Ruoppolo M, Moutiez M, Mazzeo M F, Pucci P, Ménez A, Marino G, Quéméneur E
Dipartimento di Chimica, Università degli Studi di Salerno, Italy.
Biochemistry. 1998 Nov 17;37(46):16060-8. doi: 10.1021/bi981492j.
Snake curaremimetic toxins are short all-beta proteins, containing several disulfide bonds which largely contribute to their stability. The four disulfides present in snake toxins make a "disulfide beta-cross"-fold that was suggested to be a good protein folding template. Previous studies on the refolding of snake toxins (Ménez, A. et al. (1980) Biochemistry 19, 4166-4172) showed that this set of natural homologous proteins displays different rates of refolding. These studies suggested that the observed different rates could be correlated to the length of turn 2, one out of five turns present in the toxins structure and close to the "disulfide beta-cross". To demonstrate this hypothesis, we studied the refolding pathways and kinetics of two natural isotoxins, toxin alpha (Naja nigricollis) and erabutoxin b (Laticauda semifasciata), and two synthetic homologues, the alpha mutants, alpha60 and alpha62. These mutants were designed to probe the peculiar role of the turn 2 on the refolding process by deletion or insertion of one residue in the turn length that reproduced the natural heterogeneity at that locus. The refolding was studied by electrospray mass spectrometry (ESMS) time-course analysis. This analysis permitted both the identification and quantitation of the population of intermediates present during the process. All toxins were shown to share the same sequential scheme for disulfide bond formation despite large differences in their refolding rates. The results presented here demonstrate definitely that no residues except those forming turn 2 accounted for the observed differences in the refolding rate of toxins.
蛇类箭毒样毒素是短的全β蛋白，含有几个二硫键，这些二硫键对其稳定性有很大贡献。蛇毒中存在的四个二硫键形成了一种“二硫键β-交叉”折叠，有人认为这是一种良好的蛋白质折叠模板。先前关于蛇毒重折叠的研究（梅内斯，A.等人（1980年）《生物化学》19卷，4166 - 4172页）表明，这组天然同源蛋白表现出不同的重折叠速率。这些研究表明，观察到的不同速率可能与2号转角的长度相关，2号转角是毒素结构中五个转角之一，且靠近“二硫键β-交叉”。为了验证这一假设，我们研究了两种天然同毒素（毒素α（黑颈眼镜蛇）和海蛇毒素b（半环扁尾海蛇））以及两种合成同源物（α突变体α60和α62）的重折叠途径和动力学。设计这些突变体是为了通过在转角长度中缺失或插入一个残基来探究2号转角在重折叠过程中的特殊作用，这种缺失或插入再现了该位点的天然异质性。通过电喷雾质谱（ESMS）时间进程分析来研究重折叠。这种分析既可以鉴定又可以定量过程中存在的中间体群体。尽管所有毒素的重折叠速率差异很大，但它们都显示出相同的二硫键形成顺序模式。此处给出的结果明确表明，除了形成2号转角的残基外，没有其他残基导致观察到的毒素重折叠速率差异。
