Hewson R, Newbold R J, Whitford D
Department of Biochemistry, Queen Mary and Westfield College, London, UK.
Protein Eng. 1993 Nov;6(8):953-64. doi: 10.1093/protein/6.8.953.
The DNA sequence of bovine microsomal cytochrome b5 has been amplified from a liver cDNA library using a polymerase chain reaction. The amplified cDNA when cloned into plasmids that support the high-level production of cytochrome b5 in E.coli leads to protein overexpression and results in cell colonies bearing a strong red colouration. Using cassette mutagenesis, truncated versions of the cytochrome b5 cDNA have been made that encode the first 90 amino acid residues (Ala1-Lys90), the first 104 amino acids (Ala1-Ser104) and the complete protein (Ala1-Asn133). The location of the overexpressed cytochrome b5 within prokaryotic cells is dependent on the overall length of the protein. Expression of the Ala-Lys90 and Ala1-Ser104 variants leads to a location in the cytoplasmic phase of the bacteria whereas the whole protein, Ala1-Asn133, is found within the bacterial membrane fraction. The last 30 residues of cytochrome b5 therefore contain all of the necessary information to insert the protein into E.coli membranes. The solubility of the Ala1-Ser104 variant permits the solution structure and stability of this protein to be measured using 1- and 2-D 1H-NMR methods and electronic spectroscopy. 1-D NMR studies show that the chemical shifts of the haem and haem ligand resonances of the Ala1-Ser104 variant exhibit only very slight perturbations to their magnetic microenvironments when compared with the tryptic fragment of ferricytochrome b5. These results indicate an arrangement of residues in the haem pocket that is very similar in both the Ala1-Ser104 variant and the tryptic fragment and by 2-D NMR it is shown that this similarity extends to the conformations of the polypeptide backbone and side chains. Electronic spectroscopy of this variant shows absorbance maxima for the Soret peaks at 423 nm (reduced) and 413 nm (oxidized). From absorbance spectra the relative thermal stabilities of the Ala1-Ser104 variant and the tryptic fragment were measured. In the oxidized state the Ala1-Ser104 variant denatures in a single cooperative transition with a midpoint temperature (Tm) of 73 degrees C that is significantly higher than that of 'tryptic' ferricytochrome b5. The reduced form of the protein shows increased transition temperatures (Tm approximately 78 degrees C) reflected in the values of delta Hm, delta Sm and delta(delta G) of 420 kJ/mol, 1096 J/mol/K and 12.38 kJ/mol respectively, estimated for this variant. The increased stability of the Ala1-Ser104 variant and other recombinant forms of cytochrome b5 is correlated with the presence of additional residues at the N- and C-termini.(ABSTRACT TRUNCATED AT 400 WORDS)
利用聚合酶链反应从牛肝脏cDNA文库中扩增出了微粒体细胞色素b5的DNA序列。将扩增得到的cDNA克隆到能在大肠杆菌中高效表达细胞色素b5的质粒中，会导致蛋白质过度表达，并使细胞菌落呈现出强烈的红色。利用盒式诱变技术，构建了细胞色素b5 cDNA的截短版本，它们分别编码前90个氨基酸残基（Ala1-Lys90）、前104个氨基酸（Ala1-Ser104）以及完整的蛋白质（Ala1-Asn133）。原核细胞中过表达的细胞色素b5的定位取决于蛋白质的全长。Ala-Lys90和Ala1-Ser104变体的表达导致其定位于细菌的细胞质相中，而完整的蛋白质Ala1-Asn133则存在于细菌膜组分中。因此，细胞色素b5的最后30个残基包含了将该蛋白质插入大肠杆菌膜中的所有必要信息。Ala1-Ser104变体的溶解性使得可以使用一维和二维1H-NMR方法以及电子光谱来测定该蛋白质的溶液结构和稳定性。一维NMR研究表明，与高铁细胞色素b5的胰蛋白酶片段相比，Ala1-Ser104变体的血红素和血红素配体共振的化学位移对其磁性微环境仅表现出非常轻微的扰动。这些结果表明，Ala1-Ser104变体和胰蛋白酶片段的血红素口袋中的残基排列非常相似，并且二维NMR显示这种相似性延伸到多肽主链和侧链的构象。该变体的电子光谱显示，还原态和氧化态的Soret峰的最大吸收波长分别为423 nm和413 nm。通过吸收光谱测量了Ala1-Ser104变体和胰蛋白酶片段的相对热稳定性。在氧化状态下，Ala1-Ser104变体在单一协同转变中变性，中点温度（Tm）为73℃，明显高于“胰蛋白酶”高铁细胞色素b5的中点温度。该蛋白质的还原形式显示出转变温度升高（Tm约为78℃），该变体的ΔHm、ΔSm和Δ（ΔG）值分别估计为420 kJ/mol、1096 J/mol/K和12.38 kJ/mol。Ala1-Ser104变体和细胞色素b5的其他重组形式稳定性的提高与N端和C端存在额外的残基有关。（摘要截短至400字）
