Maximum Emission Wavelength Research Articles

Calcium Carbonate as an Ionic Molecular Lock for Ultrastrong Fluorescence of Single Organic Molecules

Locking molecular conformation are widely applied in molecular engineering for improved performance. However, locking via organic functional groups often changes the original molecular properties. Following the rigidity and stability of ionic interaction in ionic compounds, we suggested the use of a molecular‐scale ionic compound, calcium carbonate oligomer, as a robust molecular segment to functionalize organic molecules. The rigid structure of the ionic molecular segments locked the organic molecules, which could remarkably limit the intramolecular motion and intermolecular interactions. This ensured a stable and ultrastrong fluorescence of the single organic molecule while preserving its original maximum emission wavelength. The locking strategy was general and extendable to multiple organic molecules. Additionally, the ultrastrong single‐molecular fluorescence can be maintained in inorganic solids with even higher quantum yields and almost unchanged maximum emission wavelength. The highest quantum yield of the investigated molecules reached 99.9%, superior to all reported organic‒inorganic fluorescent composite under air conditions. This work demonstrates a general strategy to restrict intramolecular motion and intermolecular interactions by using ionic oligomers as molecular locks, providing an alternative method for realizing ultraemissive molecules. This further demonstrates a fascinating example of molecular engineering in the presence of inorganic ionic molecules.

Calcium Carbonate as an Ionic Molecular Lock for Ultrastrong Fluorescence of Single Organic Molecules.

Locking molecular conformation are widely applied in molecular engineering for improved performance. However, locking via organic functional groups often changes the original molecular properties. Following the rigidity and stability of ionic interaction in ionic compounds, we suggested the use of a molecular-scale ionic compound, calcium carbonate oligomer, as a robust molecular segment to functionalize organic molecules. The rigid structure of the ionic molecular segments locked the organic molecules, which could remarkably limit the intramolecular motion and intermolecular interactions. This ensured a stable and ultrastrong fluorescence of the single organic molecule while preserving its original maximum emission wavelength. The locking strategy was general and extendable to multiple organic molecules. Additionally, the ultrastrong single-molecular fluorescence can be maintained in inorganic solids with even higher quantum yields and almost unchanged maximum emission wavelength. The highest quantum yield of the investigated molecules reached 99.9%, superior to all reported organic‒inorganic fluorescent composite under air conditions. This work demonstrates a general strategy to restrict intramolecular motion and intermolecular interactions by using ionic oligomers as molecular locks, providing an alternative method for realizing ultraemissive molecules. This further demonstrates a fascinating example of molecular engineering in the presence of inorganic ionic molecules.

Changes in Texture and Collagen Properties of Pork Skin during Salt-Enzyme-Alkali Tenderization Treatment.

The effects of salt-enzyme-alkali progressive tenderization treatments on porcine cortical conformation and collagen properties were investigated, and their effectiveness and mechanisms were analyzed. The tenderization treatment comprised three treatment stages: CaCl2 (25 °C/0-30 min), papain (35 °C/30-78 min), and Na2CO3 (25 °C/78-120 min). The textural, microscopic, and collagenous properties (content, solubility, and structure) of pork skin were determined at the 0th, 30th, 60th, 90th, and 120th min of the treatment process. The results showed that the shear force, hardness, and chewability of the skin decreased significantly (p < 0.05), and the elasticity exhibited a gradual increase with the progression of tenderization. The content and solubility of collagen showed no significant change at the CaCl2 treatment stage. However, the soluble collagen content increased, the insoluble collagen content decreased, and the collagen solubility increased by 18.04% during the subsequent treatment with papain and Na2CO3. Meanwhile, the scanning electron microscopy results revealed that the regular, wavy structure of the pig skin collagen fibers gradually disappeared during the CaCl2 treatment stage, the overall structure revealed expansion, and the surface microscopic pores gradually increased during the papain and Na2CO3 treatment stages. The findings of the Fourier transform infrared spectroscopy analysis indicated that the hydrogen bonding interactions between the collagen molecules and the C=O, N-H and C-N bonds in the subunit structure of collagen were substantially altered during treatment and that the breakage of amino acid chains and reduction in structural ordering became more pronounced with prolonged treatment. In the tertiary structure, the maximum emission wavelength was blue-shifted and then red-shifted, and the fluorescence intensity was gradually weakened. The surface hydrophobicity was slowly increased. The salt-enzyme-alkali tenderization treatment considerably improved the physical properties and texture of edible pork skins by dissolving collagen fibers and destroying the structure of collagen and its interaction force.

