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首页> 外文期刊>Biophysical Chemistry: An International Journal Devoted to the Physical Chemistry of Biological Phenomena >The effect of mutation at valine-45 on the stability and redox potentials of trypsin-cleaved cytochrome b5.
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The effect of mutation at valine-45 on the stability and redox potentials of trypsin-cleaved cytochrome b5.

机译：缬氨酸45突变对胰蛋白酶切割的细胞色素b5的稳定性和氧化还原电位的影响。

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摘要

In an attempt to elucidate the determinants of redox potential and protein stability in cytochrome b5, three mutants at a highly conserved residue Val45, which is a member of heme hydrophobic pocket residues have been characterized. The V45Y mutant was designed to introduce a bulkier residue and a hydroxyl group to the heme pocket. The mutants V45H and V45E were constructed to test the effect of positive and negative charge on the stability and redox potential of proteins. The influence of these mutants on the protein stability towards thermal, urea, acid, ethanol and on the redox potential were studied. It is concluded that the decrease of hydrophobic free energy and the larger volume of the tyrosine make the phenylhydroxyl group of tyrosine still sitting inside the hydrophobic pocket, while the side chain of the mutant V45E and V45H shift away from the heme pocket. The redox potentials of mutants V45Y, V45H, V45E and wild-type of cytochrome b5 are -35 mV, 8 mV, -26 mV and -3 mV, respectively. The bigger change of the V45Y on redox potential is due to the close contact between the hydroxyl group and the heme, while the changes of the V45E and V45H result from the alteration of charge density and distribution around the heme. Different relative stability of these mutants towards heat have been observed with the order: WT > V45Y-V45H > V45E being both in the oxidized and reduced state. The relative stability induced by addition of urea decreases in the order: WT > V45Y > V45H > V45E. These results suggest that the difference in the hydrophobic free energy is a major factor contributing to the stability of the Val45 mutants. Also the loose of the helix III in the mutant V45E makes it more unstable. These results indicate that residue Val45 plays an important role in the stability and redox potential of the protein.
机译：为了阐明决定性的决定因素在细胞色素b5中的氧化还原电位和蛋白质稳定性，已鉴定了高度保守的残基Val45（血红素疏水性口袋残基的一员）上的三个突变体。 V45Y突变体旨在向血红素袋中引入更大的残基和羟基。构建突变体V45H和V45E以测试正电荷和负电荷对蛋白质稳定性和氧化还原电位的影响。研究了这些突变体对蛋白质对热，尿素，酸，乙醇的稳定性以及对氧化还原电位的影响。结论是，疏水性自由能的降低和酪氨酸的较大体积使酪氨酸的苯基羟基仍位于疏水性口袋内，而突变体V45E和V45H的侧链却从血红素口袋移开。突变体V45Y，V45H，V45E和野生型细胞色素b5的氧化还原电位分别为-35 mV，8 mV，-26 mV和-3 mV。 V45Y在氧化还原电势上的较大变化是由于羟基和血红素之间的紧密接触，而V45E和V45H的变化是由于电荷密度和血红素周围分布的改变而引起的。已经观察到这些突变体对热的不同相对稳定性，其顺序为：WT> V45Y-V45H> V45E均处于氧化态和还原态。通过添加尿素引起的相对稳定性按以下顺序降低：WT> V45Y> V45H> V45E。这些结果表明，疏水性自由能的差异是促成Val45突变体稳定性的主要因素。而且，突变体V45E中的螺旋III松散，使其更加不稳定。这些结果表明残基Val45在蛋白质的稳定性和氧化还原电位中起重要作用。

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