ANALYSIS OF THE SECONDARY STRUCTURE OF THE CATALYTIC DOMAIN OF MOUSE RAS EXCHANGE FACTOR CDC25(MM)

The minimal active domain (GEF domain) of the mouse Ras exchange facto r CDC25(Mm) was purified to homogeneity from recombinant Escherichia c oli culture. The 256 amino acids polypeptide shows high activity in vi tro and forms a stable complex with H-ras p21 in absence of guanine nu cleotides. Circular dichroism (CD) spectra in the far UV region indica te that this domain is highly structured with a high content of alpha- helix (42%). Near UV CD spectra evidenced good signal due to phenylala nine and tyrosine while a poor contribution was elicited by the three tryptophan residues contained in this domain. The tryptophan fluoresce nce signal was scarcely affected by denaturation of the protein or by formation of the binary complex with H-ras p21, suggesting that the Tr p residues, which are well conserved in the GEF domain of several Ras- exchange factors, were exposed to the surface of the protein and they are not most probably directly involved in the interaction with Ras pr oteins. (C) 1998 Elsevier Science B.V.