Denaturant-dependent folding of bovine pancreatic trypsin inhibitor mutants with two intact disulfide bonds.
Denaturant-dependent folding of bovine pancreatic trypsin inhibitor mutants with two intact disulfide bonds.
复制标题
具有两个完整二硫键的牛胰腺胰蛋白酶抑制剂突变体的变性依赖性折叠。
DOI：
10.1021/bi00470a021
复制
发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Kuntz,ID
中科院分区：
文献类型：
--
作者：
Hurle,MR;Marks,CB;Kosen,PA;Anderson,S;Kuntz,ID
加州大学旧金山分校药物化学系，San Francisco, California 94143， Genentech心血管研究系，South San Francisco, California 94080摘要：用紫外差谱法研究了盐酸胍（Gdn-HCl）诱导4个牛胰蛋白酶抑制剂（BPTI）突变体展开/再折叠的平衡和动力学行为。在其中三个突变体中，我们用51位的丙氨酸和30位的缬氨酸（Val30/Ala51）、丙氨酸（Ala30/Ala51）或苏氨酸（Thr30/Ala51）取代了隐藏的30-51二硫键。对于第四个突变体，溶剂暴露的14-38二硫被一对丙氨酸取代（Alal4/Ala38）。所有突变体都保留了5-55二硫化物。实验在氧化条件下进行；因此，未展开和折叠的形式都保留了两个天然二硫键。平衡实验表明，这四种突变体相对于野生型BPTI都是不稳定的。然而，30-51突变体的稳定性随着30号位置取代的残基的疏水性而增加。动力学实验表明，这4个突变体都包含两个具有脯氨酸异构化特征的缓慢重折叠阶段。相的具体行为取决于二硫键的位置。每个30-51突变体的主要展开/重折叠阶段比Alal4/Ala38或BPTI慢一个多量级，其中14-38二硫键被碘乙酰胺特异性地减少和阻断[Jullien， M., & Baldwin， R. L.(1981) J. Mol. Biol. 145, 262 -280]，因为这种影响与蛋白质的稳定性无关。这与一个模型是一致的，在这个模型中，蛋白质内部残基的适当对接是这些突变体折叠的限速步骤。
Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94143, and Department of Cardiovascular Research, Genentech, South San Francisco, California 94080 Received September 14, 1989; Revised Manuscript Received December 14, 1989 abstract: The equilibrium and kinetic behavior of the guanidine hydrochloride (Gdn-HCl) induced unfolding/refolding of four bovine pancreatic trypsin inhibitor (BPTI) mutants was examined by using ultraviolet difference spectroscopy. In three of the mutants, we replaced the buried 30-51 disulfide bond with alanine at position 51 and valine (Val30/Ala51), alanine (Ala30/Ala51), or threonine (Thr30/Ala51) at position 30. For the fourth mutant, the solvent-exposed 14-38 disulfide was substituted by a pair of alanines (Alal4/Ala38). All mutants retained the 5-55 disulfide. Experiments were performed under oxidizing conditions; thus, both the unfolded and folded forms retained two native disulfide bonds. Equilibrium experiments demonstrated that all four mutants were destabilized relative to wild-type BPTI. However, the stability of the 30-51 mutants increased with the hydrophobicity of the residue substituted at position 30. Kinetic experiments showed that all four mutants contained two minor slow refolding phases with characteristics of proline isomerization. The specific behavior of the phases depended on the location of the disulfide bonds. The major unfolding/refolding phase for each of the 30-51 mutants was more than an order of magnitude slower than for Alal4/Ala38 or for BPTI in which the 14-38 disulfide bond was specifically reducedand blocked with iodoacetamide [Jullien, M., & Baldwin, R. L.(1981) J. Mol. Biol. 145, 265-280], Since this effect is independent of the stability of the protein, it is consistent with a model in which the proper docking of the interior residues of the protein is the rate-limiting step in the folding of these mutants.