Schultz L W, Hargraves S R, Klink T A, Raines R T
Department of Biochemistry, University of Wisconsin-Madison, 53706-1569, USA.
Protein Sci. 1998 Jul;7(7):1620-5. doi: 10.1002/pro.5560070716.
The peptide bonds preceding Pro 93 and Pro 114 of bovine pancreatic ribonuclease A (RNase A) are in the cis conformation. The trans-to-cis isomerization of these bonds had been indicted as the slow step during protein folding. Here, site-directed mutagenesis was used to replace Pro 93 or Pro 114 with a glycine residue, and the crystalline structure of the P93G variant was determined by X-ray diffraction analysis to a resolution of 1.7 A. This structure is essentially identical to that of the wild-type protein, except for the 91-94 beta-turn containing the substitution. In the wild-type protein, the beta-turn is of type VIa. In the P93G variant, this turn is of type II with the peptide bond preceding Gly 93 being trans. The thermal stabilities of the P93G and P114G variants were assessed by differential scanning calorimetry and thermal denaturation experiments monitored by ultraviolet spectroscopy. The value of delta deltaGm which reports on the stability lost in the variants, is 1.5-fold greater for the P114G variant than for the P93G variant. The greater stability of the P93G variant is likely due to the relatively facile accommodation of residues 91-94 in a type II turn, which has a preference for a glycine residue in its i + 2 position.
牛胰核糖核酸酶A（RNase A）中第93位脯氨酸（Pro 93）和第114位脯氨酸（Pro 114）之前的肽键呈顺式构象。这些肽键从反式到顺式的异构化被认为是蛋白质折叠过程中的慢步骤。在此，通过定点诱变将Pro 93或Pro 114替换为甘氨酸残基，并通过X射线衍射分析确定了P93G变体的晶体结构，分辨率为1.7埃。除了包含该取代的91 - 94位β-转角外，该结构与野生型蛋白质的结构基本相同。在野生型蛋白质中，β-转角为VIa型。在P93G变体中，该转角为II型，Gly 93之前的肽键为反式。通过差示扫描量热法和紫外光谱监测的热变性实验评估了P93G和P114G变体的热稳定性。报告变体中稳定性损失的ΔΔGm值，P114G变体比P93G变体大1.5倍。P93G变体具有更高的稳定性可能是由于91 - 94位残基在II型转角中相对容易容纳，II型转角在其i + 2位置偏好甘氨酸残基。
