PH-INDUCED DENATURATION OF PROTEINS - A SINGLE SALT BRIDGE CONTRIBUTES 3-5 KCAL MOL TO THE FREE-ENERGY OF FOLDING OF T4-LYSOZYME
PH-INDUCED DENATURATION OF PROTEINS - A SINGLE SALT BRIDGE CONTRIBUTES 3-5 KCAL MOL TO THE FREE-ENERGY OF FOLDING OF T4-LYSOZYME
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DOI：
10.1021/bi00461a025
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发表时间：
1990-03-06
期刊：
影响因子：
2.9
通讯作者：
DAHLQUIST, FW
中科院分区：
文献类型：
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作者：
ANDERSON, DE;BECKTEL, WJ;DAHLQUIST, FW
用核磁共振技术研究了T4溶菌酶天冬氨酸70(Asp70)和组氨酸31(His31)侧链之间形成的盐桥的能量学。天然状态下残基的pKa值与它们在未折叠蛋白质中的值相扰动，使得His31在天然状态下的pKa值为9.1，在10度时的未折叠状态下为6.8。加适量的盐。类似地，天冬氨酸PKA在自然状态下从其在展开状态下的值3.5-4.0移位到约0.5的值。PKa的这些变化表明，盐桥稳定在3-5千卡/摩尔。这意味着与未折叠状态相比，盐桥使本征状态稳定了3-5千卡/摩尔。这反映在蛋白质突变体的热力学稳定性上，在突变体中，天冬酰胺取代了Asp70和/或His31。这些观察和对质子化状态与蛋白质折叠的热力学耦合的考虑表明了一种酸变性的机制，即当pH降至4以下时，通过其酸残基的质子化来逐步稳定未折叠状态。只有当折叠状态的酸性基团的pKa值低于未折叠状态时，才能稳定未折叠状态。当pH足够低时，自然状态和展开状态的酸基都被完全质子化，表观展开平衡常数变得不依赖于pH。类似的论点也适用于碱基诱导的展开。这些观察结果表明，每个可电离基团对折叠状态稳定性的静电贡献可以通过简单地用核磁共振或其他方法测量其表观pKa来直接评估。
The energetics of a salt bridge formed between the side chains of aspartic acid 70 (Asp70) and histidine 31 (His31) of T4 lysozyme have been examined by nuclear magnetic resonance techniques. The pKa values of the residues in the native state are perturbed from their values in the unfolded protein such that His31 has a pKa value of 9.1 in the native state and 6.8 in the unfolded state at 10.degree. C in moderate salt. Similarly, the aspartate pKa is shifted to a value of about 0.5 in the native state from its value of 3.5-4.0 in the unfolded state. These shifts in pKa show that the salt bridge is stabilized 3-5 kcal/mol. This implies that the salt bridge stabilizes the native state by 3-5 kcal/mol as compared to the unfolded state. This is reflected in the thermodynamic stability of mutants of the protein in which Asp70, His31, or both are replaced by asparagine. These observations and consideration of the thermodynamic coupling of protonation state to folding of proteins suggest a mechanism of acid denaturation in which the unfolded state is progressively stabilized by protonation of its acid residues as pH is lowered below pH 4. The unfolded state is stabilized only if acidic groups in the folded state have lower pKa values than in the unfolded state. When the pH is sufficiently low, the acid groups of both the native and unfolded states are fully protonated, and the apparent unfolding equilibrium constant becomes pH independent. Similar arguments apply to base-induced unfolding. These observations suggest that the electrostatic contribution of each ionizable group to the stability of the folded state can be directly assessed by simply measuring its apparent pKa by NMR or other methods.