Near-infrared probes based on the phenanthridinium for dynamic monitoring of viscosity fluctuations during mitophagy

Abnormal changes in the mitochondrial microenvironment can cause a variety of diseases. Consequently, based on the twisted intramolecular charge transfer (TICT) theory, three hemicyanine fluorescent probes 1a-c were designed and developed by linking the phenanthridine group with different electron donor groups for the dynamic detection of mitochondrial viscosity in this paper. They have the advantages of near-infrared emission, excellent pH stability and viscosity sensitivity. Optical experiments showed that the maximum absorption and emission wavelengths of the probes in different solvents located at 411 − 529 nm and 623 − 684 nm with large Stokes shifts of 133 – 230 nm. In addition, when the viscosity increased from 0.89 cP to 856 cP, their fluorescence intensities in glycerol enhanced by 115-fold, 83-fold and 303-fold compared with water, respectively. The fluorescence intensities of probes showed excellent fit to the Forster Hoffmann equation, and exhibited nearly OFF-ON response to viscosity. Moreover, the probe 1a with good photostability and low cytotoxicity could target mitochondria. It was verified that 1a also could detect variation in mitochondrial viscosity during autophagy by modelling starvation and drug-induced (rapamycin and nystatin) in HeLa cells. Therefore, this study provides a useful probe for the analysis of mitophagy process in HeLa cells by the viscosity variation of mitochondria.

Marine eukaryote bioluminescence: a review of species and their functional biology

AbstractBioluminescence, the ability of organisms to produce visible light, has intrigued scientists for centuries. Studies have examined bioluminescence, using a wide range of approaches and organisms, from its ecological role to its underlying molecular mechanisms, leading to various applications and even a Nobel prize. Over the last ten years, an increasing amount of data has been collected leading to a growing number of recognized marine bioluminescent species. This review provides and describes a referenced listing of the eukaryotic luminous marine species, including information related to: (i) intrinsic versus extrinsic source of the bioluminescence, (ii) the color and maximum wavelength of emission, (iii) the bioluminescent system (substrate and enzyme) and the associated molecules, (iv) the availability of light organ/cell(s) pattern and histological structure, (v) the physiological control of the light production, and (vi) the demonstrated or suggested bioluminescent function(s). This listing provides basic information and references for researchers in or entering in the field of marine bioluminescence. Using a semi-quantitative approach, we then highlight major research gaps and opportunities and reflect on the future of the field.

A Water‐Soluble Fluorescent Probe Derived from Pyridine‐2,6‐Dicarboxylic Acid: Synthesis, Interaction with Ions, and Two‐Photon Microscopy

