Dye-pair reporter systems for protein-peptide molecular interactions.
Modifying a linear peptide near each terminus with a fluorescent dye can make it able to signal its own binding to a protein. As originally described, the dye pair is composed of fluorescein and tetramethylrhodamine [Wei, A.-P., Blumenthal, D. K., and Herron, J. N. (1994) Anal. Chem. 66, 1500-1506]. This paper shows that it may also be two molecules of tetramethylrhodamine. In aqueous solution, mutual affinity of the dyes causes fluorescence-quenching contact between them. When the peptide is bound by an antibody or cleaved by a proteinase, or when acetonitrile is added, dye-to-dye contact decreases and fluorescence increases 3-15-fold. When five peptides of 4-20 amino acid residues were doubly modified with tetramethylrhodamine, each product had the absorption spectrum of a tetramethylrhodamine dimer. As the peptides were not known to have special conformational features, self-affinity of the dye appeared to be the main cause of dimerization. Disruption of the dye dimers by acetonitrile suggested that dimerization of the dye(s) in aqueous solution was largely an effect of hydrophobicity. Dye-tagged peptides were used in fluorometric assays for two peptide-protein interactions. First, a peptide from type II collagen recognized by a monoclonal antibody was derivatized with two different dye pairs. The monoclonal bound each modified peptide, disrupting dye-to-dye contact and increasing fluorescence up to 4-fold. Second, a phosphopeptide recognized by an SH2 domain was tagged with fluorescein and tetramethylrhodamine, and its binding to the SH2 domain was detected through fluorescence. Doubly dye-tagged peptides offer a direct, solution-phase assay for protein-peptide binding.
Geoghegan
K F
KF
Central Research Division, Pfizer Inc., Eastern Point Road, Groton, Connecticut 06340, USA. kieran_f_geoghegan@groton.pfizer.com
Rosner
P J
PJ
Hoth
L R
LR
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry; D003094:Collagen / Q000737:chemistry; D010450:Endopeptidases / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D018909:src Homology Domains
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D010455:Peptides; D010748:Phosphopeptides; D011506:Proteins; D012235:Rhodamines; C005358:tetramethylrhodamine; D003094:Collagen; D010450:Endopeptidases; D019793:Fluorescein