La découverte des Protéines Fluorescentes Phototransformables (PTFPs) issuesd’espèces anthozoaires a ouvert, grâce à leurs propriétés photophysiques particulières, unvaste champ d’investigation pour l'imagerie biologique de fluorescence. L'un des sousgroupesdes PTFPs est formé des protéines fluorescentes réversiblement photocommutables(RSFPs), qui peuvent être commutées réversiblement entre des états non-fluorescent etfluorescent. Le photoblanchiment est la perte définitive d’émission de fluorescence sousexcitation et est un phénomène commun à toutes les molécules fluorescentes. Lephotoblanchiment a un impact important sur la qualité des images de microscopie, notammenten imagerie de super-résolution. Les RSFPs ont tendance à perdre de leur performance àchaque cycle de commutation, un processus dénommé “photofatigue”. Notre intérêt est centrésur l'étude des mécanismes de photofatigue des RSFPs.Nous avons rapporté les structures cristallographiques d’IrisFP photoblanchie par uneforte et une basse intensité d’illumination à température ambiante ainsi que les modificationsspectroscopiques associées. Nos résultats démontrent que différentes intensités d'excitationpeuvent donner lieu à différentes voies de photoblanchiment. Sous faible intensité d'excitation,une voie de photoblanchiment dépendante de l'oxygène a été mise en évidence. Lesmodifications structurales induites par la production d'oxygène singulet à l'intérieur de lapoche du chromophore ont révélé l'oxydation de deux résidus soufrés, Met159 et Cys171,piégeant le chromophore dans un état protoné non-fluorescent. Sous haute intensitéd'excitation, une voie de photoblanchiment oxygène-indépendante totalement différente a ététrouvée. Le Glu212, strictement conservé, subit une décarboxylation associée à un importantréarrangement du réseau de liaisons hydrogènes autour du chromophore, et un changementd’hybridation sp2 vers sp3 du carbone reliant les cycles du chromophore est observé. En tantque résidu clé impliqué dans le photoblanchiment induit par faible intensité d'excitation, nousavons muté Met159 en alanine afin d'éviter une sulfoxydation. Nous avons trouvé que lemutant IrisFP-M159A démontre une photostabilité améliorée en solution, en gel PVA et dansdes cellules E. coli. / The discovery of phototransformable FPs (PTFPs) from Anthozoa species, thanks totheir photophysical properties, has opened a large field in biological fluorescence imaging.One of the PTFPs’ sub-groups consists of Reversible Photoswitchable Fluorescent Proteins(RSFPs), which can be reversibly switched between nonfluorescent and fluorescent states.Photobleaching is the permanent loss of the fluorescence-emitting capacity under excitation,which is a common phenomenon among all the fluorescent molecules. Photobleaching has alarge impact on the microscopy image quality, notably on super-resolution imaging.Photoswitchable fluorescent proteins have a tendency to lose performance within everyswitching cycle, a process referred to as “photofatigue”. Our interest of study is focused onthe photobleaching mechanisms of RSFPs.We have reported the crystallographic structure of photobleached IrisFP under highand low illumination intensity at room temperature as well as its spectroscopic modifications.We found that different illumination intensities can result in different photobleachingpathways. Under low illumination intensity, an oxygen-dependent photobleaching pathwaywas evidenced. Structural modifications induced by singlet-oxygen production within thechromophore pocket revealed the oxidation of two sulfur-containing residues, Met159 andCys171, locking the chromophore in a nonfluorescent protonated state. Under highillumination intensity, a completely different, oxygen-independent photobleaching pathwaywas found. The conserved Glu212 underwent decarboxylation concomitantly with anextensive rearrangement of the H-bond network around the chromophore, and an sp2-to-sp3hybridization change of the carbon atom bridging the chromophore cyclic moieties wasobserved. As Met159 is the key residue involved in low-intensity illumination photobleaching,we have then mutated Met159 into Alanine in order to avoid sulfoxidation. We found that theIrisFP-M159A mutant display an enhanced photostability in solution, in PVA gel and inE.coli cells.

