BODIPY® FL NTPs are multipurpose tools for
Scheme 1: BODIPY® FL NTPs as fluorescent probes for protein-NTP-interactions. Binding equilibria of BODIPY®-NTP/protein complexes as well as their conversion by hydrolysis or phosphorothioate transfer can be analyzed by numerous fluorescence-based, chromatographic, electrophoretic and imaging approaches. Most strikingly, GTPγS - BDP-FL shows an increase of fluorescence by a factor of 5 upon protein binding.
Regulatory ATPase and GTPase are the ubiquitous regulators of cellular signaling in all domains of life. Their activity is modulated by binding and cleavage of their cognate NTP. For the characterization of protein-nucleotide interactions and subsequent conversion steps, BODIPY® FL NTPs have been established as fluorescent multipurpose tools. Applications include:
|Application||Protein examples||Analytical technique|
|Qualitative/quantitative analysis of protein-nucleotide equilibria (KD, kon, koff)[1-4]||Cdc42, Gα0/i, Rac, Ras, Rho, RNAse L||Fluorescence increase
Fluorescence polarization (FP)
Capillary electrophoresis (CE)
|Kinetic analysis of nucleotide cleavage[5-6]||FHIT, Gα0/i||Fluorescence increase
Thin layer chromatography (TLC)
|Activity-based kinase labeling||Two-component system||Imaging|
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 Huang et al. (2014) Dimeric structure of pseudokinase RNase L bound to 2-5A reveals a basis for interferon-induced antiviral activity. Mol. Cell. 53 (2):221.
 Korlach et al. (2004) Spontaneous nucleotide exchange in low molecularweight GTPases by fluorescently labeled gamma-phosphate-linked GTPanalogs. Proc. Natl. Acad. Sci. USA 101 (9):2008.
 McEwen et al. (2001) Fluorescent BODIPY-GTP analogs: real-time measurement of nucleotide binding to G proteins. Anal. Biochem. 291 (1):109.
 Whelan et al. (2004) Affinity Assays Using Fluorescence Anisotropy with Capillary Electrophoresis Separation. Anal. Chem. 76 (24):7380.
 Draganescu et al. (2000) Fhit-nucleotide specificity probed with novel fluorescent and fluorogenic substrates. J. Biol. Chem. 275 (7):4555.
 Jameson et al. (2007) Capillary Electrophoresis Assay for G Protein-Coupled Receptor-Mediated GTPase Activity. Anal. Chem. 79 (3):1158.
 Wilke et al. (2012) Activity-based probe for histidine kinase signaling. J. Am. Chem. Soc. 134 (22):9150.