Molecular Mechanism of Protein Folding and Functioning by Means of Deletions and Insertions

通过删除和插入实现蛋白质折叠和功能的分子机制

基本信息

批准号：
10480182
负责人：
KATAOKA Mikio
金额：
$ 7.49万
依托单位：
NARA INSTITUTE OF SCIENCE AND TECHNOLOGY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10480182/
关键词：
staphylococcal nuclease photoactive yellow protein deletion mutant Insertion mutant enzyme activity protein folding compact denatured state 変性構造 Photoactive Yellow Protein 欠損変異体蛋白質折り畳み光反応遺伝子操作 Staphy lococcal nuclease タンパク質折

项目摘要

The tertiary fold of the internal deletion mutant, D44-49, of Staphylococcal nuclease (SNase) is identical to that of wild type, but D44-49 is more stable against heat and acid than wild type. D114-119 forms a dimmer with domain swapping. The overall fold of D114-119 is quite similar to that of wild type but is less stable than wild type. Both deletion mutants show now enzymatic activity. D114-119 lacks a substrate-binding ability, while D44-49 can bind a substrate. D44-49 lacks a catalytic activity. Based on the crystal structure analysis of D44-49, it is clarified that the loop flexibility is essential for the catalytic activity.Systematic deletions from the C terminus of SNase were performed to reveal the role of C-terminal region in folding and functioning. D140-149 is in compact denatured structure, which is identical to D137-149. D140-149 shows enzymatic activity as well as D137-149, and these two mutants possess the substrate-induced folding ability. D142-149 and D143-149 are id … More entical to wild type. D141-149 shows intermediate properties of wild type and D140-149. D141-149/W140A is indistinguishable with D140-149. On the other hand, the substitutions of S141 into A or N show no effects on the structure and activity of D142-149. We can conclude the followings : 1) W140 is a key residue to maintain the native conformation stably under a physiological condition ; 2) the side chain of the 141th residue is not important but main chain information is important to take a native conformation ; 3) if the polypeptide shows an enzymatic activity, the polypeptide can fold into a native conformation, but the foldability is not necessarily related to the activity.In the case of photoactive yeIIow protein, V122 is the essential residue to form holo-protein and to keep native structure stably. If the polypeptide contains up to K123, the polypeptide can behave as PYP in terms of structure and function. R124 makes photoreaction efficient. V125 makes native structure stable.These results also suggest that the folding in sequence space is two-state transition. Less

葡萄球菌核酸酶（SNase）的内部缺失突变体D44-49的第三折与野生型相同，但D44-49比野生型更稳定。 D114-119与域交换形成调光器。 D114-119的整体折叠与野生型非常相似，但不如野生型稳定。两个缺失突变体现在均显示酶活性。 D114-119缺乏底物结合能力，而D44-49可以结合基板。 D44-49缺乏催化活性。基于D44-49的晶体结构分析，澄清的是，循环灵活性对于催化活性至关重要。进行SNase的C末端的系统缺失以揭示C末端区域在折叠和功能中的作用。 D140-149处于紧凑的变性结构，与D137-149相同。 D140-149显示了酶活性以及D137-149，这两个突变体具有底物诱导的折叠能力。 D142-149和D143-149是ID…更适合野生型。 D141-149显示了野生型和D140-149的中间特性。 D141-149/W140A与D140-149无法区分。另一方面，将S141的取代对D142-149的结构和活动没有影响。可以包含以下内容：1）W140是在身体状况下保持稳定的本地会议的关键保留； 2）第141保留的侧链并不重要，但主链信息对于参加本地会议很重要； 3）如果多肽显示出酶促活性，多肽可以折叠成天然会议，但可折叠性不一定与活性有关。在光活性YEIOW蛋白的情况下，V122是形成全蛋白并保持天然结构稳定的基本残留物。如果多肽最多包含K123，则多肽可以在结构和功能方面表现为PYP。 R124使光反应有效。 V125使天然结构稳定。这些结果还表明，序列空间中的折叠是两态跃迁。较少的