Ž . Biochimica et Biophysica Acta 1383 1998 292–300 Analysis of the secondary structure of the catalytic domain of mouse Ras exchange factor CDC25 Mm Paola Coccetti a , Enrico Monzani b , Lilia Alberghina a , Luigi Casella b , Enzo Martegani a,c, ) a Sezione di Biochimica Comparata, Dipartimento di Fisiologia e Biochimica Generali, UniÕersita’ di Milano, Via Celoria 26, 20133 Milano, Italy b Dipartimento di Chimica Generale, UniÕersita di PaÕia, 27100 PaÕia, Italy ` c III Facolta’ di Scienze M.F. Naturali, UniÕersita di Milano, 21100 Varese, Italy ` Received 24 September 1997; revised 3 December 1997; accepted 9 December 1997 Abstract Ž . Mm The minimal active domain GEF domain of the mouse Ras exchange factor CDC25 was purified to homogeneity from recombinant Escherichia coli culture. The 256 amino acids polypeptide shows high activity in vitro and forms a stable Ž . complex with H-ras p21 in absence of guanine nucleotides. Circular dichroism CD spectra in the far UV region indicate Ž . 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. q 1998 Elsevier Science B.V. Keywords: GDPrGTP exchange factor; Ras protein; p21 ras-GEF interaction; Bacterial expression; Protein purification 1. Introduction Ras proteins play an essential role in a variety of signal transduction pathways relevant for growth reg- w x ulation and differentiation in eukaryotic cells 1,2 . They cycle between the active GTP-bound and inactive GDP-bound state; the intracellular levels of Abbreviations: MBP, maltose-binding-protein; ME, 2 b-mer- captoethanol; IPTG, isopropyl b D-thiogalactopyranoside; GEF, guanine nucleotide exchange factor; CD, circular dichroism ) Corresponding author. Fax: q39-2-70632811; E-mail: marteg@imiucca.csi.unimi.it the Ras-GDP and Ras-GTP complexes are carefully regulated by two classes of proteins: Ras-GEF, which catalyzes GDPrGTP exchange generating the active GTP-bound form, and Ras-GAP, which stimulates the intrinsic RAS-GTPase activity promoting the hy- drolysis to the inactive GDP-bound form. To date two different classes of Ras-GEFs have been identi- w x fied in mammals: Sos proteins 3,4 and Mm w x CDC25 rRas-GRF 5–7 whose Ras-activating do- main is homolog to the C-terminal domain of the Saccharomyces cereÕisiae Ras activator CDC25 pro- wx Ž tein 5 . Sos proteins h-Sos1 and h-Sos2 in human . and mSos1rmSos2 in mouse are ubiquitously ex- pressed in all tissues while full-length CDC25 Mm has 0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. Ž . PII S0167-4838 97 00212-4