Huyghues-Despointes B M, Nelson J W
Department of Biochemistry, Louisiana State University, Baton Rouge 70803-1806.
Biochemistry. 1992 Feb 11;31(5):1476-83. doi: 10.1021/bi00120a026.
Apamin is an 18-residue bee venom peptide with the sequence CNCKAPETALCARRCQQH-amide and contains 2 disulfide bonds connecting C-1 to C-11 and C-3 to C-15. In the folding of reduced, unfolded apamin to native apamin with two disulfide bonds, the one-disulfide folding intermediate states are not populated to significant levels. To study the properties of the one-disulfide intermediates, we have synthesized two peptide models to mimic the one-disulfide intermediates, Apa-1 and Apa-2, in which two cysteines in the sequence have been replaced by alanines. These peptides can form only one of the native disulfide bonds, C-1 to C-11 in the case of Apa-1 and C-3 to C-15 in the case of Apa-2. The stabilities of these disulfide bonds have been measured as a function of pH, concentration of urea, and temperature, in order to understand which contributions stabilize the disulfide-bonded structures. Using oxidized and reduced glutathione, the equilibrium constants for forming the disulfide bonds at 25 degrees C and pH 7.0 are 0.018 M for Apa-1 and 0.033 M for Apa-2 and show little dependence on pH or temperature. Both disulfide bonds are destabilized slightly (by approximately a factor of 2) between 0 and 8 M urea. Circular dichroism spectra indicate that although both Apa-1 and Apa-2 exhibit some structure, Apa-2 exhibits more than Apa-1. The results suggest that in the folding of apamin, the one-disulfide intermediate containing the C-3 to C-15 disulfide bond, as in Apa-2, is favored slightly. Secondary structure provides modest stabilization to this intermediate.
蜂毒明肽是一种由18个氨基酸残基组成的蜂毒肽，序列为CNCKAPETALCARRCQQH - 酰胺，含有两条二硫键，分别连接C - 1与C - 11以及C - 3与C - 15。在还原的、未折叠的蜂毒明肽折叠成具有两条二硫键的天然蜂毒明肽的过程中，单二硫键折叠中间态的含量未达到显著水平。为了研究单二硫键中间体的性质，我们合成了两个肽模型来模拟单二硫键中间体，即Apa - 1和Apa - 2，其中序列中的两个半胱氨酸被丙氨酸取代。这些肽只能形成天然二硫键中的一条，Apa - 1形成C - 1与C - 11之间的二硫键，Apa - 2形成C - 3与C - 15之间的二硫键。已测定这些二硫键的稳定性随pH、尿素浓度和温度的变化情况，以便了解哪些因素有助于稳定二硫键连接的结构。使用氧化型和还原型谷胱甘肽，在25℃和pH 7.0条件下形成二硫键的平衡常数，Apa - 1为0.018 M，Apa - 2为0.033 M，且对pH或温度的依赖性较小。在0至8 M尿素之间，两条二硫键的稳定性均略有降低（约为2倍）。圆二色光谱表明，尽管Apa - 1和Apa - 2都呈现出一些结构，但Apa - 2的结构比Apa - 1更多。结果表明，在蜂毒明肽的折叠过程中，如Apa - 2中那样含有C - 3至C - 15二硫键的单二硫键中间体略占优势。二级结构为该中间体提供了适度的稳定性。
