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Four overlapping peptide fragments of human c-Raf-1 (residues 55-132, 55-117, 77-132, and 77-117) were expressed in Escherichia coli as carboxyl-terminal extensions of maltose binding protein (MBP). The MBP-Raf fusions were purified by affinity chromatography on amylose resin and tested for binding to Ras.GTP indirectly by measuring their ability to inhibit the stimulation of Ras GTPase activity by GTPase activating protein (GAP120) in vitro. MBP-Raf(55-132) was a potent inhibitor in this assay (50% inhibition at 100 nM concentration), but the other fusion proteins had no measurable effect. The fusion partners were cleaved with Factor Xa protease and separated by gel filtration. The 8960-dalton Raf(55-132) fragment retained full activity as a competitive inhibitor of GAP120. It also blocked Ras-stimulated germinal vesicle breakdown in frog oocytes. Raf(55-132) was further characterized by circular dichroism and nuclear magnetic resonance spectroscopy. The results indicate that this fragment of c-Raf-1 adopts a highly structured, monomeric conformation in solution.

A combination of one- and two-dimensional NMR experiments has been used to identify and spatially locate the heme pocket residues in the paramagnetic, low spin, met-cyano complex of elephant myoglobin. In addition to assigning resonances of the conserved residues, we have also assigned Gln64 (E7) and an aromatic ring designated PheA whose side chain is inserted into the heme pocket, as found earlier for elephant carbonmonoxy-myoglobin and oxy-myoglobin (Yu, L. P., La Mar, G. N., and Mizukami, H. (1990) Biochemistry 29, 2578-2585). The assigned conserved proximal side residues (Leu89(F4), Ala90(F5), His93(F8), His97(FG3), Ile99(FG5), Leu104(G5), Phe138(H15), and Tyr146(H23)) and conserved distal side residues (Phe43(CD1), Thr67(E10), Val68(E11), and Ala71(E14)) in elephant met-cyano-myoglobin are found to have orientations similar to those in sperm whale met-cyano-myoglobin. The observed dipolar connectivities and dipolar shift pattern for the substituted Gln64(E7) place the Gln in the heme pocket oriented toward the iron, as found for His64(E7). The conserved structural elements demand that the inserted PheA originate from the B-helix (i.e. Phe27 or Phe33). Dipolar contacts between the inserted PheA and the conserved residues Phe43(CD1), Val68(E11), Ile107(G8), and Gln64(E7), place PheA in the position occupied by the B10 residue in sperm whale myoglobin (Mb), with the larger size of the PheA side chain as compared to the replaced Leu being accommodated by the vacancy that occurs in sperm whale Mb. The paramagnetic induced relaxation places PheA in van der Waals contact with the bound ligand. Hence we conclude that the B10 position of elephant Mb is occupied by a Phe, and this substitution relative to sperm whale Mb is responsible for the low autoxidation rate and low reduction potential of elephant Mb. A reduced autoxidation rate has been reported for a sperm whale synthetic point mutant Leu29(B10) --> Phe (Carver, T. E., Brantley, R. E., Jr., Singleton, E. W., Arduini, R. M., Quillin, M. L., Phillips, G. N., and Olson, J. S. (1992) J. Biol. Chem. 267, 14443-14450). The published sequence of elephant Mb places B-helix Phe residues at position 27(B8) and 33(B14), but a Phe at neither of these positions can account for the observed NMR properties. Since a large proportion of the substitutions in elephant relative to sperm whale Mb, and some of the least conservative, occur in the B-helix, neither a structurally perturbed B-helix nor an error in the sequence can be discounted.