Foram utilizadas proteínas da famÌlia das GFPs e nanocristais fluorescentes de CdSe/ZnS para caracterização da interação e verificação de transferência de energia por ressonância (FRET) entre estes compostos. Formou-se dois pares doador-receptor onde ora uma proteína figurava como doadora, ora um nanocristal ocupava este papel. Verificou-se que, em ambos os casos, o doador sofre supressão da fluorescência após a formação de complexo com o receptor, complexo este motivado por interação eletrostática e dependente de pH. Foi possível comprovar, através da observação de emissão sensitizada e redução da anisotropia, que entre o par formado por nanocristal com emissão no verde e proteína HcRed1 como receptora, de fato ocorre FRET. As distâncias aparentes entre doador e receptor foram determinadas a partir da eficiência da supressão da fluorescência do doador e da distância de Förster. As distâncias assim obtidas são compatíveis com as dimensões das proteínas e dos nanocristais / Proteins belonging to the GFP family were used to characterize their interaction with fluorescent CdSe/ZnS nanocrystals and to verify the occurrence of resonance energy transfer (FRET) among these elements. Two donor-acceptor pairs were established, one having a protein as donor and the other having a nanocrystal as donor. In both cases the donor suffers quenching of its fluorescence after the formation of a complex with the acceptor. The complex formation is dependent on pH and is due to electrostatic interaction. It was possible to prove the occurrence of FRET between CdSe/ZnS nanocrystals emitting green fluorescence as donors and the protein HcRed1 as acceptor, through the detection of sensitized emission and anisotropy reduction. Apparent donor-acceptor distances were determined from efficiency measurements and Förster distances. The obtained distances agreed with the protein and nanocrystal dimensions

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Red fluorescent proteins (RFPs) are used extensively in biological research because their longer emission wavelengths are less phototoxic and allow deeper imaging of animal tissue. However, far-red RFPs generally display low brightness, emphasizing the need to develop brighter variants. Here, we investigate three approaches to rigidify the RFP chromophore to increase the quantum yield, and thereby brightness. We first used computational protein design on a maturation-efficient mRojo-VHSV variant previously engineered in our lab to introduce a Superdecker motif, a parallel pi-stack comprising aromatic residue side chains and the phenolate moiety of the chromophore, which we hypothesized would enhance chromophore packing and reduce non-radiative decay. The best mutants identified showed up to 1.7-fold higher quantum yield at pH 9, relative to their parent protein. We next postulated that brightness could be further increased by rigidifying the chromophore via branched aliphatic residues. Computational protein design was performed on a dim mCherry variant, mRojoA, followed by directed evolution on the brightest mutant. The combination of these methodologies yielded mSandy2, the brightest Discosoma-derived monomeric RFP with an emission maximum above 600 nm. Finally, we aimed to increase brightness by focusing on positions where residue rigidity correlated to quantum yield in mCherry-related RFPs according to NMR data that had been previously acquired in our lab. Combinatorial site-saturation mutagenesis was performed on two different surface patches of mCherry at positions 144/145/198 and 194/196/220. Our results demonstrated that surface residues may not be adequate targets for this approach. Altogether, the work herein presents unique rational design methodologies that can be used to increase brightness in RFPs.

L'epicocconone est un produit naturel tricyclique, de la famille des azaphilones, isolé en 2003 d'un champignon Epicoccum nigrum. Ce composé se lie de façon covalente aux amines, conduisant à la formation d'une énamine fluorescente. Cette réaction, réversible en fonction du pH, fait de ce composé un excellent marqueur de protéines pour la détection sur gels d'électrophorèse compatible avec une analyse de spectrométrie de masse. La synthèse de ce produit naturel a été débutée au sein de notre laboratoire en s'appuyant sur les travaux réalisés précédemment et mettant en jeu une étape clé de désaromatisation oxydante à l'aide d'iode hypervalent. Une étude méthodologique de réaction clé a permis de comparer l'efficacité et la diastéréosélectivité de l'oxydation effectuée par l'iode (III) ou l'iode (V). / Epicocconone is a tricyclic natural product of the azaphilone family, isolated from the fungus Epicoccum nigrum. This compound covalently binds to primary amines, leading to a protein conjugate which is highly fluorescent. This reaction, reversible according to the pH, make this compound an excellent protein stain compatible with mass spectrometry analysis. The synthesis of this natural product has been started in our laboratory based on the previous work in involves a key oxidative dearomatization using hypervalent iodine. Methodological studies of this key reaction allowed us to compare the efficiency and diastereoselectivity of iodine (III) and iodine (V) mediated oxidations.

