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Heme Pocket Structural Properties of a Bacterial Truncated Hemoglobin from Thermobifida fusca

机译：嗜热栖热菌细菌截短的血红蛋白的血红素口袋结构性质

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摘要

An acidic surface variant (ASV) of the “truncated” hemoglobin from Thermobifida fusca wasndesigned with the aim of creating a versatile globin scaffold endowed with thermostability and a high level ofnrecombinant expression in its soluble form while keeping the active site unmodified. This engineered proteinnwas obtained by mutating the surface-exposed residues Phe107 and Arg91 to Glu. Molecular dynamicsnsimulations showed that the mutated residues remain solvent-exposed, not affecting the overall proteinnstructure. Thus, the ASV was used in a combinatorial mutagenesis of the distal heme pocket residues in whichnone, two, or three of the conserved polar residues [TyrB10(54), TyrCD1(67), and TrpG8(119)] werensubstituted with Phe. Mutants were characterized by infrared and resonance Raman spectroscopy andncompared with the wild-type protein. Similar Fe-proximalHis stretching frequencies suggest that none of thenmutations alters the proximal side of the heme cavity. Two conformers were observed in the spectra of the COncomplexes of both wild-type and ASV protein: form 1 with ν(FeC) and ν(CO) at 509 and 1938 cm-1nand formn2 with ν(FeC) and ν(CO) at 518 and 1920 cm-1n, respectively.Molecular dynamics simulationswere performednfor the wild-type and ASV forms, as well as for the TyrB10 mutant. The spectroscopic and computationalnresults demonstrate that CO interacts with TrpG8 in form 1 and interacts with both TrpG8 and TyrCD1 innform 2. TyrB10 does not directly interact with the bound CO.
机译：设计了来自Thermobifida fusca的“截短”血红蛋白的酸性表面变异体（ASV），目的是创建一种通用的球蛋白支架，该支架具有热稳定性和可重组形式的高水平非重组表达，同时保持活性位点不变。通过将表面暴露的残基Phe107和Arg91突变为Glu，可以获得这种工程蛋白。分子动力学模拟表明突变的残基保持溶剂暴露状态，而不影响整体蛋白结构。因此，ASV用于末端血红素袋残基的组合诱变，其中没有，两个或三个保守的极性残基[TyrB10（54），TyrCD1（67）和TrpG8（119）]被Phe取代。突变体的特征在于红外和共振拉曼光谱，与野生型蛋白相比。相似的Fe近端His的拉伸频率表明没有突变会改变血红素腔的近端。在野生型和ASV蛋白的COn配合物的光谱中观察到两个构象异构体：509和1938 cm-1n处的ν（FeC）和ν（CO）的形式1和于250℃处的ν（FeC）和ν（CO）的formn2。分别对野生型和ASV形式以及TyrB10突变体进行了分子动力学模拟，分别为518 cm-1n和1920 cm-1n。光谱和计算结果表明，CO与形式1的TrpG8相互作用，并与TrpG8和TyrCD1信息2相互作用。TyrB10不直接与结合的CO相互作用。

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  • 来源
    《Biochemistry》 |2010年第49期|p.10394-10402|共9页
  • 作者

    Enrica Droghetti Francesco Paolo Nicoletti Alessandra Bonamore Leonardo Boechi Pau Arroyo Maez Dario A. Estrin Alberto Boffi Giulietta Smulevich and Alessandro Feis;

  • 作者单位

    ‡Dipartimento di Chimica “Ugo Schiff ”, Universitu0002 a di Firenze, Via della Lastruccia 3, I-50019 Sesto Fiorentino (FI), Italy,§InstitutePasteur, Fondazione Cenci Bolognetti, Department of Biochemical Sciences and CNR Institute ofMolecular Biology and Pathology,University of Rome “La Sapienza”, Piazzale Aldo Moro 5, I-00185 Rome, Italy, and ) Departamento de Quı ´mica Inorgu0003 anica,Analı ´ tica y Quı ´mica Fı ´sica/INQUIMAE-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,Ciudad Universitaria, Pabellu0003 on II (C1428EHA), Buenos Aires, Argentina;

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