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

葡萄球菌核酸酶（SNase）的内部缺失突变体D44-49的三级折叠与野生型相同，但D44-49对热和酸的稳定性比野生型高。D114-119形成具有结构域交换的二聚体。D114-119的总体折叠与野生型非常相似，但不如野生型稳定。两种缺失突变体现在都显示酶活性。D114-119缺乏底物结合能力，而D44-49可以结合底物。D44-49缺乏催化活性。通过对D44-49的晶体结构分析，阐明了其环的柔性对催化活性的重要性，并对SNase的C端进行了系统的缺失，以揭示C端区域在折叠和功能中的作用。D140-149为致密变性结构，与D137-149相同。D140-149与D137-149一样具有酶活性，这两个突变体都具有底物诱导的折叠能力。D142-149和D143-149为id 关于我们 对野生型感兴趣。D141-149显示野生型和D140-149的中间性质。D141-149/W140 A与D140-149无法区分。另一方面，S141被A或N取代对D142-149的结构和活性没有影响。结果表明：1）W140是在生理条件下保持天然构象稳定的关键残基，2）第141位残基的侧链信息对天然构象的形成并不重要，而主链信息对天然构象的形成起重要作用; 3）如果多肽显示酶活性，则多肽可以折叠成天然构象，但折叠性与活性没有必然的关系，在光敏黄蛋白中，V122是形成完整蛋白和稳定保持天然结构的必需残基。如果多肽含有高达K123，则多肽在结构和功能方面可以表现为PYP。R124使光反应有效。V125使天然结构稳定，这些结果也表明序列空间中的折叠是两态跃迁。少

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