Characterization of the second conserved domain in the heme uptake protein HtaA from Corynebacterium diphtheriae.
Characterization of the second conserved domain in the heme uptake protein HtaA from Corynebacterium diphtheriae.
复制标题
白喉棒杆菌血红素摄取蛋白 HtaA 第二个保守结构域的表征。
DOI：
10.1016/j.jinorgbio.2016.11.027
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发表时间：
2017
影响因子：
3.9
通讯作者：
Dixon,DabneyW
中科院分区：
文献类型：
--
作者：
Uluisik,RizvanC;Akbas,Neval;Lukat-Rodgers,GudrunS;Adrian,SethA;Allen,CourtniE;Schmitt,MichaelP;Rodgers,KentonR;Dixon,DabneyW
HtaA是一种血红素结合蛋白，是白喉棒状杆菌血红素摄取系统的一部分。HtaA含有两个保守区（CR 1和CR2）。先前已经报道，这两个结构域都可以结合血红素; CR2结构域比CR 1结构域更强地结合血红蛋白。在这项研究中，我们报告的HtaA-CR2的生物物理特性。紫外-可见光谱和共振拉曼实验是一致的，这个域包含一个单一的血红素，通过轴向酪氨酸配体结合到蛋白质。保守的酪氨酸和组氨酸残基（Y361，H412和Y 490）的突变体进行了研究。与野生型蛋白质（~ 20%）相比，这些突变体分离出的血红素非常少（≤ 5%）。用丁酮除去血红素后的重构得到蛋白质的另一种形式。HtaA-CR2折叠非常稳定;有必要在盐酸胍存在下进行热变性实验。HtaA-CR2的展开速度非常缓慢;即使在37 °C的6.8 M GdnHCl中，半衰期也只有5小时。相比之下，WT HtaA-CR 2和上述突变体的apo形式在低得多的GdnHCl浓度下解折叠，表明血红素在稳定结构中的作用，并暗示血红素转移仅在体内对伴侣蛋白产生影响。
HtaA is a heme-binding protein that is part of the heme uptake system in Corynebacterium diphtheriae. HtaA contains two conserved regions (CR1 and CR2). It has been previously reported that both domains can bind heme; the CR2 domain binds hemoglobin more strongly than the CR1 domain. In this study, we report the biophysical characteristics of HtaA-CR2. UV–visible spectroscopy and resonance Raman experiments are consistent with this domain containing a single heme that is bound to the protein through an axial tyrosine ligand. Mutants of conserved tyrosine and histidine residues (Y361, H412, and Y490) have been studied. These mutants are isolated with very little heme (≤ 5%) in comparison to the wild-type protein (~ 20%). Reconstitution after removal of the heme with butanone gave an alternative form of the protein. The HtaA-CR2 fold is very stable; it was necessary to perform thermal denaturation experiments in the presence of guanidinium hydrochloride. HtaA-CR2 unfolds extremely slowly; even in 6.8 M GdnHCl at 37 °C, the half-life was 5 h. In contrast, the apo forms of WT HtaA-CR2 and the aforementioned mutants unfolded at much lower concentrations of GdnHCl, indicating the role of heme in stabilizing the structure and implying that heme transfer is effected only to a partner protein in vivo.