Iimveliso

I-membrane topology ye-colicin E1 ye-channel domain yaphononongwa yi-fluorescence resonance energy transfer (FRET).I-FRET ibandakanya i-amino acid yemfuzo efakwe kwi-amino acid (coumarin) njengomnikezeli kunye nentsalela ebhalwe ngokukhethiweyo ye-cysteine ​​​​ebotshelelwe nge-DABMI (4-(dimethylamino)phenylazophenyl-4'-maleimide) njengomamkeli we-FRET.Intsalela ye-fluorescent ye-coumarin ifakwe kwiprotheni nge-orthogonal tRNA/aminoacyl-tRNA isibini se-synthetase esivumela ukufakwa okukhethiweyo kuyo nayiphi na indawo ngaphakathi kwendawo ye-colicin channel.I-variant nganye ine-stop (TAG) yokuguqulwa kwe-coumarin incorporation kunye ne-cysteine ​​​​(TGT) yokuguqulwa kwe-DABMI.Imigama emithandathu ye-interhelical ngaphakathi kwe-helices ye-1-6 yagqitywa ngokusebenzisa uhlalutyo lwe-FRET kuzo zombini iindawo ezinyibilikayo kunye ne-membrane.Idatha ye-FRET ibonise utshintsho olukhulu kwimida ye-interhelical phakathi kwe-helices 3-6 phezu kobudlelwane be-membrane enika ingqiqo entsha kwi-membrane-bound bound structure ye-channel domain.Ngokubanzi, i-coumarin-DABMI FRET i-interhelical efficiencies yehla kwi-membrane ebophelelayo, ukwakha phezu kwemodeli yeambrela ye-colicin channel.Imodeli yovavanyo yemeko evaliweyo yesizinda sesiteshi yaphuhliswa ngokusekelwe kwidatha yeFRET yangoku kunye nepapashwe ngaphambili.Imodeli icebisa ukucwangciswa kweesetyhula ze-helices 1-7 kwicala lewotshi ukusuka kwicala le-extracellular kunye ne-membrane interfacial association ye-helices 1, 6, 7, kunye ne-10 ejikeleze i-transmembrane hairpin ephakathi eyenziwe yi-helices 8 kunye ne-9.