AbstractTwo‐photon excited fluorescence is a process in which the excited fluorophore relaxes and emits fluorescence after two‐photon absorption. Combined with appropriate two‐photon fluorescent probes, two‐photon microscopy enables observation deep inside tissue without damage. Former reports showed that the two‐photon absorption cross sections of donor–acceptor (D–A) dipoles, as well as D‐π‐D and D–A–D quadrupoles can be strengthened by using strong D–A groups. In this study, a novel water‐soluble fluorescent probe with D‐π‐A structure containing pyridine‐2,6‐dicarboxylic acid unit was synthesized. The maximum excitation wavelength and emission wavelength of the probe in aqueous solution were 328 nm and 471 nm, respectively. The fluorescence emitted by the probe can be quenched by various metal cations. The pyridyl N and carboxyl O in the probe formed chelates with Cu2+, which resulted in a fluorescence quenching response, and showed a linear relationship between fluorescent intensity and Cu2+ concentration with wide linear range and low limit of detection. CCK‐8 tests showed the probe had no obvious toxicity to 4T1 cells in the range of 0.4–50 μmol L−1, and the cell survival rate was above 86 %. The probe had a large two‐photon absorption cross section ranging from 680 nm to 820 nm. The two‐photon absorption cross section reached 100 GM at 760 nm of excitation wavelength. By means of microscopic imaging of 4T1 cells stained with the probe, one‐photon fluorescence represented blue emission, while two‐photon fluorescence represented bright green emission. The probe was proved to have good cell permeability, and preferred to target nucleus, showing application potential in two‐photon fluorescence imaging technology.

Glycosylated gelatin prepared based on electron beam irradiation and its physicochemical properties

The influence of electron beam irradiation (EBI) treatment on the modification of gelatin-galactose glycosylation was thoroughly examined. The results of the degree of grafting and browning revealed that EBI triggered the glycosylation reaction of gelatin. The degree of glycosylation exhibited a gradual increase with the rising irradiation dose, reaching a maximum of 25 kGy. Moreover, the irradiation process opened up gelatin's internal structure, exposing its hydrophobic groups. This exposure led to an enhancement in sample surface hydrophobicity. The fluorescence intensity at the maximum emission wavelength of the fluorescence spectra decreased; Fourier infrared spectroscopy demonstrated a new absorption peak at 1074 cm−1 for the glycosylation product. These findings substantiate that gelatin formed a new product through covalent bonding with galactose. Glycosylation boosted the emulsification stability of gelatin from 1.92 min to 10.42 min and improved its emulsification and rheological properties. These outcomes affirm that EBI can effectively induce the glycosylation reaction of gelatin, thereby enhancing its functional properties. In addition, EBI has the potential to supplant the conventional heating glycosylation method. This study lays a solid theoretical foundation for the future application of glycosylation and gelatin.

Post-functionalization of alternating π-conjugated copolymers containing fluorene moieties via anodic chlorination using AlCl3

AbstractFluorene (Fl) derivatives are representative emitting motifs; thus, they are often installed into alternating π-conjugated copolymers (P(Fl-Ar)) as soluble polymeric emitters. Many researchers have focused on modifying the combined arylene units in P(Fl-Ar) derivatives to tune their optoelectronic properties; however, P(Fl-Ar) derivatives that contain fluorene units with functional groups at their sp2 carbons remain limited. Here, we synthesize P(Fl-Ar) derivatives comprising sp2-chlorinated fluorene units via anodic chlorination using aluminum chloride (AlCl3). The introduced chlorine atoms affect the optoelectronic properties of the pristine P(Fl-Ar) derivatives. Compared with the precursor P(Fl-Ar) derivatives, chlorinated P(Fl-Ar) derivatives exhibit longer maximum emission wavelengths.

Green synthesis of carbon dots from Hakka yellow wine lees: Characterization and applications for Fe3+ and NaFeEDTA sensing

In this study, Hakka yellow wine lees carbon dots (HYWL-CDs) with an average diameter of 1.75 nm are synthesized from Hakka yellow wine lees (HYWL) via a one-pot hydrothermal method. Results of X-ray photoelectron spectroscopy and FTIR reveal the presence of C=C and –OH in the HYWL-CDs. Additionally, the HYWL-CDs present excitation-wavelength dependence, with maximum excitation and emission wavelengths of 360 and 440 nm, respectively. The fluorescence of the HYWL-CDs can be quenched by Fe3+. The Fe3+ concentration is correlated linearly with the quenching rate over a linear range of 0.6–1.1 mg/mL, and the limit of detection is 0.18 μg/mL. And phenols have been shown to be present in HYWL-CDs, and the color reaction between phenols and FeCl3 is most likely the mechanism by which FeCl3 causes fluorescence burst in HYWL-CDs. Moreover, the HYWL-CDs demonstrate significant potential for detecting NaFeEDTA in iron-fortified soy sauces, the linear-fit equation was y = 185.38x + 1024.6 (R2 = 0.9995) The HYWL-CDs are loaded onto paper strips, iron-fortified straw mushroom dark soy sauce showed noticeable color changes, which allows one to differentiate normal soy sauce from iron-fortified soy sauce more conveniently and provides a new perspective for the high-value utilization of yellow wine lees.

