Annotation of the results obtained in 2018
The efficiency of labeling for number of model proteins was evaluated using bacterial coiled-coil helices and a fluorescent protein (or photo-activated fluorescent protein) with a pair helix. The most efficient pairs of helixes were selected with the help of fluorescence recovery after photobleaching and super-resolution localization fluorescence microscopy experiments. The reversibly photoactivated red fluorescent proteins based on FusionRed (rsFusionRed1, rsFusionRed2, rsFusionRed3) developed within the framework of the project have been used for RESOLFT nanoscopy of cytoskeleton proteins in living cells. It has been established that rsFusionRed2 and rsFusionRed3 proteins provide the fastest RESOLFT imaging among all published reversibly photoswitching red fluorescent proteins. Also, the possibility of using a combination of lasers with low phototoxicity (510 nm and 590 nm) for RESOLFT was shown for the first time. https://www.nature.com/articles/s41592-018-0052-9 In the framework of the developed two-component protein labeling system using fluorogen-protein pairs, a new combination of fluorogen and lipocalin was detected. A new fluorogen-protein pair exhibits fluorescence in the red region and brightness comparable to that of spectrally similar fluorescent proteins. The use of a new labeling system for fluorescence microscopy as well as for single molecule localization microscopy is shown. https://www.mdpi.com/1422-0067/19/12/3778 Under conditions of single-molecule localization microscopy, experiments were performed on the tracking of single protein molecules with membrane localization, labeled using mutant variants of photoactivated PA-GFP protein developed in this project. The PA-GFP variant providing the longest uninterrupted tracks of the labeled protein molecule movement was selected under sptPALM conditions.

 

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Annotation of the results obtained in 2016
Genetically engineered constructs, coding for fusion proteins of the proteins of interest - elements of cytoskeleton (vimentin, actin) with different sorts of bacterial coiled-coils have been constructed; as well as a palette of fluorescent reporters also carrying complement spirals within their structure. On a cell culture model, the performance of different pairs and the exchange rate were determined. The labelling efficiency of best-performing coiled-coil pairs was similar to the fluorescent protein fusions. For different photoactivatable fluorescent proteins on the FusionRed basis with substitutions in key positions 69, 148, 179 the photoconversion contrast and fatigue were assessed. Two selected reversibly switchable fluorescent proteins outperformed some of popular red photoswitchable fluorescent proteins. Fluorogen-protein pairs suitable for live-cell protein labelling were selected. Lipocalin mutants capable of reverse binding of synthetic fluorogenic dye, exhibit high photostability in fusions with cytoskeleton proteins, outperforming fluorescent proteins. Genetically engineered constructs for the evaluation of single-molecule and photostability performance of PA-GFP mutants were constructed. The photostability and the photon budgets were estimated in live-cell experiments.

 

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Annotation of the results obtained in 2017
Under the conditions of single-molecule localization microscopy, the efficiency of caveolin labeling with bacterial coiled-coils and photoactivated fluorescent protein with a complement helix was evaluated. Due to the transient nature of the interaction of the helices, a constant exchange of the fluorescent protein occurs between the target protein and the intracellular pool. As a result, a high density of labeling of the target structure is achieved, sufficient for the reconstruction of super-resolution images. Developed reversibly photo-activated red fluorescent proteins based on FusionRed showed effective switching between dark and fluorescent states under confocal and wide-field fluorescence microscopy. It has also been found that some widely used red fluorescent proteins, including FusionRed and TagRFP, exhibit reversible photoconversion to the non-fluorescent state. In this case, the return to the fluorescent state occurs spontaneously, and at steady-state irradiation of tens of watts per square centimeter, stable fluctuations in the signal can be maintained. Such fluctuations turned out to be possible to use for reconstruction of super-resolution imaging under moderate irradiation intensities, suitable for live cells. http://pubs.rsc.org/en/content/articlelanding/2017/cc/c6cc09200d A two-component protein labeling system was developed utilizing pairs of fluorogen and protein. In this case, the genetically coded reporter based on the bacterial lipocalin reversibly binds the low molecular weight fluorogen, which easily penetrates into the living cell. The constant fluorogen exchange allows for controlling the density of labeling of target structures in single-molecule localization microscopy by changing the fluorogen concentration. Also, the fluorogen exchange improves photostability in wide-field fluorescence microscopy, localization super-resolution microscopy, as well as STED-microscopy http://www.ibch.ru/en/press/news/science/2143 http://pubs.rsc.org/en/content/articlelanding/2017/sc/c7sc01628j#!divAbstract Target proteins have been selected and corresponding vectors have been created with them to test the developed photoactivated fluorescent proteins optimized for quantitative modalities of super-resolution localization microscopy, including single particle tracking PALM (sptPALM).

 

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