus sp.PCC7335 can non-covalently bind to PEB/PCB autocatalytically,thus emitting the red-shift fluorescence.The pigment was extracted by two protein denaturation methods of urea or enzyme reaction and two extraction methods of butanone or chloroform,which results in a less toxicity preparation method with low expenses,high extraction efficiency and simple experimental operation.