Bioimaging tests of the prepared biarsenical probes

Before any experiments with living cells could be conducted, it was necessary to check the binding ability of the SplAsH derivatives in vitro. Therefore, a TetCys-mut-GFP-TetCys protein was chosen as the test substrate. This represents an isolated Green Fluorescent Protein with two point mutations that disrupt its ability to emit light and with additional tetracysteine motifs attached to N- and C-Terminals.

Figure 70. Absorption spectra of the collected fractions from size exclusion chromatography performed after incubation of TetCys-mut-GFP-TetCys-protein with the compound SplAsH-RhS-OF. The numbers on the right side of the spectra are the fraction numbers (every fraction was 0.5 mL). At the beginning we can observe the fractions which contain the unknown substrates (for example fraction number 4 has in the absorption spectrum a band at 550 nm, that can indicate the presence of SplAsH-RhS-OF (or products of its decomposition) in this fraction). Then, in the absorption spectra of the fractions 13–18, where the substances with higher molecular weight should be present, a band at 280 nm was observed (this band is marked with green arrows). These fractions contain the TetCys-mut-GFP-TetCys-protein.

We tried to isolate the conjugate of the above mentioned protein with the compound SplAsH-RhS-OF using size exclusion chromatography with a spectroscopic control.

Unfortunately, we did not find any fractions displaying the absorption bands of the protein

TetCys-mut-GFP-TetCys (at 280 nm) and the compound SplAsH-RhS-OF (at 550 nm) (see Figure 70).

As a positive control, a similar experiment was done with the commercially available FlAsH (Lumio-Green/Invitrogen). In this experiment, the FlAsH was clearly bound to the tetracysteine containing protein (see Figure 71).

-0,005 0 0,005 0,01 0,015 0,02 0,025 0,03

250 350 450 550 650

λ^{, nm}

Abs.

Fraktion1 Fraktion2 Fraktion3 Fraktion4 Fraktion5 Fraktion6 Fraktion7 Fraktion8 Fraktion9 Fraktion10 Fraktion11 FlAsH

TetCys-mut-GFP-TetCys

Figure 71. Absorption spectra of the collected fractions from the size exclusion chromatography column obtained after the conjugation experiment between TetCys-mut-GFP-TetCys-protein and FlAsH. The fractions 4 and 5 both contain characteristic bands attributed to TetCys-mut-GFP-TetCys-protein, and to FlAsH; their maxima are marked with black arrows).

After that, we performed another conjugation experiment, in which a preliminary treatment of the protein with the TCEP reagent (for reduction of the disulfide bonds) was performed. As an analytical method, the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was chosen.

Figure 72. SDS-PAGE of TetCys-mut-GFP-TetCys-protein labelled with FlAsH and the compound SplAsH-RhS-OF. The picture A is taken under UV light (left) and the picture B after staining with Roti^®-Blue (right). The number of lanes is the same in both pictures. By the special Roti^®-Blue dye we visualized the protein bands. Lanes 1 and 10 contain a set of molecular weight markers, lanes 8 and 9 contain big bands which are visible only under UV light and can be attributed to SplAsH-RhS-OF (and its decomposition products), 7 – the band of mutated non-fluorescent GFP, 6 – the band of TetCys-mut-GFP-TetCys-protein (also mutated GFP, but with two tetracysteine tags attached to the N- and C-terminals), lane 5 corresponds to the experiment with mutated GFP and SplAsH-RhS-OF [there are two bands:

the first one (on picture B) is attributed to the non-fluorescent mutated GFP, the second one (on picture A) is a big fluorescent band of SplAsH-RhS-OF that indicates the absence of binding of the biarsenical probe SplAsH-RhS-OF to the protein without a tetracysteine tag), 4 – the same experiment as in the lane 5, but the biarsenical probe FlAsH was used for the labelling (in this lane, only the band of the mutated GFP-protein is presented, because the non-bound FlAsH is non-fluorescent), 3 – the main experiment with TetCys-mut-GFP-TetCys-protein and biarsenical probe SplAsH-RhS-OF (two fluorescent bands can be seen:

the low band corresponds to the trace of the initial biarsenical probe SplAsH-RhS-OF, the upper band corresponds to the complex of TetCys-mut-GFP-TetCys-protein with the biarsenical probe SplAsH-RhS-OF, the protein nature of which was confirmed by the blue band in the left picture), 2 – additional control experiment with TetCys-mut-GFP-TetCys-protein and biarsenical probe FlAsH (only one fluorescent band is seen, namely the complex of TetCys-mut-GFP-TetCys-protein with the biarsenical probe FlAsH, the protein nature of which was confirmed by the blue band in the left picture).

The presence of the bright fluorescent band (visible upon UV light illumination) in the expected area of the SDS-PAGE gel indicate, that a complex between TetCys-mut-GFP-TetCys-protein and the compound SplAsH-RhS-OF was formed and could be separated by the SDS-PAGE method.

