The development of higher wavelength Forster (Fluorescence) Resonance Energy Transfer (FRED substrates has provided a useful enabling tool for the purposes of studying the interactions of drugs, and potential drugs, with biologically active proteins in vitro, overcoming many of the problems such as poor sensitivity and interference associated with traditional fluorescent substrates. In particular these substrates have proven highly useful to the study of peptidases (the proteins responsible for hydrolytic cleavage of the peptide bonds between amino acids during peptide/protein catabolism) where the hydrolysis of fluorophore labelled FRET peptide substrates can be monitored with great sensitivity, as a result of the relatively large changes in fluorescence generated by low levels of substrate turnover. Despite these advantages, to date little investigation has been undertaken into the potential applications for such FRET peptides to the characterisation of physiological interactions. Within this study the potential applications of the higher wavelength FRET pairs fluorescein (fAM) and tetramethylrhodamine (TAMRA) in the characterisation of disintegrin metallopeptidases was investigated with a focus on two areas; .applications within the design of homogenous real-time cellular assays and applications within the characterisation of b!.Qp101ecule binding interactions in vitro. Both areas of study have previously been dem~nstrated to be unsuited to investigation using traditional lower wavelength FRET peptides. For the purposes of evaluating applications to cellular assay design, a FAM-TAMRA substrate for ADAM-17 (TACE) was investigated as a tool to measure TACE surface activity in cultured cell lines. To investigate applications to the study of biomolecule interactions in vitro, a FAM-TAMRA substrate for ADAMTS-4 was. used to characterise exosite binding interactions between ADAMTS-4, its physiological inhibitor, TIMP-3 and its substrate aggrecan. Based on the observations of this study, the use of higher wavelength fluorophores successfully overcomes many of the problems traditionally associated with FRET peptides in these areas, and as such provides a useful tool for use in the characterisation ofphysiological interactions.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:490592 |
| Date | January 2007 |
| Creators | Wayne, Gareth |
| Publisher | University of East Anglia |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
Page generated in 0.0018 seconds