Effect of electron beam irradiation on glycosylation reaction and structural characterization of whey isolate protein.

Whey protein isolate (WPI) is a high-quality animal protein resource. The modification of WPI through physical, chemical and biological methods can substantially improve the functional properties of proteins. This study investigated the effect of electron beam irradiation (EBI) on the modification of WPI-xylose glycosylation. The degree of grafting and browning revealed that EBI promoted WPI glycosylation. The maximum emission wavelength of intrinsic fluorescence was red-shifted and the fluorescence intensity was reduced, suggesting that irradiation induced the unfolding of the WPI structure, thereby promoting glycosylation. Fourier-transformed infrared spectroscopy revealed that the covalent binding of the conjugates occurred on the introduction of the hydrophilic groups, resulting in decreased surface hydrophobicity. When compared with conventional wet-heat glycosylation, irradiation-assisted glycosylation improved the emulsifying activity of WPIfrom 179.76 ± 0.83 to 277.83 ± 1.44 m2 g-1, and the emulsifying and rheological properties improved. These results confirmed that EBI can increase the degree of WPI glycosylation and improve the functional properties of proteins, thereby laying a theoretical foundation for the further application of WPI. © 2024 Society of Chemical Industry.

Red/near-infrared (NIR) difluoroboron β-diketonate derivatives with reversible mechanochromism for cellular imaging

Near-infrared (NIR) fluorophores have promoted the development of materials for bioimaging, but traditional NIR dyes usually suffer from aggregation-caused quenching (ACQ), impeding their applications. Herein, we propose two difluoroboron β-diketonate complexes TBO and TBS, consisting a donor–acceptor (D-A) structure with triphenylamine (TPA) moiety as an electron donors and difluoroboron as well as furan or thiophene building block as an electron acceptor. The theoretical calculation and optical data shows that both of them have intramolecular charge transfer (ICT) characteristics. Such ICT characteristics endow them with both solvatochromism and dual-state emission (DSE) properties. In the solvent CH2Cl2, the emission wavelength of TBO ranges from 550 nm to 750 nm, with a low fluorescence quantum yield (Φ = 7.0 %). However, in the less polar solvent hexane, the emission wavelength blue-shifts, with an increased Φ reaching up to 18 %. Moreover, TBO and TBS exhibit mechanochromic characteristics and rare multi-channel fluorescence emission phenomena at solid-state. Their solid-state samples can emit fluorescence in four spectral bands with maximum emission wavelengths at 300 nm, 400 nm, 600 nm, and 770 nm under excitation at 240 nm. These unique optical properties are expected to be utilized for detecting polarity of system and deformation. Moreover, according to the results of cell imaging and flow cytometry, TBO molecular were easily internalized into Hela cells and distributed in the cytoplasm with strong red fluorescence. Therefore, this research inspires more insight into development of NIR luminogens for biomedical imaging.

