Method Construction Of Vps4 Depolymerization Of ESCRT? By Single Molecule Fluorescence

ESCRT(Endosomal Sorting Complex Required for Transport)which was first discovered as an important macromolecular multiprotein machinary involved in protein sorting pathways of multivesicular bodies,and facilitate various roles in cell division,HIV budding,neurodegenerative disorders and plasma membrane repair.Intracellularly,the ESCRT? complex is continuously being assembled and disassembled by an AAA+ ATPase Vps4.These two together constitute a molecular shearing machine,which plays an important role in the cleavage and separation of vesicles.In recent years,great progress has been achieved in resolving the structural aspects of the ESCRT-Vps4 related protein complexes but the dynamics and molecular mechanism of Vps4 depolymerization of ESCRT? complex still remains elusive.Few questions like.the arrangement of the individual protein components in the ESCRT? complex,their binding order and mechanistic details of Vps4 mode of action on the ESCRT? complexes remains to be understood.Therefore,in this report the dynamic process of ESCRT? complex depolymerization has been studied.Our main focus was to construct an ESCRT? system suitable for the singlemolecule assay using biochemical methods,negative electron microscopy and singlemolecule fluorescence.The structure of the ESCRT? protein from PDB was analyzed,and the amino acids which have no predicted affect on the structural stability and filament formation were selected for mutagenesis.The mutant proteins only have one Cys.The mutant proteins were extracted and the stabilities were investigated.The efficiency of fluorescence labeling of mutant protein was investigated by absorption spectroscopy.The ability of the mutant protein to form filaments and its depolymerization by Vps4 was checked by negative staining electron microscopy and sedimentation assay.Finally,Five sites on ESCRT? protein Snf7-K57,G58,N59,G120 and D122,were selected to be mutated to cysteine.K57 C mutant protein was unstable.G58 C mutant protein had low labeling efficiency.Mutatants N59 C and G120 C affected the formation of filament.Only D122 C mutant protein was stable with high labeling efficiency.The labeled protein formed filaments that could be depolymerized by Vps4.The Snf7 D122 C mutant was screened out,to carry out single molecule studies on the ESCRT?-Vps4 system.These results serve as a basis for establishing a standard single molecule assay for elucidating the mechanistic aspects of ESCRT? formation and its depolymerization by Vps4.