Characterisation of the dominant oxidative folding intermediate of hen lysozyme.
Characterisation of the dominant oxidative folding intermediate of hen lysozyme.
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DOI：
10.1006/jmbi.1999.2915
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发表时间：
1999
影响因子：
5.6
通讯作者：
B. van den Berg;E. Chung;C. Robinson;C. Dobson
中科院分区：
文献类型：
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作者：
B. van den Berg;E. Chung;C. Robinson;C. Dobson
用含8.0M尿素的缓冲液稀释还原变性溶菌酶，使其在pH值接近中性时被有效地氧化和复性，并在pH为2的条件下用反相高效液相色谱分离复性中间体。用胃酸消化结合高分辨傅立叶变换离子回旋共振(FT-ICR)质谱仪和串联MS/MS鉴定了主要中间体为DES-[76-94]。该物种具有四个天然二硫键中的三个，但缺乏连接天然蛋白质中两个折叠结构域的Cys76-Cys94二硫键。DES-[76-94]的二维核磁共振表征表明，它具有高度天然的结构。这为该物种在复性过程中的积累提供了一个解释，因为对完全天然蛋白质的直接氧化将受到蛋白质内部埋藏Cys94的限制。
Reduced denatured lysozyme has been oxidised and refolded at pH values close to neutral in an efficient way by dilution from buffers containing 8.0 M urea, and refolding intermediates were separated by reverse-phase HPLC at pH 2. By using peptic digestion in combination with high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tandem MS/MS the dominant intermediate was identified to be des-[76-94]. This species has three of the four native disulphide bonds, but lacks the Cys76-Cys94 disulphide bond which connects the two folding domains in the native protein. Characterisation of des-[76-94] by 2D1H NMR shows that it has a highly native-like structure. This provides an explanation for the accumulation of this species during refolding as direct oxidation to the fully native protein will be restricted by the burial of Cys94 in the protein interior.