Hebert E J, Giletto A, Sevcik J, Urbanikova L, Wilson K S, Dauter Z, Pace C N
Department of Medical Biochemistry, Center for Macromolecular Design, Texas A&M University, College Station 77843-1114, USA.
Biochemistry. 1998 Nov 17;37(46):16192-200. doi: 10.1021/bi9815243.
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 --> Ser mutant of RNase Sa was determined at 1.7 A 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 Asn 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.
研究了肽基团的氢键作用对球状蛋白质构象稳定性的贡献。微生物核糖核酸酶（RNase）家族中的一个保守残基是RNase Sa中第39位的天冬酰胺、RNase T1中第44位的天冬酰胺以及RNase Ba（芽孢杆菌RNA酶）中第58位的天冬酰胺。该天冬酰胺的酰胺基团被掩埋，并与相邻的肽基团形成两个相似的分子内氢键，以固定所有三种蛋白质表面的一个环。因此，它是肽基团氢键作用的一个良好模型。当保守的天冬酰胺被丙氨酸取代时，突变蛋白稳定性的降低分别为2.2（Sa）、1.8（T1）和2.7（Ba）千卡/摩尔。当保守的天冬酰胺被天冬氨酸取代时，RNase Sa和T1的突变蛋白稳定性分别降低1.5和1.8千卡/摩尔，但RNase Ba的突变蛋白稳定性增加0.5千卡/摩尔。当保守的天冬酰胺被丝氨酸取代时，RNase Sa和T1的突变蛋白稳定性分别降低2.3和1.7千卡/摩尔。RNase Sa的Asn 39→Ser突变体的结构在1.7埃分辨率下被确定。在突变位点附近有显著的构象变化：（1）Ser 39的侧链取向与Asn 39不同，并与两个保守水分子形成氢键；（2）突变体中Ser 42的肽键改变构象，使得侧链与主链形成三个新的分子内氢键，以取代野生型结构中与水分子形成的三个氢键；（3）锚定氢键的丧失使突变体中的表面环比野生型RNase Sa中的更灵活。结果表明，保守天冬酰胺的掩埋和氢键作用对微生物RNase的稳定性有很大贡献，并强调了结构信息在解释突变蛋白稳定性研究中的重要性。