Constructing soybean protein isolate /bacterial cellulose co-assemblies by pH shifting treatment: Molecular conformation and physicochemical properties

The study elucidates that the pH shifting treatment unfolds the conformation of soybean protein isolate (SPI), enabling it to intertwine with bacterial cellulose (BC) and form SPI/BC co-assemblies. Results from intrinsic fluorescence spectroscopy and surface hydrophobicity indicate that the SPI with pH shifting treatment shows a notable blue shift in maximum emission wavelength and increased surface hydrophobicity. It demonstrates that pH shifting treatment facilitates the unfolding of SPI's molecular conformation, promoting its entanglement with high aspect ratio BC. Particle size distribution and microstructural analysis further demonstrate that the pH shifting treatment facilitates the formation of SPI/BC co-assemblies. Evaluation of processing properties reveals that the SPI/BC co-assemblies exhibited exceptional gel and emulsification properties, with gel strength and emulsifying activity respectively six and two times higher than natural SPI. This enhancement is attributed to the thickening properties of BC with a high aspect ratio and the superior hydrophobicity of SPI in its molten globule state.

Development of a Pyrone-Fused Tricyclic Scaffold-based Ratiometric Fluorescent Probe for Al3+ Detection.

Aluminum (Al3+) is environmentally abundant and can harm living organisms in various ways, such as by inhibiting root growth, damaging faunal nervous systems, and promoting tumor cell proliferation. However, the dynamics of Al3+ in living organisms are largely unknown; thus, detecting Al3+ in the environment and organisms is crucial. Fluorescent probes are useful tools for the selective detection of metal ions. In particular, ratiometric fluorescent probes exhibit a detection response at two different maximum fluorescence emission wavelengths; which is advantageous for avoiding the influence of background fluorescence. A novel pyrone-fused tricyclic scaffold-based ratiometric fluorescent probe for detecting Al3+, ethyl 11-imino-1-oxo-3-phenyl-1H,11H-pyrano[4,3-b] quinolizine-5-carboxylate (PQ), was developed in this study. The PQ fluorescence blue shifted from 505 to 457nm upon the addition of Al3+. The blue shift was accompanied by a change in the fluorescence color of the PQ solution from green to blue. Fluorescence titration experiments demonstrated that the fluorescence intensity ratio at the two peaks of interest (457/505nm) increased in a concentration-dependent manner upon the addition of Al3+. Moreover, this study demonstrated that a PQ-soaked paper displays a visible color change under ultraviolet light upon exposure to Al3+. The above results suggest that PQ is an effective ratiometric probe for the detection of Al3+ in the environment. Future studies will be conducted to introduce various substituents and develop fluorescent probes by leveraging the fluorescence property of a pyrone-fused tricyclic scaffolds.

Enhancement of non-covalent interaction between soy protein isolate and quercetin by sodium alginate

Effects of sodium alginate (SA) on the non-covalent interaction between soybean protein isolate (SPI) and quercetin (Que) were investigated by multispectral technology, molecular docking and dynamics simulation technology. Structural alterations of the binary complexes were observed after SA addition, characterized by a red shift of maximum fluorescence emission wavelength. The introduction of 0.1% (w/v) SA led to a reduction of 12.3% in the α-helix and β-sheet structures, accompanied by 12.6% increase in the β-turn and random coil conformations. The binding of SA to SPI provided electrostatic interactions and facilitated the subsequent binding of SPI to Que. Molecular docking confirmed that hydrophobic interactions and electrostatic interactions were also the main driving force. Molecular dynamics simulation emphasized that the ternary complexes with SA exhibited greater stability compared to the binary ones. The foaming and emulsifying properties of SPI-Que complexes were enhanced by 33.76% and 68.28%, respectively, due to the addition of SA.

Near-infrared polarity fluorescent probe for wash-free imaging lipid droplets and the application in the diagnosis of non-alcoholic fatty liver tissue