Figure 73. SDS-PAGE of isolated mitochondria from a yeast strain that expresses a triple tetracysteine tagged Por1 protein and is labelled with compound SplAsH-RhS-OF and compound SplAsH-TAMRA (negative control – wild type mitochondria, positive control – FlAsH). Lanes: 6 – experiment with the isolated mitochondria with the triple tetracysteine tagged version of Por1 (one of the proteins of the outer mitochondrial membrane) and biarsenical probe SplAsH-RhS-OF (the red arrow points to the fluorescent band of the labelled mitochondrial protein that indicates the binding of the biarsenical probe SplAsH-RhS-OF with the tetracysteine tagged version of the mitochondrial protein Por1), 5 – experiment with wild-type mitochondria and SplAsH-RhS-OF (the red arrow points to the very pale fluorescent band of the unspecifically labelled mitochondrial protein; the absence of the fluorescent band at the same place as in lane 6 confirms the binding ability of SplAsH-RhS-OF), 4 – the same experiment as in the case of lane 6, but with biarsenical probe SplAsH-TAMRA (the red arrow points to the fluorescent band of the labelled mitochondrial protein that indicates binding of the biarsenical probe SplAsH-TAMRA to the tetracysteine tagged version of the mitochondrial protein Por1), 3 – experiment with a wild-type mitochondria and SplAsH-TAMRA (the red arrow points to the very pale fluorescent band of the unspecifically labelled mitochondrial protein; the absence of the fluorescent band at the same place as in the lane 4 confirms the binding ability of SplAsH-TAMRA), 2 – additional control experiment with the triple tetracysteine tagged version of the mitochondrial protein Por1 and biarsenical probe FlAsH (the red arrow points to the fluorescent band of labelled

mitochondrial protein that confirms the presence of the tetracysteine tags in the used mitochondrial protein Por1 to which the biarsenical probe FlAsH are bound), 1 – experiment with wild-type mitochondria and FlAsH (the red arrow points to the very pale fluorescent band of the unspecifically labelled mitochondrial protein; the absence of the fluorescent band at the same place as in lane 2 confirms the binding ability of FlAsH).

The labeling experiments with the isolated mitochondria expressing a three TetCys-tagged version of Por1 (one of the proteins in the outer mitochondrial membrane), were also successful. These mitochondria were successfully labelled with the two biarsenical probes:

SplAsH-RhS-OF and SplAsH-TAMRA. These facts were confirmed in the same manner as earlier, i. e. by observation of the bright fluorescent lanes upon UV light illumination in the required area of an SDS-PAGE plate (Figure 73).

Therefore, we have confirmed, that the specific binding of our SplAsH derivatives to the tetracysteine tags in proteins could indeed be achieved.

Unfortunately, the attempted in vivo labelling of the mitochondrial proteins containing tetracysteine tags failed to produce positive results.

Figure 74. Confocal image of yeast cells, stained by means of electroporation with SplAsH-RhS-OF (left picture) and the recorded fluorescence of the same cells caused by the mitochondrial targeted Blue Fluorescent Protein (m-BFP) (right picture). In the right picture, we can see mitochondrial network (blue), but in the left picture we can see only unspecific background staining, because only random red spots inside the cells are seen.

A yeast strain expressing the mitochondrial targeted, mutated and tetracysteine tagged GFP and additional expressing a mitochondrial targeted Blue Fluorescent Protein was the first object of staining. These yeast cells were stained by electroshock according to a standard protocol^[142] with FlAsH (as a positive control) or with SplAsH-RhS-OF. While the yeast

mitochondria were very well stained with FlAsH, SplAsH-RhS-OF (see Figure 74) showed nearly no mitochondrial staining at all. A similar experiment which was carried out with SplAsH-TAMRA did not show any improvements compared to SplAsH-RhS-OF.

Figure 75. Confocal image of PFA (polyformaldehyde) fixed yeast cells, which were permeabilized by treatment with Triton – X100 and stained with SplAsH-RhS-OF (left picture) and the detected fluorescence of the same cells from the mitochondrial targeted m-BFP (right picture). In the right picture, we can see the bright blue mitochondrial network, which could be attributed to the cell mitochondria, but in the left picture we cannot see clear mitochondrial staining, because in this picture the entire cell is red-colored inside (without any separations of the organells).

The same yeast strain employed for the electrostaining was now fixed with 8 % PFA and afterwards permeabilized with 0.5 % Triton – X100. Neither SplAsH-RhS-OF (Figure 75) nor SplAsH-TAMRA showed any significant degree of mitochondrial staining.

Figure 76. Confocal image of the PFA-fixed yeast cells permeabilized by treatment with Triton – X100, treated with TCEP treatment and stained with the SplAsH-TAMRA. In this

picture, also the entire cells are red-colored inside (without any separations of the organelles, besides the strange tubulin-like artefacts, which look like a tubular structure inside the cells).

In the next series of experiments, the yeast cells expressing triple tetracysteine-tagged α-Tubulin was used. Then, these cells were fixed and permeabilized as before, but additionally treated with 1 mM TCEP to reduce disulfide bonds. They were afterwards stained with FlAsH, SplAsH-RhS-OF, or SplAsH-TAMRA (see Figure 76). As a negative control, a wild type strain was used. Unexpectedly, the TCEP treatment induced an α-Tubulin-like staining artefact in the yeast cells regardless of the applied dye that can also be seen in wild-type cells.

Incubation of the TetCys-mut-GFP-TetCys expressing cells mentioned before with SplAsH-RhS-OF and SplAsH-TAMRA did not result in a clear mitochondrial staining, too.

Thus, higher numbers of tetracysteine tags in the one protein and the change of the type of protein in the combination with the TCEP pre-treatment also did not help to increase the selectivity of labelling by our biarsenical probes in vivo.

Experimental Part