Synthesis and application of fluorescent ras proteins for live-cell imaging.
Semisynthetic Ras proteins are efficient probes for cell-biology experiments. With a Bodipy FL fluorophore introduced at an appropriate site on the Ras peptide by solid-phase synthesis, the resulting Ras chimera is processed by the cellular machinery and the intracellular localization of the protein can then be visualized by means of confocal laser fluorescence microscopy at relatively low concentrations. The absence of a large N-terminal protein tag overcomes possible interferences in the interaction with cellular partner proteins. The fluorescence emission from Bodipy FL is continuous and disappears only after irreversible bleaching. These characteristics make Ras proteins with nonprotein fluorophores suitable for biophysical analysis. The easy accessibility of the lipopeptide moiety by chemical synthesis opens up numerous options for further biological investigations.
Reents
Reinhard
R
Max-Planck-Institut für Molekulare Physiologie, Abteilung Chemische Biologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Wagner
Melanie
M
Schlummer
Stefanie
S
Kuhlmann
Jürgen
J
Waldmann
Herbert
H
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry*; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D018631:ras Proteins / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry*
D005456:Fluorescent Dyes; D018631:ras Proteins