Helix proximity and ligand-induced conformational changes in the lactose permease of Escherichia coli determined by site-directed chemical crosslinking.
Helix proximity and ligand-induced conformational changes in the lactose permease of Escherichia coli determined by site-directed chemical crosslinking.
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通过定点化学交联测定大肠杆菌乳糖通透酶的螺旋接近度和配体诱导的构象变化。
DOI：
10.1006/jmbi.1997.1099
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Kaback,HR
中科院分区：
文献类型：
--
作者：
Wu,J;Kaback,HR
N和C-末端的乳糖通透酶，每个与一个单一的半胱氨酸残基的一半，共表达，交联进行了研究。碘或N，N′-邻苯二马来酰亚胺（o-PDM;刚性6 μ m），交联Asn 245 →Cys（螺旋VII）和Ile 52 →Cys或Ser 53 →Cys（螺旋II）。N，N′-对亚苯基二马来酰亚胺（p-PDM;刚性10 μ m）较弱地交联245/53 Cys对，但不交联245/52 Cys对，而1，6-双马来酰亚胺己烷（BMH;柔性16 μ m）交联这两对Cys对的效率低于o-PDM。因此，245与52和53的距离几乎相等，最多约为6 μ m。BMH或p-PDM交联Gln 242 →Cys和Ser 53 →Cys，但o-PDM无效，表明距离变化高达10 μ m。配体结合增加了245/53与p-PDM或BMH的交联，对o-PDM几乎没有影响，并降低了碘交联。在245/52中观察到类似的效果。配体增加与p-PDM或BMH的242/53交联，但与o-PDM没有观察到交联。因此，配体诱导螺旋平移或剪刀样位移3-4 π。交联245/53抑制转运，表明构象柔性对功能是重要的。
N and C-terminal halves of lactose permease, each with a single-Cys residue, were co-expressed, and crosslinking was studied. Iodine or N, N′-o-phenylenedimaleimide (o-PDM; rigid 6 Å), crosslinks Asn245→Cys (helix VII) and Ile52→Cys or Ser53→Cys (helix II). N,N′-p-phenylenedimaleimide (p-PDM; rigid 10 Å) crosslinks the 245/53 Cys pair weakly, but does not crosslink 245/52, and 1,6-bis-maleimidohexane (BMH; flexible 16 Å) crosslinks both pairs less effectively than o-PDM. Thus, 245 is almost equidistant from 52 and 53 by up to about 6 Å. BMH or p-PDM crosslinks Gln242→Cys and Ser53→Cys, but o-PDM is ineffective, indicating that distance varies by up to 10 Å. Ligand binding increases crosslinking of 245/53 with p-PDM or BMH, has little effect with o-PDM and decreases iodine crosslinking. Similar effects are observed with 245/52. Ligand increases 242/53 crosslinking with p-PDM or BMH, but no crosslinking is observed with o-PDM. Therefore, ligand induces a translational or scissors-like displacement of the helices by 3-4 Å. Crosslinking 245/53 inhibits transport indicating that conformational flexibility is important for function.