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Title
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Characterization of a 78-residue fragment of c-Raf-1 that comprises a minimal binding domain for the interaction with Ras-GTP.
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Authors
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J.E.Scheffler,
D.S.Waugh,
E.Bekesi,
S.E.Kiefer,
J.E.LoSardo,
A.Neri,
K.M.Prinzo,
K.L.Tsao,
B.Wegrzynski,
S.D.Emerson.
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Ref.
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J Biol Chem, 1994,
269,
22340-22346.
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PubMed id
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Abstract
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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.
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