Lipid droplets (LDs), as primary cellular energy storage organelles, play essential roles not only in energy conservation but also in inflammatory responses, apoptosis, and the progression of diseases such as non-alcoholic fatty liver disease (NAFLD). Studies indicate that alterations in the polarity of LDs are closely linked to pathological factors like oxidative stress, ferroptosis, and lipophagy. Therefore, developing biological probes for precisely monitoring LDs polarity is crucial for understanding these biological phenomena and devising new therapeutic strategies. In this context, we synthesized four fluorescent probes (CNL1-CNL4) to precisely monitor the polarity of LDs using triphenylamine and carbazole derivatives as electron donors and isophorone and benzopyranonitrile units as electron acceptors, employing intramolecular charge transfer (ICT) mechanism. Our findings demonstrated that all four probes exhibited high sensitivity and specificity for polarity changes. Probes CNL2 and CNL4 were sensitive to polarity, and their maximum fluorescence intensity and maximum emission wavelength demonstrated a good linear relationship with polarity parameters within a certain range. Biological experiments demonstrated that both CNL2 and CNL4 possess excellent wash-free imaging capabilities and superior LDs targeting abilities but also enable dynamic monitoring of LDs and respond well to changes in LDs polarity under varying concentrations of oleic acid stimulation. Due to its longer emission wavelength (>650 nm), boasting excellent photostability, and featuring a larger Stokes shift (≈168 nm in acetonitrile), probe CNL4 was selected for further studies. Furthermore, probe CNL4 effectively monitors changes in LDs polarity under conditions of ferroptosis and oxidative stress. It has shown potential in the diagnosis of NAFLD tissue and during the process of lipophagy, highlighting its value in disease diagnostics and biological research. These findings not only deepen our understanding of the mechanisms underlying NAFLD progression but also provide new tools for diagnosing and treating related diseases.

Stapling Cysteine[2,4] Disulfide Bond of α-Conotoxin LsIA and Its Potential in Target Delivery.

α-Conotoxins, as selective nAChR antagonists, can be valuable tools for targeted drug delivery and fluorescent labeling, while conotoxin-drug or conotoxin-fluorescent conjugates through the disulfide bond are rarely reported. Herein, we demonstrate the [2,4] disulfide bond of α-conotoxin as a feasible new chemical modification site. In this study, analogs of the α-conotoxin LsIA cysteine[2,4] were synthesized by stapling with five linkers, and their inhibitory activities against human α7 and rat α3β2 nAChRs were maintained. To further apply this method in targeted delivery, the alkynylbenzyl bromide linker was synthesized and conjugated with Coumarin 120 (AMC) and Camptothecin (CPT) by copper-catalyzed click chemistry, and then stapled between cysteine[2,4] of the LsIA to construct a fluorescent probe and two peptide-drug conjugates. The maximum emission wavelength of the LsIA fluorescent probe was 402.2 nm, which was essentially unchanged compared with AMC. The cytotoxic activity of the LsIA peptide-drug conjugates on human A549 was maintained in vitro. The results demonstrate that the stapling of cysteine[2,4] with alkynylbenzyl bromide is a simple and feasible strategy for the exploitation and utilization of the α-conotoxin LsIA.

The controllable synthesis of multi-color carbon quantum dots modified by polythiophene and their application in fluorescence detection of Au3+ and Hg2+

Herein, hydrothermal method was used to prepare a series of multi-color polythiophene modified carbon quantum dots. Under UV excitation, fluorescence their maximum emission wavelengths appear at 612 nm, 570 nm, and 540 nm respectively. The prepared CD-BTH and CD-BN can have specific detection of Au3+ and Hg2+ through fluorescence quenching effect. The detection limits for Au3+ are 3 nM and 5.4 nM respectively, and for Hg2+ are 23 nM and 90 nM respectively. CD-KN detects Au3+ specifically through fluorescence resonance, with a detection limit of 33 nM. Under the interference of other metal ions, three types of polythiophene modified quantum carbon dots exhibit excellent selectivity for the responsive ions. Meanwhile, this article also elucidates the law that as the electron withdrawing ability of the side chains of polythiophene derivatives increasing, the fluorescence emission peaks of the prepared polythiophene modified carbon dots shifts red and the fluorescence quantum yield is higher.

A ratiometric fluorescence and colorimetry dual-signal sensing strategy based on o-phenylenediamine and AuNCs for determination of Cu2+ and glyphosate.

