Vik Steven B, Ishmukhametov Robert R
Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275-0376, USA.
J Bioenerg Biomembr. 2005 Dec;37(6):445-9. doi: 10.1007/s10863-005-9488-6.
The structure of subunit a of the Escherichia coli ATP synthase has been probed by construction of more than one hundred monocysteine substitutions. Surface labeling with 3-N-maleimidyl-propionyl biocytin (MPB) has defined five transmembrane helices, the orientation of the protein in the membrane, and information about the relative exposure of the loops connecting these helices. Cross-linking studies using TFPAM-3 (N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimido-propionamide) and benzophenone-4-maleimide have revealed which elements of subunit a are near subunits b and c. Use of a chemical protease reagent, 5-(-bromoacetamido)-1,10-phenanthroline-copper, has indicated that the periplasmic end of transmembrane helix 5 is near that of transmembrane helix 2.
通过构建一百多个单半胱氨酸取代突变体,对大肠杆菌ATP合酶的a亚基结构进行了研究。用3-N-马来酰亚胺基丙酰生物素(MPB)进行表面标记,确定了五个跨膜螺旋、蛋白质在膜中的方向,以及有关连接这些螺旋的环的相对暴露情况的信息。使用TFPAM-3(N-(4-叠氮基-2,3,5,6-四氟苄基)-3-马来酰亚胺基丙酰胺)和二苯甲酮-4-马来酰亚胺进行交联研究,揭示了a亚基的哪些部分靠近b亚基和c亚基。使用化学蛋白酶试剂5-(-溴乙酰胺基)-1,10-菲咯啉-铜表明,跨膜螺旋5的周质端靠近跨膜螺旋2的周质端。