Analysis of the Secondary Structure of the Catalytic Domain of Mouse Ras Exchange Factor CDC25Mm

The minimal active domain GEF domain. of the mouse Ras exchange factor CDC25Mm was purified to homogeneity
from recombinant Escherichia coli culture. The 256 amino acids polypeptide shows high activity in vitro and forms a stable
complex with H-ras p21 in absence of guanine nucleotides. Circular dichroism CD. spectra in the far UV region indicate
that this domain is highly structured with a high content of a-helix 42%.. Near UV CD spectra evidenced good signal due
to phenylalanine and tyrosine while a poor contribution was elicited by the three tryptophan residues contained in this
domain. The tryptophan fluorescence signal was scarcely affected by denaturation of the protein or by formation of the
binary complex with H-ras p21, suggesting that the Trp 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 proteins. q1998 Elsevier Science B.V