Fluorescent labeling, generations fluorophores

This chapter describes methods of fluorophore labeling to D residues with improvements over the previous ones, and methods to introduce fluorophores to the CCA sequence. Both sequence motifs are general to tRNAs and are located in the hot spots for binding interactions, suggesting that these site-specific fluorescent labeling methods will have a broad range of utility in generating important information on tRNA dynamics. 

As a comparison, fluorescent labeling of tRNA with PyC is achieved in one step by the CCA enzyme, and thus is conceptually and technically simpler than labeling of tRNA with proflavin, rhodamine, or Cy3 and Cy5-hydrazides via D residues. However, the fluorescence emission intensity of PyC is not as high as those of the other fluorophores and thus may not be suitable for single-molecule experiments. Nonetheless, enzymatic labeling of tRNA with PyC is easy to implement and should be applicable to all tRNA sequences (both wild type and mutants), which can be generated by in vitro transcription without the requirement for a specific modification or for native tRNA species. 

Elucidation of the thermodynamic basis of Rab membrane targeting requires analysis of interaction between prenylated Rab proteins (GDP/GTP-bound) and REP/GDI. Such analysis is made possible by generation of fluorescent labelled prenylated Rab proteins (Scheme 10a) [30, 54]. A series of Rab7-based protein probes with one or two isoprenyl moieties and fluorophores on the lipid moiety or the lysine side chain were prepared using the EPL technique. The semisynthetic method enables precise installation of GDP/GTP into Rab proteins to generate the off and on states, yielding for the first time homogeneous preparations of functionalized prenylated proteins in a well-defined nucleotide bound state [87]. 

Fluorescent probes are relatively small molecules that are used to label biomolecules such as proteins, antibodies and nucleic acids. They contain functional groups and specific physical and chemical characteristics that confer suitability for their use as detection moieties. To date, thousands of fluorescent probes are known each with varying spectral properties. Fluorophores may be intrinsic or extrinsic in nature. Intrinsic fluorophores are naturally occurring whereas extrinsic fluorophores are added to generate a fluorescence signal to facilitate measurement of a specific target molecule. Fluorescent labels have provided excellent sensitivity for a range of assay systems that can be applied to the determination of almost any analyte. 

Light at the excitation maximum of one fluorophore does not usually further excite shorter wavelength fluorescent labels. Thus, no crosstalk is generated by them. 

FIGURE 2.6 Fluorescence polarization readout principle. In the intact peptidic substrate (amino acids symbolized by X, Y and Z) labeled with a fluorophore (dye) and a molecular weight enhancer (MWE) at the opposite sites of the scissile bond, a high polarization is observed (long black arrow). After cleavage of the substrate between amino acids X and Y by a protease, the MWE and dye are decoupled and a low polarization of the labeled cleavage product is generated (short black arrows). A decrease of the polarization dependent on the enzymatic velocity is recorded. 

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