Completely buried, non-ion-paired glutamic acid contributes favorably to the conformational stability of pyrrolidone carboxyl peptidases from hyperthermophiles
Completely buried, non-ion-paired glutamic acid contributes favorably to the conformational stability of pyrrolidone carboxyl peptidases from hyperthermophiles
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DOI：
10.1021/bi052610n
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发表时间：
2006-06-13
期刊：
影响因子：
2.9
通讯作者：
Yutani, Katsuhide
中科院分区：
文献类型：
--
作者：
Kaushik, Jai K.;Iimura, Satoshi;Yutani, Katsuhide
来自超嗜热菌的吡咯烷酮羧基肽酶（PCP）在疏水核心中具有结构保守且完全掩埋的Glu 192;相比之下，嗜温蛋白中的相应残基是疏水残基Ile。埋藏的可电离残留物是否有助于超嗜热PCP的稳定或不稳定？为了阐明埋藏的谷氨酸在稳定超嗜热菌PCP中的作用，我们构建了五个PCP-0 SH的Glu 192突变体（C142 S/C188 S，PCP的Cys-free双突变体），并研究了它们的热和pH诱导的解折叠和晶体结构，并将它们与PCP-0 SH进行了比较。非极性突变体（E192 A/I/V）和极性突变体（E192 D/Q）在酸性条件下的稳定性均低于PCP-0 SH。在碱性区，除E192 D外，突变蛋白均比PCP-0 SH稳定。热稳定性数据和理论计算表明Glu 192的表观pK（a）值>= 7.3。目前的结果证实，PCP-0 SH中的质子化Glu 192与Pro 168的羰基氧和肽氮形成强氢键。在E -> A/D突变体中，通过将水分子引入位置192周围产生的空腔中形成新的分子间氢键，而在E -> I/V突变体中氢键消失。基于结构的突变蛋白质的经验稳定性与实验结果吻合良好。结果表明：（1）完全掩埋的Glu 192通过形成强的分子内氢键而有助于PCP-0 SH的稳定化;（2）非电离和掩埋的Glu所形成的氢键比疏水基团的掩埋对蛋白质构象稳定性的贡献更大。
Pyrrolidone carboxyl peptidases ( PCPs) from hyperthermophiles have a structurally conserved and completely buried Glu192 in the hydrophobic core; in contrast, the corresponding residue in the mesophile protein is a hydrophobic residue, Ile. Does the buried ionizable residue contribute to stabilization or destabilization of hyperthermophile PCPs? To elucidate the role of the buried glutamic acid in stabilizing PCP from hyperthermophiles, we constructed five Glu192 mutants of PCP-0SH ( C142S/C188S, Cys-free double mutant of PCP) from Pyrococcus furiosus and examined their thermal and pH-induced unfolding and crystal structures and compared them with those of PCP-0SH. The stabilities of apolar ( E192A/I/V) and polar ( E192D/Q) mutants were less than PCP-0SH at acidic pH values. In the alkaline region, the mutant proteins, except for E192D, were more stable than PCP-0SH. The thermal stability data and theoretical calculations indicated an apparent pK(a) value >= 7.3 for Glu192. Present results confirmed that the protonated Glu192 in PCP-0SH forms strong hydrogen bonds with the carbonyl oxygen and peptide nitrogen of Pro168. New intermolecular hydrogen bonds in the E -> A/D mutants were formed by a water molecule introduced into the cavity created around position 192, whereas the hydrogen bonds disappeared in the E -> I/V mutants. Structure-based empirical stability of mutant proteins was in good agreement with the experimental results. The results indicated that ( 1) completely buried Glu192 contributes to the stabilization of PCP-0SH because of the formation of strong intramolecular hydrogen bonds and ( 2) the hydrogen bonds by the nonionized and buried Glu can contribute more than the burial of hydrophobic groups to the conformational stability of proteins.