Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1
Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1
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DOI：
10.1021/bi9815243
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发表时间：
1998-11-17
期刊：
影响因子：
2.9
通讯作者：
Pace, CN
中科院分区：
文献类型：
--
作者：
Hebert, EJ;Giletto, A;Pace, CN
研究了多肽基团之间的氢键对球状蛋白构象稳定性的贡献。微生物核糖核酸酶(RNase)家族的保守残基之一是天冬酰胺，分别位于RNase Sa的第39位、RNase T1的44位和RNase BA(Barnase)的58位。这种天冬酰胺的酰胺基团被掩埋，并与邻近的多肽基团形成两个类似的分子内氢键，在所有三种蛋白质的表面锚定一个环。因此，它是一个很好的多肽基团氢键模型。当保守的天冬酰胺被丙氨酸取代时，突变蛋白的稳定性降低了2.2(Sa)、1.8(T1)和2.7(Ba)kcal/mol。当天冬氨酸取代保守的天冬酰胺时，突变蛋白的稳定性对RNase Sa和T1分别降低了1.5和1.8kcal/mol，而对RNase Ba则增加了0.5kcal/mol。当保守的天冬酰胺被丝氨酸取代时，突变蛋白对核糖核酸酶Sa和T1的稳定性分别降低了2.3kcal/mol和1.7kcal/mol。用1.7埃分辨率测定了核糖核酸酶Sa的ASN-39双右箭丝突变体的结构。在突变位点附近有一个显著的构象变化：(1)Ser39的侧链与Bsn 39的侧链方向不同，并与两个保守的水分子形成氢键；(2)Ser42的肽键改变了突变体的构象，使侧链与骨架形成三个新的分子内氢键，取代了野生型结构中存在的三个与水分子的氢键；以及(3)锚定氢键的丧失使突变体的表面环比野生型RNaseSa更灵活。结果表明，保守的天冬酰胺的埋藏和氢键对微生物核糖核酸酶的稳定性有很大的贡献，强调了结构信息在解释突变蛋白质稳定性研究中的重要性。
The contribution of hydrogen bonding by peptide groups to the conformational stability of globular proteins was studied. One of the conserved residues in the microbial ribonuclease (RNase) family is an asparagine at position 39 in RNase Sa, 44 in RNase T1, and 58 in RNase Ba (barnase). The amide group of this asparagine is buried and forms two similar intramolecular hydrogen bonds with a neighboring peptide group to anchor a loop on the surface of all three proteins. Thus, it is a good model for the hydrogen bonding of peptide groups. When the conserved asparagine is replaced with alanine, the decrease in the stability of the mutant proteins is 2.2 (Sa), 1.8 (T1), and 2.7 (Ba) kcal/mol. When the conserved asparagine is replaced by aspartate, the stability of the mutant proteins decreases by 1.5 and 1.8 kcal/mol for RNases Sa and T1, respectively, but increases by 0.5 kcal/mol for RNase Ba. When the conserved asparagine was replaced by serine, the stability of the mutant proteins was decreased by 2.3 and 1.7 kcal/mol for RNases Sa and T1, respectively. The structure of the Asn 39 double right arrow Ser mutant of RNase Sa was determined at 1.7 Angstrom resolution. There is a significant conformational change near the site of the mutation: (1) the side chain of Ser 39 is oriented differently than that of Bsn 39 and forms hydrogen bonds with two conserved water molecules; (2) the peptide bond of Ser 42 changes conformation in the mutant so that the side chain forms three new intramolecular hydrogen bonds with the backbone to replace three hydrogen bonds to water molecules present in the wild-type structure; and (3) the loss of the anchoring hydrogen bonds makes the surface loop more flexible in the mutant than it is in wild-type RNase Sa. The results show that burial and hydrogen bonding of the conserved asparagine make a large contribution to microbial RNase stability and emphasize the importance of structural information in interpreting stability studies of mutant proteins.