Fluoresceination of FepA during colicin B killing: effects of temperature, toxin and TonB.
Fluoresceination of FepA during colicin B killing: effects of temperature, toxin and TonB.
复制标题
FepA 在大肠杆菌素 B 杀灭过程中的荧光作用：温度、毒素和 TonB 的影响。
DOI：
10.1111/j.1365-2958.2009.06715.x
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发表时间：
2009
影响因子：
3.6
通讯作者：
Klebba,PhillipE
中科院分区：
文献类型：
--
作者：
Smallwood,ChuckR;Marco,AmparoGala;Xiao,Qiaobin;Trinh,Vy;Newton,SaleteMC;Klebba,PhillipE
我们研究了35个基因工程半胱氨酸巯基在大肠杆菌FepA中不同位置的反应性。荧光素马来酰亚胺（FM）表面环残基的修饰在体内具有强烈的温度依赖性，而其他位点的反应性受影响较小。与牛血清白蛋白的对照反应显示，环残基反应性的温度依赖性异常高，表明多个环（L2、L3、L4、L5、L7、L 8、L10）中的构象变化在37°C下将受体转化为更易接近的形式。在0°C下，大肠杆菌素B结合在10个表面环位点中的8个位点处削弱或阻断标记，推测是通过空间位阻。总体而言，大肠杆菌素B吸附降低了FepA N-和C-结构域中35个位点中一半以上的反应性。然而，大肠杆菌素B在37°C下渗透到细胞中并不增加FepA中任何残基的化学修饰。FM修改模式同样不受thetonBlocus的影响。FepA在atonB宿主菌株中以较低水平表达，但当我们考虑到这种降低时，无论TonB存在与否，其FM标记都是相当的。因此，我们没有检测到FepA中的TonB依赖性结构变化，无论是单独的还是当它在37°C与大肠杆菌素B相互作用时。化学修饰的唯一变化是细菌素与受体蛋白结合时空间位阻的减少。N结构域残基的反应性没有增加，这与大肠杆菌素B多肽穿过FepA通道的观点相反。
We studied the reactivity of 35 genetically engineered Cys sulphydryl groups at different locations inEscherichia coliFepA. Modification of surface loop residues by fluorescein maleimide (FM) was strongly temperature‐dependentin vivo, whereas reactivity at other sites was much less affected. Control reactions with bovine serum albumin showed that the temperature dependence of loop residue reactivity was unusually high, indicating that conformational changes in multiple loops (L2, L3, L4, L5, L7, L8, L10) transform the receptor to a more accessible form at 37°C. At 0°C colicin B binding impaired or blocked labelling at 8 of 10 surface loop sites, presumably by steric hindrance. Overall, colicin B adsorption decreased the reactivity of more than half of the 35 sites, in both the N‐ and C‐ domains of FepA. However, colicin B penetration into the cell at 37°C did not augment the chemical modification of any residues in FepA. The FM modification patterns were similarly unaffected by thetonBlocus. FepA was expressed at lower levels in atonBhost strain, but when we accounted for this decrease its FM labelling was comparable whether TonB was present or absent. Thus we did not detect TonB‐dependent structural changes in FepA, either alone or when it interacted with colicin B at 37°C. The only changes in chemical modification were reductions from steric hindrance when the bacteriocin bound to the receptor protein. The absence of increases in the reactivity of N‐domain residues argues against the idea that the colicin B polypeptide traverses the FepA channel.