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Experimental procedure to predict the folding units of an unknown protein structure

预测未知蛋白质结构折叠单位的实验程序

基本信息

批准号：
03680235
负责人：
SEGAWA Shin-ichi
金额：
$ 1.34万
依托单位：
School of Science, Kwansei Gakuin University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03680235/
关键词：
Protein folding Folding units Fragments of cytochrome c Fragment of a helix-forming propensity 蛋白質の折りたたみチトクロームc 分子構造の進化

项目摘要

The protein structure is generally composed of several folding units, which are smaller substructures. The purpose of our research is to determine such folding units based on experimental data.We prepared several lysozyme fragments by tryptic digestion, and measured their CD spectra. When they are dissoled in aqueous solution, they generally show typical CD spectra of random coiled peptide chain. However, the additon of trifluoroethanol (TFE) to the solution (up to about 50 volume %) cause the change in CDspectra according to the inherent property of the peptide fragment. It induces a group of peptide fragments to show a CD spectrum typical of the helical conformation. On the other hand, it has little influence on the CD spectra of another group of peptide fragments. We call the former group the peptide fragment of a helix-forming propensity and the latter the peptide fragment of a helix-breaking propensity. An interesting fact was found, that is, the lysozyme fragments of a helix-forming propensity are just located in helical parts in the whole lysozyme structure. The fragments of a helix-breaking propensity serve to interrupt the propagation of helix formation, and comactly to assemble some substructures folded locally. If this rule is general, we can use it to predict helical parts of unknown protein structure. Therefore, we applied the abovementioned method to cytochrome c. Several peptide fragments of tunacytochrome c were prepared by trypsin or V8-protease digestion. Peptide fragments (1-21)H, which has a covalently bonded heme group, (56-73) and (91-103) have remarkable helix-forming propensity. On the contrary, peptide fragments of (22-44), (40-53) have the helix-breaking propensity. This strngly supports the rule found by us in lysozyme fragments that the peptide fragment of a helix-forming propensity is just located in the helical art in the whole protein structure.

蛋白质结构通常由几个折叠单元组成，这些折叠单元是较小的子结构。我们研究的目的是根据实验数据确定这样的折叠单元。我们通过胰蛋白酶消化制备了几种溶菌酶片段，并测量了它们的CD光谱。当它们溶解在水溶液中时，通常显示出典型的无规卷曲肽链的CD光谱。然而，根据肽片段的固有特性，向溶液中添加三氟乙醇（TFE）（高达约 50 体积%）会导致 CD 谱发生变化。它诱导一组肽片段显示出典型的螺旋构象的圆二色光谱。另一方面，它对另一组肽片段的圆二色光谱影响不大。我们将前一组称为具有螺旋形成倾向的肽片段，将后者称为具有螺旋断裂倾向的肽片段。发现了一个有趣的事实，即具有螺旋形成倾向的溶菌酶片段恰好位于整个溶菌酶结构中的螺旋部分。螺旋断裂倾向的碎片用于中断螺旋形成的传播，并紧凑地组装一些局部折叠的子结构。如果这个规则是通用的，我们可以用它来预测未知蛋白质结构的螺旋部分。因此，我们将上述方法应用于细胞色素c。通过胰蛋白酶或V8-蛋白酶消化制备金藻细胞色素c的几个肽片段。肽片段 (1-21)H 具有共价键合的血红素基团，(56-73) 和 (91-103) 具有显着的螺旋形成倾向。相反，(22-44)、(40-53)的肽片段具有螺旋断裂倾向。这有力地支持了我们在溶菌酶片段中发现的规则，即具有螺旋形成倾向的肽片段恰好位于整个蛋白质结构的螺旋区中。