Fluorescence Experiments Research Articles

Structural studies of human Fis1 reveals a dynamic region important for Drp1 recruitment and mitochondrial fission

Fission protein 1 (Fis1) and dynamin‐related protein 1 (Drp1) were initially described as being evolutionarily conserved for mitochondrial fission, yet the role of Fis1 in human fission is unclear and disputed by many. In budding yeast where Fis1 helps to recruit Drp1 from the cytoplasm to mitochondria for fission, an N‐terminal “arm” of Fis1 is required for function. The yeast Fis1 arm interacts intramolecularly with a conserved surface that governs in vitro interactions with yeast Drp1. In human Fis1, NMR and x‐ray structures show a different arm conformation and its importance for human Drp1 recruitment is unknown. Here, we use MD simulations and comparisons to experimental NMR chemical shifts to show the human Fis1 arm can adopt an intramolecular conformation akin to that observed with the yeast molecule. This finding is further supported through intrinsic tryptophan fluorescence and NMR experiments on human Fis1 with and without the arm. Using NMR, the human Fis1 arm is also observed to be sensitive to environmental changes. The importance of these findings are revealed in cellular studies where the removal of the hFis1 arm reduces Drp1 recruitment and mitochondrial fission suggesting an important role for Fis1 in human mitochondrial fission.Support or Funding InformationThis project was supported by the following National Institutes of Health grants: R01GM067180 (to RBH), and R01HL128240 (to MEW).

Catalytic and structural effects of flexible loop deletion in organophosphorus hydrolase enzyme: A thermostability improvement mechanism

Thermostability improvement of enzymes used industrially or commercially would develop their capacity and commercial potential due to increased enzymatic competence and cost-effectiveness. Several stabilizing factors have been suggested to be the base of thermal stability, like proline replacements, disulfide bonds, surface loop truncation and ionic pair networks creation. This research evaluated the mechanism of increasing the rigidity of organophosphorus hydrolase enzyme by flexible loop truncation. Bioinformatics analysis revealed that the mutated protein retains its stability after loop truncation (five amino acids deleted). The thermostability of the wild-type (OPH-wt) and mutated (OPH-D5) enzymes were investigated by half-life, Delta Gi, and fluorescence and far-UV CD analysis. Results demonstrated an increase half-life and Delta Gi in OPH-D5 compared to OPH-wt. These results were confirmed by extrinsic fluorescence and circular dichroism (CD) spectrometry experiments, therefore, as rigidity increased in OPHD5 after loop truncation, half-life and Delta Gi also increased. Based on these findings, a strong case is presented for thermostability improvement of OPH enzyme by flexible loop truncation after bioinformatics analysis.

In Situ High-Resolution AFM Imaging and Force Probing of Cell Culture Medium-Forming Nanogranular Surfaces for Cell Growth.

Utilizing cell culture medium to grow cells in vitro has been widely studied in the past decades and has been recognized as an acknowledged way for investigating cell activities. However, due to the lack of adequate observation tools, the detailed mechanisms regulating cell growth in cell culture medium are still not fully understood. In this work, atomic force microscopy (AFM), a powerful tool for observing native biological systems under near-physiological conditions with high resolution, was applied to reveal the nanogranular surfaces formed in cell culture medium in situ for promoting cell growth. First, AFM imaging of glass slides (glass slides were previously incubated in cell culture medium) in aqueous environment clearly visualized the cell culture medium-forming nanogranular surfaces on glass slides. By altering the incubation time of glass slides in cell culture medium, the dynamic formation of nanogranular surfaces was remarkably observed. Next, fluorescent labeling experiments of the cell culture medium-treated glass slides showed that bovine serum proteins were contained in the nanogranular surfaces. Further, the adhesive interactions between cells and nanogranular surfaces probed by AFM force spectroscopy and the cell growth experiments showed that cell culture medium-forming nanogranular surfaces promote cell attachment and growth. The study provides novel insights into nanotopography-regulated molecular mechanisms in cell growth and demonstrates the outstanding capabilities of AFM in addressing biological issues with unprecedented spatial resolution under aqueous conditions, which will have potential impacts on the studies of cell behaviors and cell functions.

