Structural characterization of a 14-residue peptide ligand of the retinoblastoma protein: comparison with a nonbinding analog.
The retinoblastoma protein, a 110-kDa nuclear anti-oncoprotein, complexes specifically with transforming proteins of several oncogenic DNA viruses. A peptide [NLFCSEEMPSSDDE] derived from one of the viral proteins (simian virus 40 large T antigen) is known to competitively bind retinoblastoma protein, but a mutant analog [NLFCSKEMPSSDDE] does not. We studied the T peptide with HPLC to determine whether it can dimerize, and we employed circular dichroism spectroscopy to determine whether both peptides can exist in stable secondary structural conformations. HPLC analyses revealed that the T peptide is subject to oxidation and readily dimerizes. Circular dichroism analyses showed that both peptides can be induced to form stable secondary structural conformations under conditions that stabilize intramolecular hydrogen bonding in short peptides (90% 2,2,2-trifluoroethanol; 4 degrees C). The circular dichroism spectra of both peptide species were similar except for a statistically significant difference in the contour near 210 nm. Spectral analysis of the T-derived peptide species predicted elements of alpha-helix (18%), antiparallel beta-sheet (21%), beta-turn (22%) and unordered conformations (41%). An analysis of the mutant peptide species also predicted elements of alpha-helix (8%), antiparallel beta-sheet (28%), beta-turn (22%) and unordered conformations (40%). Thus, a small difference in the stabilized secondary structural conformations of the two sets of peptide species might partly explain their differential binding affinities for retinoblastoma protein, but it is likely that electrostatic charge differences resulting from the glutamic acid to lysine substitution play a dominant role.
Breese
K
K
Albany Medical College.
Friedrich
T
T
Andersen
T T
TT
Smith
T F
TF
Figge
J
J
D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D016160:Retinoblastoma Protein / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; D008024:Ligands; D010446:Peptide Fragments; D016160:Retinoblastoma Protein