Heme-bound SiaA from Streptococcus pyogenes: Effects of mutations and oxidation state on protein stability.
Heme-bound SiaA from Streptococcus pyogenes: Effects of mutations and oxidation state on protein stability.
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DOI：
10.1016/j.jinorgbio.2015.10.016
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发表时间：
2016-05
影响因子：
3.9
通讯作者：
Dixon DW
中科院分区：
文献类型：
--
作者：
Akbas N;Draganova EB;Block DR;Sook BR;Chan YF;Zhuo J;Eichenbaum Z;Rodgers KR;Dixon DW
蛋白质SiaA（HtsA）是酿脓链球菌中血红素摄取途径的一部分。在这份报告中，我们提出了血红素结合的丙氨酸突变体的轴向组氨酸（H229 A）和甲硫氨酸（M79 A）配体，以及赖氨酸（K61 A）和半胱氨酸（C58 A）位于附近的血红素丙酸（基于同源建模）和控制突变体（C47 A）。pH滴定得到的pKa值范围为9.0至9.5，接近WT SiaA的9.7值。突变体的共振拉曼光谱表明，铁血红素环境可能是不同的野生型;光谱的亚铁态是相似的。通过光谱电化学滴定法测定K61 A突变体的中点还原电位为61 ± 3 mV（相对于SHE），与野生型蛋白（68 ± 3 mV）相似。盐酸胍的加入显示了两个蛋白质变性过程，与血红素从蛋白质形式中的损失一致，不同的是血红素在结合口袋中的方向（较慢过程的半衰期为1至3天）。蛋白质展开的容易程度与残基与血红素的相互作用强度有关。我们推测，动力学上容易，但只有部分展开，随后是一个非常缓慢的方法完全展开的状态，可能是血红素运输蛋白质的一个基本属性。释放/转移血红素的小运动伴随着对广泛展开的抵抗，可以保持蛋白质的三维形式以供进一步摄取和释放。
The protein SiaA (HtsA) is part of a heme uptake pathway in Streptococcus pyogenes. In this report, we present the heme binding of the alanine mutants of the axial histidine (H229A) and methionine (M79A) ligands, as well as a lysine (K61A) and cysteine (C58A) located near the heme propionates (based on homology modeling) and a control mutant (C47A). pH titrations gave pKa values ranging from 9.0 to 9.5, close to the value of 9.7 for WT SiaA. Resonance Raman spectra of the mutants suggested that the ferric heme environment may be distinct from the wild-type; spectra of the ferrous states were similar. The midpoint reduction potential of the K61A mutant was determined by spectroelectrochemical titration to be 61 ± 3 mV vs. SHE, similar to the wild-type protein (68 ± 3 mV). The addition of guanidine hydrochloride showed two processes for protein denaturation, consistent with heme loss from protein forms differing by the orientation of the heme in the binding pocket (the half-life for the slower process was one to three days). The ease of protein unfolding was related to the strength of interaction of the residues with the heme. We hypothesize that kinetically facile but only partial unfolding, followed by a very slow approach to the completely unfolded state, may be a fundamental attribute of heme trafficking proteins. Small motions to release/transfer the heme accompanied by resistance to extensive unfolding may preserve the three dimensional form of the protein for further uptake and release.