Hodges R S, Zhou N E, Kay C M, Semchuk P D
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Pept Res. 1990 May-Jun;3(3):123-37.
The objective of this study was to evaluate quantitatively the contribution that hydrophobic residues and disulfide bonds make to protein stability using a model protein with well-defined secondary, tertiary and quaternary structure. This de novo-designed protein consists of two identical 35-residue alpha-helical polypeptide chains arranged in a coiled-coil structure, which are stabilized by nine pairs of interchain hydrophobic interactions from leucine residues and an interchain disulfide bond. The mutant proteins differ from the Leu-protein only at positions 16 and 19 of each chain which contain either Ile, Val, Ala, Phe or Tyr instead of Leu residues. The stability of each protein was determined in its reduced and oxidized form from guanidine hydrochloride denaturation experiments using circular dichroism measurements. The Leu-, Ile-, Val- and Phe-proteins containing the disulfide bond were essentially 100% alpha-helical in benign medium (0.05 M phosphate buffer, pH 7, containing 0.1 M KCl) with transition midpoints of 5.3, 4.1, 2.9 and 2.4 M denaturant, respectively. Reduction of the disulfide bond had little effect on the Leu-protein but resulted in significant decreases in helicity of the other mutants. The order of protein stability of these analogs remains the same in the absence or presence of the disulfide bond (Leu- greater than Ile- greater than Val- greater than Phe- greater than Tyr- greater than Ala-protein). Interestingly, the more stable the protein was in the absence of the disulfide bond, the larger the contribution the disulfide bond made to protein stability. The identical mutation in the disulfide bridge protein had a much greater effect on protein stability than in the reduced protein. This suggests that the disulfide bond controls the conformation of the coiled-coil some 14-17 residues further along the polypeptide chain (the disulfide bond is between positions 2 and 2' and the closest mutation site in the coiled-coil is at position 16 and 16'). In contrast, in the absence of the disulfide bond, the coiled-coil is more flexible and can accommodate the mutations more easily by adjusting the interchain packing around the mutation sites. The helix-forming tendency of nonpolar residues was deduced in this study by comparing the alpha-helicity of the mutant polypeptides in aqueous buffer containing 50% trifluoroethanol where they exist as single-stranded helices. Hydrophobicity is an important factor in determining the alpha-helix forming tendency of aliphatic nonpolar residues in amphipathic alpha-helices.
本研究的目的是使用一种具有明确二级、三级和四级结构的模型蛋白，定量评估疏水残基和二硫键对蛋白质稳定性的贡献。这种从头设计的蛋白质由两条相同的35个残基的α-螺旋多肽链组成，排列成卷曲螺旋结构，通过来自亮氨酸残基的九对链间疏水相互作用和一个链间二硫键得以稳定。突变蛋白与亮氨酸蛋白的不同之处仅在于每条链的第16和19位，这些位置含有异亮氨酸、缬氨酸、丙氨酸、苯丙氨酸或酪氨酸，而非亮氨酸残基。通过圆二色性测量，利用盐酸胍变性实验测定了每种蛋白质还原态和氧化态的稳定性。在良性介质（0.05 M磷酸盐缓冲液，pH 7，含0.1 M KCl）中，含有二硫键的亮氨酸、异亮氨酸、缬氨酸和苯丙氨酸蛋白基本上100%呈α-螺旋结构，变性剂的转变中点分别为5.3、4.1、2.9和2.4 M。二硫键的还原对亮氨酸蛋白影响不大，但导致其他突变体的螺旋度显著降低。在有或无二硫键的情况下，这些类似物的蛋白质稳定性顺序相同（亮氨酸蛋白＞异亮氨酸蛋白＞缬氨酸蛋白＞苯丙氨酸蛋白＞酪氨酸蛋白＞丙氨酸蛋白）。有趣的是，在无二硫键时蛋白质越稳定，二硫键对蛋白质稳定性的贡献就越大。二硫键桥连蛋白中的相同突变对蛋白质稳定性的影响比还原态蛋白中的影响大得多。这表明二硫键控制着卷曲螺旋在多肽链上大约再往后14 - 17个残基处的构象（二硫键位于第2和2'位之间，卷曲螺旋中最接近的突变位点在第16和16'位）。相反，在无二硫键的情况下，卷曲螺旋更具柔韧性，能够通过调整突变位点周围的链间堆积更轻松地容纳突变。本研究通过比较突变多肽在含有50%三氟乙醇的水性缓冲液中的α-螺旋度，推导了非极性残基的螺旋形成倾向，其中它们以单链螺旋形式存在。疏水性是决定两亲性α-螺旋中脂肪族非极性残基α-螺旋形成倾向的一个重要因素。