Eight new coordination polymers containing rigid 4-(4-carboxy-phenyl)-pyridine-2-carboxylic acid: Synthesis, structural diversity, fluorescence and magnetic properties

Eight new coordination polymers (CPs) were successfully synthesized through the solvo-thermal reactions of H2cppc ligand and a series of transition metal salts, [M(cppc)(H2O)2]n [M = Co, (1); Ni, (2); Zn, (3); Cd, (4)], [M(Hcppc)2]n [M = Cd, (5); Mn, (6); Cu, (7)], [Pb(cppc)(H2O)]n (8), [H2cppc = 4-(4-carboxy-phenyl)-pyridine-2-carboxylic acid]. These compounds were all characterized by elemental analysis, IR spectra, single-crystal X-ray diffraction, powder X-ray diffraction and thermal analysis. Compounds 1–4 have similar molecular formula except for different metal centers, notably, compounds 1–3 with isomorphic frameworks display two-dimensional layer structure, in which contain left- and right-handed chiral chains constructed from M2(H2O)2 dimers and cppc2− anions. However, compound 4 exhibits a three-dimensional network with Cd-O-Cd inorganic chains, different from that of compounds 1–3. Compounds 5–7 also have similar molecular formula except for different metal centers, the isomorphic compounds 5 and 6 exhibit two-dimensional undulating layers, while compound 7 displays one-dimensional chain. Compound 8 is a 2D layer structure with Pb2O2 secondary building units. In general, coordination geometries of metal centers and coordination modes of the ligands have an important effect on the structures of targeted compounds. Fluorescent experiments indicate that compounds 3–5 and 8 display apparently blue light emission in the solid state. Magnetic properties of compounds 1, 2, 6 and 7 are also investigated.

Organic-Inorganic Composite Nanorods as an Excellent Mimicking Peroxidases for Colorimetric Detection and Evaluation of Antioxidant.

N,N'-Dicarboxy methyl perylene diimide-coated CeO2 nanorods (PDI/CeO2 NR) were synthesized via an ultrasonic-assisted method. PDI/CeO2 exhibits the superb mimic peroxidase functions confirmed by the catalyzed oxidation of TMB by hydrogen peroxide in less than 60 s along with color transformation from colorless to blue. The catalytic mechanism was confirmed to be an electronic transfer mechanism by fluorescence experiment. Thus, a visual colorimetric sensor was constructed for hydrogen peroxide detection with a low detection limit (LOD = 2.23 μM). Comparing the inhibition effects of l-cysteine (Cys), ascorbic acid (AA) and glutathione (GSH) on the catalytic oxidation of TMB, it can be found that they possessed different types of inhibition on the oxidation of TMB by H2O2 using PDI/CeO2, and AA has better antioxidant effect, followed by Cys and GSH. On the basis of the excellent antioxidant effect of AA, a low-cost colorimetric sensor was also used to detect AA, and a lower LOD value (0.68 μM) was obtained in the linear range of 5.0-30 μM.

Studies on binding of single-stranded DNA with reduced graphene oxide-silver nanocomposites.

The binding reaction of reduced graphene oxide-silver nanocomposites (rGO-AgNCs) with calf thymus single-stranded DNA (ssDNA) was studied by ultraviolet-visible absorption, fluorescence spectroscopy and circular dichroism (CD), using berberine hemisulphate (BR) dye as a fluorescence probe. The absorbance of ssDNA increases, but the fluorescence intensity is quenched with the addition of rGO-AgNCs. The binding of rGO-AgNCs with ssDNA was able to increase the quenching effects of BR and ssDNA, and induce the changes in CD spectra. All of the evidence indicated that there was a relatively strong interaction between ssDNA and rGO-AgNCs. The data obtained from fluorescence experiments revealed that the quenching process of ssDNA caused by rGO-AgNCs is primarily due to complex formation, i.e. static quenching. The increasing trend of the binding equilibrium constant (Ka) with rising temperature indicated that the binding process was an endothermic reaction. The calculated thermodynamic parameters showed that the binding process was thermodynamically spontaneous, and hydrophobic association played predominant roles in the binding of ssDNA to the surface of rGO-AgNCs.

