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，它们是较小的亚结构。本研究的目的是在实验数据的基础上确定这种折叠单元，我们用胰蛋白酶消化法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，它们通常显示出典型的无规卷曲肽链的CD谱。然而，向溶液中添加三氟乙醇（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的螺旋构象的CD光谱。另一方面，它对另一组肽片段的CD谱几乎没有影响。我们称前者为具有螺旋形成倾向的肽片段，而后者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的螺旋部分。螺旋断裂倾向的片段用于中断螺旋形成的传播，并且有助于组装局部折叠的一些亚结构。如果这个规则是普遍的，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。用胰蛋白酶或V8-蛋白酶消化金枪鱼细胞色素c的几个肽段。具有共价键合的血红素基团的肽片段（1-21）H、（56-73）和（91-103）具有显著的螺旋形成倾向。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律，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。

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.