A ratiometric fluorescence sensing strategy has been developed for the determination of Cu2+ and glyphosate with high sensitivity and specificity based on OPD (o-phenylenediamine) and glutathione-stabilized gold nanoclusters (GSH-AuNCs). Water-soluble 1.75-nm size GSH-AuNCs with strong red fluorescence and maximum emission wavelength at 682nm were synthesized using GSH as the template. OPD was oxidized by Cu2+, which produced the bright yellow fluorescence oxidation product 2,3-diaminophenazine (DAP) with a maximum fluorescence emission peak at 570nm. When glyphosate existed in the system, the chelation between glyphosate and Cu2+ hindered the formation of DAP and reduced the fluorescence intensity of the system at the wavelength of 570nm. Meanwhile, the fluorescence intensity at the wavelength of 682nm remained basically stable. It exhibited a good linear relationship towards Cu2+ and glyphosate in water in the range 1.0-10 µM and 0.050-3.0µg/mL with a detection limit of 0.547 µM and 0.0028µg/mL, respectively. The method was also used for the semi-quantitative determination of Cu2+ and glyphosate in water by fluorescence color changes visually detected by the naked eyes in the range 1.0-10 µM and 0.30-3.0µg/mL, respectively. The sensing strategy showed higher sensitivity, more obvious color changes, and better disturbance performance, satisfying with the detection demands of Cu2+ and glyphosate in environmental water samples. The study provides a reliable detection strategy in the environment safety fields.

Interaction of major saffron constituent safranal with trypsin: An experimental and computational investigation

Trypsin is a serine protease, an important digestive enzyme that digests the proteins in the small intestine. In the present study, we have investigated the interaction of safranal, a major saffron metabolite, with trypsin using spectroscopic and molecular docking analyses. Fluorescence emission spectra of trypsin were largely affected by the inner filter effect from safranal; that's why these were corrected using the standard procedure. The corrected fluorescence spectra have shown that the safranal quenched the intrinsic fluorescence of trypsin with a blue shift in the wavelength of emission maximum, which revealed that the microenvironment of the fluorophore became more hydrophobic. There was approximately 1: 1 fair binding between them, which increased with a rise in temperature. The interaction was favored, principally, by hydrophobic forces, and there was an efficient energy transfer from the fluorophore to the safranal. Synchronous fluorescence spectra suggested that the tryptophan residues were the major ones taking part in the fluorescence quenching of trypsin. Safranal also influenced the secondary structure of trypsin and caused partial unfolding. Molecular Docking and the Molecular Dynamics simulation of the free and complexed trypsin was also carried out. Safranal formed a stable, non-covalent complex within the S2′-S5′ subsite. Moreover, two nearby tyrosine residues (Tyr39 and Tyr151) stabilized safranal through π-π interactions. Additionally, the presence of safranal led to changes in the protein flexibility and compactness, which could indicate changes in the surrounding of tryptophan residues, impacting their fluorescence. Furthermore, a loss in compactness is in line with the partial unfolding observed experimentally. Thus, both experimental and computational studies were in good agreement with each other.

Photoactivatable Blue Fluorescent Protein.

Photoactivatable and photoswitchable fluorescent proteins (FPs) are sophisticated molecular tools that in combination with super-resolution microscopy are helping to elucidate many biological processes. Through the Y66H mutation in the chromophore of the violet fluorescent protein SumireF, we created the first photoactivatable blue fluorescent protein (PA-BFP). This protein is rapidly activated over ordinary UV transilluminators at 302 or 365 nm in irreversible mode and by direct exposition to sunlight. The maximum excitation and emission wavelengths of this protein, centered at 358 and 445 nm, respectively, resemble the values of DAPI-the blue stain widely used in fluorescence microscopy to visualize nucleic acids in cells. Therefore, the immediate use of PA-BFP in cellular biology is clear because the technology required to follow this new genetically encoded reporter at the microscopic level has already been established. PA-BFP can potentially be used together with other photoactivatable fluorescent proteins of different colors to label multiple proteins, which can be simultaneously tracked by advanced microscopic techniques.