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首页> 外文期刊>Biochemistry >'Designing out' disulfide bonds: thermodynamic properties of 30-51 cystine substitution mutants of bovine pancreatic trypsin inhibitor.
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'Designing out' disulfide bonds: thermodynamic properties of 30-51 cystine substitution mutants of bovine pancreatic trypsin inhibitor.

机译：“设计”二硫键：牛胰胰蛋白酶抑制剂的30-51个胱氨酸取代突变体的热力学性质。

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摘要

We have used a combination of spectroscopic and calorimetric techniques to assess the thermodynamic and extrathermodynamic consequences of paired amino acid substitutions at positions 30 and 51 in bovine pancreatic trypsin inhibitor (BPTI). Correctly folded, wild type BPTI contains a disulfide at the 30-51 positions, with the nonbackbone atoms of this cystine being relatively solvent inaccessible. Mutants missing this buried 30-51 disulfide adopt a conformation very similar to that of the native state of wild type BPTI (Eigenbrot et al., 1990, 1992), although they are severely destabilized relative to the wild type molecule (Hurle et al., 1990). We have conducted a systematic effort to find the energetically most favorable substitution for this buried 30-51 disulfide in BPTI. To this end, we have studied and characterized the thermally induced and guanidine hydrochloride-induced denaturation transitions for a family of mutants in which the amino acid residue(s) at positions 30 and/or 51 have been systematically altered. Specifically, we studied the unfolding transitions of the following series of residue 30/residue 51 paired substitution mutants: C30A/C51A, C30V/C51A, C30G/C51A, C30S/C51A, C30T/C51A, C30A/C51S, C30S/C51S, and C30G/C51M. For this series of mutants, comparisons between the relative stabilization free energies, derived from analysis of the denaturation profiles, allow us to reach the following conclusions: (a) side chains containing polar moieties (Ser and Thr) are destabilizing, with this effect being position dependent (i.e., a serine substitution is more destabilizing at position 51 than at position 30); (b) the destabilizing effects of two serine substitutions are approximately additive, suggesting that side chain-side chain hydrogen bonds between the two serine hydroxyl groups probably are weak or nonexistent; (c) the thermodynamic impact of a Gly30 substitution is consistent with a glycine-induced increase in the configurational entropy of the unfolded state; (d) the C30G/C51M mutant is highly destabilized relative to the C30A/C51A mutant despite the fact that, based on considerations of hydrophobicity and steric fit, substitution of a buried disulfide by Gly30 and Met51 would be expected to be optimal. Methionine may be particularly ill-suited as a buried disulfide substitute due to the large loss of side chain conformational entropy it undergoes during the transition from the unfolded to the native state. In the aggregate, our data provide insight into the residue-, position-, and context-dependent consequences on protein stability of "designing out" the buried 30-51 disulfide bond in the BPTI molecule. These data also suggest that a previously unrecognized component of disulfide bridge stabilization of proteins is the relatively minor penalty in side chain conformational entropy incurred by cystine residues during folding due to their severely restricted rotation even in the unfolded state.
机译：我们已经使用光谱和量热技术的组合来评估牛胰胰蛋白酶抑制剂（BPTI）中30和51位氨基酸配对配对的热力学和热力学后果。正确折叠的野生型BPTI在30-51位含有一个二硫键，该胱氨酸的非主链原子相对来说是溶剂难以接近的。缺少这种埋藏的30-51二硫键的突变体的构象与野生型BPTI的天然状态非常相似（Eigenbrot等，1990,1992），尽管相对于野生型分子而言，它们的稳定性极差（Hurle等。 （1990年）。我们进行了系统的努力，以找到在能量上最有利的替代BPTI中埋藏的30-51二硫化物的方法。为此，我们已经研究并表征了突变体家族的热诱导和盐酸胍诱导的变性转变，其中突变体在位置30和/或51处的氨基酸残基已被系统地改变。具体而言，我们研究了以下系列的残基30 /残基51对配对突变体的展开转变：C30A / C51A，C30V / C51A，C30G / C51A，C30S / C51A，C30T / C51A，C30A / C51S，C30S / C51S和C30G / C51M。对于这一系列突变体，通过对变性曲线的分析得出的相对稳定自由能之间的比较，使我们得出以下结论：（a）含有极性部分（Ser和Thr）的侧链不稳定，这种影响是位置依赖性（即，丝氨酸取代在位置51处比在位置30处更不稳定）; （b）两个丝氨酸取代基的去稳定作用是近似加和的，表明两个丝氨酸羟基之间的侧链-侧链氢键可能是弱的或不存在的; （c）Gly30取代的热力学影响与甘氨酸诱导的展开状态构型熵的增加一致; （d）C30G / C51M突变体相对于C30A / C51A突变体是高度不稳定的，尽管事实是，基于疏水性和空间拟合的考虑，用Gly30和Met51取代埋藏的二硫键被认为是最佳的。蛋氨酸可能特别不适合用作掩埋的二硫键替代物，因为它在从展开状态到天然状态的过渡过程中，侧链构象熵损失很大。总体而言，我们的数据可洞悉“设计” BPTI分子中埋藏的30-51二硫键对蛋白质稳定性的残基，位置和上下文相关影响。这些数据还表明，蛋白质的二硫键桥稳定的一个先前无法识别的组成部分是胱氨酸残基在折叠过程中由于即使在未折叠状态下也受到严格限制的旋转而在侧链构象熵中相对较小的损失。

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