Labeling

short summary

As part of the toolbox , the labeling of a protein in vivo is a useful tool that allows the investigation of a protein in its native environment. As a label for our target protein we use the fluorescent amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine (CouAA) that is incorporated by an orthogonal t-RNA /aminoacyl-synthethase pair at a defined position.
To demonstrate this tool we want to find out if the Ribulose 1,5-bisphosphat Carboxylase Oxygenase (RuBisCo) is located in the carboxysome , an artificial compartment surrounded by proteins and used by the iGEM Team CeBiTec 2014 to increase the activity of the RuBisCo. The carboxysome has already been labeled with green fluorescent protein (GFP) and we want to co-localizate the RuBisCo labeled with an genetically encoded fluorescent amino acid L-(7-hydroxycoumarin-4-yl) ethylglycine and in comparison labeled with red fluorescent protein (RFP).

Labeling of a protein in vivo

Protein localization in vivo can be performed by labeling the target protein with a fluorescent protein like green fluorescent protein (GFP) or red fluorescent protein (RFP). The labeling is done by a translational fusion of the CDS from the fluorescent protein C- or N- terminal with a short linker to the CDS from the target protein. But the labeling is limited to the C- or N-terminus and due to its size GFP (29 kDa [FLUO3]) could be bigger than the target protein and be a hindrance. Both could cause a significant change of the structure of the target protein or a loss of function,especially if the protein is part of an assembly in a larger complex or oligomer [FLUO1,2].
The usage of a genetically encoded fluorescent amino acid would circumvent these problems and deliver a tool to study protein localization and function in vivo and in vitro. An orthogonal t-RNA/aminoacyl-tRNA synthetase pair allows the incorporation of amino acids in response to the amber stop codon (TAG) selectively at a defined position in the protein [FLUO1].

L-(7-hydroxycoumarin-4-yl) ethylglycine (CouAA)

The fluorescent amino acid L-(7-hydroxycoumarin-4-yl) (CouAA) ethylglycine is relatively small, has a high fluorescence quantum yield and relatively large Stoke's shift. It is also solvent polar and pH-sensitive so it can indicate pH-changes in the cell [FLUO₂]. The translational incorporation of CouAA with an aaRS was shown by Schultz 2006 and Charbon 2011,2012 into different proteins. The amino acid is suitable for in vivo and in vitro localization, even for localization in SDS-PAGES. The adsorption spectrum of CouAA is shown in figure c:

Figure 1:
L-(7-hydroxycoumarin-4-yl) ethylglycine.

Figure 2:
Adsorption and fluorescence spectrum of L-(7-hydroxycoumarin-4-yl) ethylglycine. [FluO₂].

The relative fluorescence signal of CouAA decreases over time when irradiated with the excitation wavelength. This effect is called photobleaching and occurs in more or less in all fluorophores. This photobleaching effect is shown in figure d for the labeling of the bacterial tubulin FtsZ with CouAA [Fluo1].

Figure 3:
The in vivo dynamic properties of FtsZ10CouAA. The graph represents the data corrected for photobleaching due to image acquisition for unbleached (green) and bleached (blue) regions; the red line represents the theoretical recovery curve fit. FtsZ10CouAA (The labeled protein) half-time recovery is 12(+-5) s (mean +-standard deviation); 11.6 s in the example shown. [Fluo1].

Colocalisation of the ribulose 1,5-bisphosphate carboxylase oxygenase and the carboxysome

Ribulose 1,5 bisphosphate Carboxylase Oxygenase (RuBisCo)

The protein we want to label with CouAA is the 1,5- bisphosphate carboxylase oxygenase (RuBisCo). RuBisCo catalyzes the incorporation of inorganic CO₂ to ribulose-1,5-bisphosphate to form two 3-phosphoglycerate molecules. The catalyzed reaction is shown in figure b. [RuBisCo1].

Figure 4:
Reaction catalyzed by Ribulose 1,5-bisphosphat Carboxylase Oxygenase (RuBisCo). Ribulose 1,5-bisphosphate is converted in two molecules 3-phophoglycerate.