Differential roles of tryptophan residues in conformational stability of Porphyromonas gingivalis HmuY hemophore
Differential roles of tryptophan residues in conformational stability of Porphyromonas gingivalis HmuY hemophore
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DOI：
10.1186/1471-2091-15-2
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发表时间：
2014-02-10
期刊：
影响因子：
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通讯作者：
Olczak, Teresa
中科院分区：
文献类型：
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作者：
Bielecki, Marcin;Wojtowicz, Halina;Olczak, Teresa
背景：我们之前已经证明牙龈卟啉单胞菌 HmuY 血红素样蛋白结合血红素并从宿主血红素蛋白中清除血红素，以进一步将其递送至同源血红素受体 HmuR。本研究的目的是表征存在和不存在血红素时 HmuY 变体的结构特征以及色氨酸残基在构象稳定性中的作用。结果：HmuY 在第 51 和 73 位具有色氨酸残基，这些残基在多种细菌中存在的 HmuY 同系物中是保守的，并且在第 161 位具有色氨酸残基，该残基仅在牙龈卟啉单胞菌中鉴定的 HmuY 中发现。菌株。我们表达并纯化了野生型 HmuY 及其蛋白质变体，其中单个色氨酸残基被丙氨酸或酪氨酸残基取代。所有 HmuY 变体均进行热变性和荧光光谱分析。替换最埋藏的 W161 仅适度影响蛋白质稳定性。在 W73 中观察到缺乏大疏水侧链对热稳定性的最深远影响。此外，表面暴露的 W51 的替换会导致蛋白质稳定性的最大损失，甚至酪氨酸残基的大芳香族侧链也几乎没有潜力替换该色氨酸残基。血红素结合导致 51 位色氨酸残基对蛋白质表面的不同暴露。 HmuY 变体结构稳定性的差异表明血红素结合后蛋白质三级结构的变化。结论：在这里，我们证明了色氨酸残基在蛋白质构象稳定性中的不同作用。我们还提出了由允许血红素与蛋白质结合的三级变化引起的apo-和holoHmuY的不同构象。
Background: We have previously shown that the P. gingivalis HmuY hemophore-like protein binds heme and scavenges heme from host hemoproteins to further deliver it to the cognate heme receptor HmuR. The aim of this study was to characterize structural features of HmuY variants in the presence and absence of heme with respect to roles of tryptophan residues in conformational stability.Results: HmuY possesses tryptophan residues at positions 51 and 73, which are conserved in HmuY homologs present in a variety of bacteria, and a tryptophan residue at position 161, which has been found only in HmuY identified in P. gingivalis strains. We expressed and purified the wildtype HmuY and its protein variants with single tryptophan residues replaced by alanine or tyrosine residues. All HmuY variants were subjected to thermal denaturation and fluorescence spectroscopy analyses. Replacement of the most buried W161 only moderately affects protein stability. The most profound effect of the lack of a large hydrophobic side chain in respect to thermal stability is observed for W73. Also replacement of the W51 exposed on the surface results in the greatest loss of protein stability and even the large aromatic side chain of a tyrosine residue has little potential to substitute this tryptophan residue. Heme binding leads to different exposure of the tryptophan residue at position 51 to the surface of the protein. Differences in structural stability of HmuY variants suggest the change of the tertiary structure of the protein upon heme binding.Conclusions: Here we demonstrate differential roles of tryptophan residues in the protein conformational stability. We also propose different conformations of apo-and holoHmuY caused by tertiary changes which allow heme binding to the protein.