Evolution of peptides that modulate the spectral qualities of bound, small-molecule fluorophores.
BACKGROUND
Fluorophore dyes are used extensively in biomedical research to sensitively assay cellular constituents and physiology. We have created, as proof of principle, fluorophore dye binding peptides that could have applications in fluorescent dye-based approaches in vitro and in vivo.


RESULTS
A panel of Texas red, Rhodamine red, Oregon green 514 and fluorescein binding peptides, termed here 'fluorettes', was selected via biopanning of a combinatorial library of 12-mer peptides fused to a minor coat pIII protein of the filamentous bacteriophage M13. The 'best' fluorette sequences from each of the groups were subjected to further mutagenesis, followed by a second biopanning to select a new generation of improved fluorettes. Phage were selected that had higher avidity for each fluorophore except Rhodamine red. Of these, peptides were characterized that could specifically and with high affinity bind at least one dye, Texas red, in solution. In addition, the binding of certain peptides to Texas red shifted the peak excitation and/or the emission spectra of the bound dye.


CONCLUSIONS
Peptides in the context of phage display could readily be selected that could bind to small-molecule fluorophores. The affinities of selected mutant fluorettes could be increased by mutation and further selection. Only a subset of the free peptides could bind free dyes in solution, suggesting that phage context contributed to the selection and ability of certain peptidic regions to independently bind the dyes. Future screens might lead to the creation of other dye-binding peptides with novel characteristics or Texas red derivatives with cross-linking substituents might be designed to increase the utility of the system.
Rozinov
M N
MN
Department of Molecular Pharmacology Stanford University Medical Center Stanford CA 94305-5332 USA.
Nolan
G P
GP
D000595:Amino Acid Sequence; D001435:Bacteriophages; D001483:Base Sequence; D002352:Carrier Proteins / Q000138:chemical synthesis* / Q000378:metabolism; D004926:Escherichia coli; D019793:Fluorescein / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis* / Q000378:metabolism; D011485:Protein Binding; D012235:Rhodamines / Q000378:metabolism; D014966:Xanthenes / Q000378:metabolism
D002352:Carrier Proteins; D005456:Fluorescent Dyes; D019151:Peptide Library; D010455:Peptides; D012235:Rhodamines; D014966:Xanthenes; C034657:Texas red; D019793:Fluorescein