Imikhiqizo

I-membrane topology yesizinda sesiteshi se-colicin E1 yafundwa ngokudluliswa kwe-fluorescence resonance energy (FRET).I-FRET ihilela i-amino acid ye-fluorescent efakwe ngokofuzo (coumarin) njengomnikezeli kanye nezinsalela ze-cysteine ​​​​ezihlanganiswe ne-DABMI (4-(dimethylamino)phenylazophenyl-4'-maleimide) njengomamukeli we-FRET.Izinsalela ze-fluorescent coumarin zafakwa kuphrotheni nge-orthogonal tRNA/aminoacyl-tRNA synthetase pair evumela ukufakwa okukhethiwe kunoma iyiphi isayithi ngaphakathi kwesizinda sesiteshi se-colicin.Okuhlukile ngakunye kubambe ukuguqulwa kwesitobhi (TAG) sokufakwa kwe-coumarin kanye nokuguqulwa kwe-cysteine ​​(TGT) kokunamathiselwe kwe-DABMI.Amabanga ayisithupha e-interhelical ngaphakathi kwama-helices 1-6 anqunywe kusetshenziswa ukuhlaziya kwe-FRET kuzo zombili izifunda ezincibilikayo nezibopha ulwelwesi.Idatha ye-FRET ibonise izinguquko ezinkulu kumabanga we-interhelical phakathi kwama-helices 3-6 phezu kwe-membrane association enikeza ukuqonda okusha esakhiweni esiboshwe ulwelwesi sesizinda sesiteshi.Ngokuvamile, ukusebenza kahle kwe-coumarin-DABMI FRET kwe-interhelical kwehle ekubopheni ulwelwesi, kwakhela phezu kwemodeli yesambulela yesiteshi se-colicin.Imodeli yesikhashana yesimo esivaliwe sesizinda sesiteshi yakhiwe ngokusekelwe kudatha yamanje neshicilelwe ngaphambilini ye-FRET.Imodeli iphakamisa ukuhlelwa okuyisiyingi kwama-helices 1-7 ngendlela yewashi ukusuka ohlangothini olungaphandle kwe-extracellular kanye ne-membrane interfacial association yama-helices 1, 6, 7, no-10 azungeze iphini lezinwele eliphakathi nendawo elakhiwe ama-helices 8 no-9.