Probes genetically encoded

Umezawa Y (2005) Genetically encoded optical probes for imaging cellular signaling pathways. Biosens Bioelectron 20 2504-11... 

Site-specific incorporation of biophysical probes into proteins allows the structure and function of proteins to be probed with greater precision and accuracy using various biophysical means. Genetically encoded biophysical probes in the form of unnatural amino acids further extend the potential of these studies into live cells, the... 

Cell membranes are two-dimensional fluids that exhibit a wide range of dynamic behaviors. Recent technical advances have enabled unprecedented views of membrane dynamics in living cells. In this technical review, we provide a brief overview of three well-studied examples of membrane dynamics lateral diffusion of proteins and lipids in the plane of the membrane, vesicular trafficking between intracellular compartments, and exchange of proteins on and off membranes. We then discuss experimental approaches to monitor membrane protein and lipid dynamics, and we place a special emphasis on the use of genetically encoded fluorescent probes and live cell-imaging techniques. 

Until recently, experiments that probe protein diffusion relied on fluorescent antibody-based probes, and thus were limited to plasma membrane proteins with extracellular epitopes. In contrast, the mechanisms that regulate diffusion of intracellular membrane proteins remained unexplored because of their inaccessibility to labeling. With the development of genetically encoded fluorescent probes, such studies have become tractable because proteins targeted to a particular organelle can be flu-orescently labeled directly with GEP. In addition, improved technology has now made it possible to monitor the movement of multiple types of proteins or lipids, tagged with different markers simultaneously. Some examples of the types of questions it is now possible to address include ... 

In addition, a genetically encoded ratiometric sensor for H2O2 is now available. This probe, named HyPer, incorporates a YEP variant into the prokaryotic H2O2-sensing protein (OxyR) and was used successfully to monitor the formation of H2O2 in single mitochondria within a living cell [8]. 

Ugwumba, I.N., Ozawa, K., de la Cruz, L., Xu, Z.-Q. et al. (2011) Using a genetically encoded fluorescent amino acid as a site-specific probe to detect binding of low-molecular-weight compounds. Assay Drug Dev. Technol, 9, 50- 57. 

In principle, any couple of fluorophores can be used for FRET, provided that the emission spectrum of the donor overlaps with the absorption of the acceptor. For a review of FRET-couples (and RO values) of chemical dyes see [62]. Furthermore, donors with a high fluorescence quantum-yield and acceptors with a high molar absorbance will display increased FRET. For FLIM it will be important to tune the instrument-performance to ensure maximal sensitivity to small changes in lifetimes at the control donor lifetime. Usually this is easily achieved. Many FRET-pairs have been used for FRET-FLIM including chemical probes as Fluorescein-Rhodamine [54,93],calcein-sulforhodamine B [94], and Cy3-Cy5, [70]. Since 1996, the availability of genetic-encoded fluorophores such as CFP, GFP, YFP has boosted application of FRET-FLIM enormously [95]. Nowadays fluorescent-tagging of proteins no longer depends on laborious protein pu-... 

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