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

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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.