Among heme proteins, the c-type cytochromes are distinguished by the fact that the heme is covalently linked to the protein by two thioether linkages. Upon removal of the heme, the resulting apo-cytochrome c (apocyt) is unfolded. However, a partly folded state of apocyt can be obtained by adding heme to the apoprotein under neutral conditions [1,2]. There is today a strong interest in the conformational features of partly folded states (or molten globules) of proteins that can be obtained at equilibrium, since these states are related to those that can be observed in kinetic experiments of protein folding. Among the techniques currently used in studying the molecular features of partly folded states, limited proteolysis can provide experimental results which are easy to obtain and nicely complement those derived from the use of other more classical physicochemical methods and approaches [3,4]. Here, the non-covalent binding of heme to apocyt to form the apocyt-heme complex was probed by proteolysis. Proteinase K and trypsin have been utilized for these experiments, which were conducted at neutral pH utilizing an enzyme:substrate and heme:apocyt ratios of 1:100 and 1:1.2, respectively. The proteolysis of apocyt, apocyt-heme complex and holocyt have been monitored by polyacrylamide gel electrophoresis and HPLC. Holocyt resists the digestion by proteinase K and trypsin, while apocyt is digested by both proteases to numerous small peptides. The apocyt-heme complex shows a resistance to proteolytic digestion intermediate between that of apocyt and that of holocyt. The results of the proteolysis experiments have been correlated with the conformational features of the three protein species, as given by far-UV circular dichroism measurements. Whereas apocyt is essentially unfolded, the apocyt-heme complex shows substantial helical secondary structure, approaching that of holocyt. It is concluded that the highly folded, native holocyt resists to proteolysis being unable to bind to the active site of the proteolytic enzyme, while the unfolded and flexible apocyt is easily degraded and the partly folded apocyt-heme complex is comparatively more resistant to proteolysis. The results of this study emphasize the utility of proteolytic enzymes for elucidating conformational features of proteins.

[1] Dumont, M.E., Corin, A.F. & Campbell, G., Biochemistry 33, 7368-7378 (1994).

[2] Goldberg, M.E., Schaeffer, F., Guillou, Y. & Djavadi-Ohaniance, L., J. Biol. Chem. 274, 16052-16061 (1999).

[3] Fontana, A., Polverino de Laureto, P., De Filippis, V., Scaramella, E. & Zambonin, M., Folding & Design 2, R17-R26 (1997).

[4] Fontana, A., Polverino de Laureto, P., De Filippis, V., Scaramella, E. & Zambonin, M., Proteolytic Enzymes (E. Sterchi & W. Stöcker, Eds.), Springer Verlag, Heidelberg, pp. 257-284 (1999).