Recognition properties of peptides hydropathically complementary to residues 356-375 of the c-raf protein.
Two peptides with hydropathic complementarity to residues 356-375 of the c-raf protein were synthesized to determine if they recognize the raf-(356-375) peptide as well as the entire protein. One peptide was deduced from the complementary mRNA for the raf protein corresponding to residues 356-375, whereas the other was deduced solely from the amino acid sequence of the 20-mer segment using a computer program able to generate peptide sequences with hydropathic complementarity to a given sequence. Specific binding of both peptides to the raf 20-mer segment was demonstrated when either the raf 20-mer peptide or the complementary peptides were immobilized on a column. Binding affinities were in the millimolar-micromolar range. Identical binding properties were observed with peptides synthesized with either all D- or all L-amino acids, suggesting a lack of conformational dependence. Binding was also unaffected by the presence of 8 M urea or detergents, was dependent on solvent characteristics of pH and ionic strength, and was abolished by the presence of competing peptides in the eluting buffer. Recognition between raf complementary peptides was accompanied by spectral changes in the far and near UV region, as monitored by circular dichroism. Proteolytic degradation was retarded by the binding of these peptides. Once immobilized on a column, these peptides proved useful for the isolation by affinity chromatography of a recombinant c-raf protein from an Escherichia coli crude cell extract.
Fassina
G
G
Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892.
Roller
P P
PP
Olson
A D
AD
Thorgeirsson
S S
SS
Omichinski
J G
JG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004926:Escherichia coli / Q000032:analysis*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D012313:RNA; D018075:RNA, Complementary; D012333:RNA, Messenger; D012689:Sequence Homology, Nucleic Acid; D012984:Software; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D018075:RNA, Complementary; D012333:RNA, Messenger; D012313:RNA; D000255:Adenosine Triphosphate; D019908:Proto-Oncogene Proteins c-raf; D014357:Trypsin