Experimental procedure to predict the folding units of an unknown 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亚结构。我们研究的目的是根据实验数据确定这样的折叠单元。通过胰消化法制备了几个溶菌酶片段，并测定了它们的CD谱。在水溶液中溶解时，它们通常表现出典型的随机卷曲肽链的CD光谱。然而，在溶液中加入三氟乙醇（TFE）（高达约50%体积%）会根据肽片段的固有性质引起cd光谱的变化。它诱导一组肽片段显示典型螺旋构象的CD谱。另一方面，它对另一组肽片段的CD谱影响不大。我们称前者为螺旋形成倾向的肽片段，后者为螺旋断裂倾向的肽片段。我们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的螺旋部分。螺旋断裂倾向的碎片中断了螺旋形成的传播，并紧密地组装了一些局部折叠的亚结构。如果这条规则是一般的，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，并通过胰蛋白酶或v8蛋白酶消化制备了几个细胞色素c的肽片段。（56-73）和（91-103）肽片段（1-21）H具有显著的螺旋形成倾向，其中H具有共价结合血红素基团。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律，即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。

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.