Title: Imaging of fluorescently labelled mitochondria using Biplane FPALM microscopy Author: Bc. Marek Dostál Department: Fyzikální ústav UK Supervisor: prof. RNDr. Jaromír Plášek, CSc. Consultants: RNDr. Petr Ježek, DrSc. Mgr. Hana Engstová, Ph.D. Abstract: In this thesis the results are presented of a search for suitable PCFP markers for a visualization inner and outer mitochondrial membranes by unique biplane FPALM microscope. We participated in debugging the measuring software for the purpose of 3D vizualization mitochondrial nets and determine their parameters. For this purpose were developed two methods. The first one can be applied to determine the parametrs of outer membranes and the second one is applicable to determine parametrs of inner membranes. On smaller statistical file we managed to confirm dependence quality of mitochondrial net on the cell culture conditions. Under stress conditions e.g. hypoxia the quality of mitochondrial net is changed. Keywords: mitochondria, Biplane FPALM, GFP, 3D visualization.

abstract: Proteins are, arguably, the most complicated molecular machines found in nature. From the receptor proteins that decorate the exterior of cell membranes to enzymes that catalyze the slowest of chemical reactions, proteins perform a wide variety of essential biological functions. A reductionist view of proteins as a macromolecular group, however, may hold that they simply interact with other chemical species. Notably, proteins interact with other proteins, other biological macromolecules, small molecules, and ions. This in turn makes proteins uniquely qualified for use technological use as sensors of said chemical species (biosensors). Several methods have been developed to convert proteins into biosensors. Many of these techniques take advantage of fluorescence spectroscopy because it is a fast, non-invasive, non-destructive and highly sensitive method that also allows for spatiotemporal control. This, however, requires that first a fluorophore be added to a target protein. Several methods for achieving this have been developed from large, genetically encoded autofluorescent protein tags, to labeling with small molecule fluorophores using bioorthogonal chemical handles, to genetically encoded fluorescent non-canonical amino acids (fNCAA). In recent years, the fNCAA, L-(7-hydroxycoumarin-4yl)ethylglycine (7-HCAA) has been used in to develop several types of biosensors. The dissertation I present here specifically addresses the use of the fNCAA L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) in protein-based biosensors. I demonstrate 7-HCAA’s ability to act as a Förster resonance energy transfer (FRET) acceptor with tryptophan as the FRET donor in a single protein containing multiple tryptophans. I the describe efforts to elucidate—through both spectroscopic and structural characterization—interactions within a 7-HCAA containing protein that governs 7-HCAA fluorescence. Finally, I present a top-down computational design strategy for incorporating 7-HCAA into proteins that takes advantage of previously described interactions. These reports show the applicability of 7-HCAA and the wider class of fNCAAs as a whole for their use of rationally designed biosensors. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020

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No description available.

Antisense RNA (asRNA) strategies are identified as an effective and specific method for gene down-regulation at the post-transcriptional level. In this study, the major purpose is to find a correlation between the expression level and minimum free energy to enable the design of specific asRNA fragments. The thermodynamics of asRNA and mRNA hybridization were computed based on the fluorescent protein reporter genes. Three different fluorescent proteins (i) green fluorescent protein (GFP), (ii) cyan fluorescent protein (CFP) and (iii) yellow fluorescent protein (YFP) were used as reporters. Each fluorescent protein was cloned into the common pUC19 vector. The asRNA fragments were randomly amplified and the resulted antisense DNA fragments were inserted into the constructed plasmid under the control of an additional inducible plac promoter and terminator. The expression levels of fluorescent reporter protein were determined in real time by plate reader. Different results have been observed according to the fluorescent protein and the antisense fragment sequence. The CFP expression level was decreased by 50 to 78% compared to the control. However, with the GFP, the down-regulation did not exceed 30% for the different constructs used. For certain constructs, the effect was the opposite of expected and the expression level was increased. In addition, the YFP showed a weak signal compared to growth media, therefore the expression level was hard to be defined. Based on these results, a thermodynamic model to describe the relationship between the particular asRNA used and the observed expression level of the fluorescent reporter was developed. The minimum free energy and binding percentage of asRNA-mRNA complex were computed by NUPACK software. The expression level was drawn as a function of the minimum free energy. The results showed a weak correlation, but linear trends were observed for low energy values and low expression levels the CFP gene. The linear aspect is not verified for higher energy values. These findings suggest that the lower the energy is, the more stable is the complex asRNA-mRNA and therefore more reduction of the expression is obtained. Meanwhile, the non-linearity involves that there are other parameters to be investigated to improve the mathematical correlation. This model is expected to offer the chance to "fine-tune" asRNA effectiveness and subsequently modulate gene expression and redirect metabolic pathways toward the desired component. In addition, the investigation of the localization of antisense binding indicates that there are some regions that favors the hybridization and promote hence the down-regulation mechanisms. / Master of Science