(19)F nuclear magnetic resonance and crystallographic studies of 5-fluorotryptophan-labeled anthrax protective antigen and effects of the receptor on stability.
(19)F nuclear magnetic resonance and crystallographic studies of 5-fluorotryptophan-labeled anthrax protective antigen and effects of the receptor on stability.
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DOI：
10.1021/bi401405s
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发表时间：
2014-02-04
期刊：
影响因子：
2.9
通讯作者：
Bann JG
中科院分区：
文献类型：
--
作者：
Chadegani F;Lovell S;Mullangi V;Miyagi M;Battaile KP;Bann JG
炭疽保护性抗原（PA）是一种83 kDa的蛋白质，是炭疽毒素（由炭疽芽孢杆菌分泌的AB毒素）的三种蛋白质组分之一。PA能够经历几种结构变化，包括寡聚成称为前孔的七聚体或八聚体结构，以及在酸性pH下主要构象变化以形成跨膜孔。为了在残基特异性水平上跟踪这些结构变化，我们进行了初步研究，在这些研究中，我们将5-氟色氨酸（5-FTrp）生物合成纳入PA中，并且我们研究了5-FTrp标记对PA结构稳定性的影响以及对使用19 F核磁共振（NMR）与宿主受体毛细血管形态发生蛋白2（CMG 2）结合的影响。PA中有7个色氨酸，但在PA的4个结构域中，只有2个含有色氨酸：结构域1（Trp 65、-90、-136、-206和-226）和结构域2（Trp 346和-477）。Trp 346是特别感兴趣的，因为它接近CMG 2结合界面，并且因为它形成跨膜孔的一部分。我们表明，19 F共振的Trp 346是敏感的pH值的变化，与晶体学研究一致，受体结合显着稳定Trp 346 pH值和温度。此外，我们提供的证据表明，共振从遥远的结合界面的Dahans也稳定的受体。我们的研究强调了受体结合对蛋白质稳定性的积极影响，以及19 F NMR在深入了解高分子量蛋白质结构变化方面的应用。
The anthrax protective antigen (PA) is an 83 kDa protein that is one of three protein components of the anthrax toxin, an AB toxin secreted by Bacillus anthracis. PA is capable of undergoing several structural changes, including oligomerization to either a heptameric or octameric structure called the prepore, and at acidic pH a major conformational change to form a membrane-spanning pore. To follow these structural changes at a residue-specific level, we have conducted initial studies in which we have biosynthetically incorporated 5-fluorotryptophan (5-FTrp) into PA, and we have studied the influence of 5-FTrp labeling on the structural stability of PA and on binding to the host receptor capillary morphogenesis protein 2 (CMG2) using 19F nuclear magnetic resonance (NMR). There are seven tryptophans in PA, but of the four domains in PA, only two contain tryptophans: domain 1 (Trp65, -90, -136, -206, and -226) and domain 2 (Trp346 and -477). Trp346 is of particular interest because of its proximity to the CMG2 binding interface, and because it forms part of the membrane-spanning pore. We show that the 19F resonance of Trp346 is sensitive to changes in pH, consistent with crystallographic studies, and that receptor binding significantly stabilizes Trp346 to both pH and temperature. In addition, we provide evidence that suggests that resonances from tryptophans distant from the binding interface are also stabilized by the receptor. Our studies highlight the positive impact of receptor binding on protein stability and the use of 19F NMR in gaining insight into structural changes in a high-molecular weight protein.