Effect of helical kink in antimicrobial peptides on membrane pore formation.

Every cell is protected by a semipermeable membrane. Peptides with the right properties, for example Antimicrobial peptides (AMPs), can disrupt this protective barrier by formation of leaky pores. Unfortunately, matching peptide properties with their ability to selectively form pores in bacterial membranes remains elusive. In particular, the proline/glycine kink in helical peptides was reported to both increase and decrease antimicrobial activity. We used computer simulations and fluorescence experiments to show that a kink in helices affects the formation of membrane pores by stabilizing toroidal pores but disrupting barrel-stave pores. The position of the proline/glycine kink in the sequence further controls the specific structure of toroidal pore. Moreover, we demonstrate that two helical peptides can form a kink-like connection with similar behavior as one long helical peptide with a kink. The provided molecular-level insight can be utilized for design and modification of pore-forming antibacterial peptides or toxins.

A mitochondrial-targeted red fluorescent probe for detecting endogenous H2S in cells with high selectivity and development of a visual paper-based sensing platform

Hydrogen sulfide (H2S) is a lipid-soluble gas signal molecule that plays a vital role in numerous abnormal pathological courses. Therefore, monitoring the changes of intracellular H2S level is undoubtedly of great scientific significance. Here, a red fluorescent probe SXR was reported, which is in view of the ICT mechanism. SXR showed rapid response (∼6 min), excellent selectivity and large Stokes shift (∼90 nm) to H2S. In addition, fluorescent imaging experiments further proved that SXR could detect endogenous and exogenous H2S in mitochondria, which provided a powerful tool for in-depth study of H2S function in various physiological courses. More importantly, a convenient visual paper-based sensor platform was designed, proving the favorable practicability for quantitative scanning and detecting of H2S with smart phone.

Biophysical and biological impact on the structure and IgE-binding of the interaction of the olive pollen allergen Ole e 7 with lipids

Ole e 7 allergen from Olea europaea pollen possesses a major clinical relevance because it produces severe symptoms, such as anaphylaxis, in allergic patients exposed to high olive pollen counts. Ole e 7 is a non-specific lipid transfer protein (nsLTP) characterized by the presence of a tunnel-like hydrophobic cavity, which may be suitable for hosting and, thus, transporting lipids -as it has been described for other nsLTPs-. The identification of the primary amino acid sequence of Ole e 7, and its production as a recombinant allergen, allowed characterizing its lipid-binding properties and its effect at air-liquid interfaces. Fluorescence and interferometry experiments were performed using different phospholipid molecular species and free fatty acids to analyse the lipid-binding ability and specificity of the allergen. Molecular modelling of the allergen was used to determine the potential regions involved in lipid interaction. Changes in Ole e 7 structure after lipid interaction were analysed by circular dichroism. Changes in the IgE binding upon ligand interaction were determined by ELISA. Wilhelmy balance measurements and fluorescence surfactant adsorption tests were performed to analyse the surface activity of the allergen. Using these different approaches, we have demonstrated the ability of Ole e 7 to interact and bind to a wide range of lipids, especially negatively charged phospholipids and oleic acid. We have also identified the protein structural regions and the residues potentially involved in that interaction, suggesting how lipid-protein interactions could define the behaviour of the allergen once inhaled at the airways.

Photochemical Study of a New Bimolecular Photoinitiating System for Vat Photopolymerization 3D Printing Techniques under Visible Light

In this work, we presented a new bimolecular photoinitiating system based on 2-amino-4,6-diphenylpyridine-3-carbonitrile derivatives as visible photosensitizers of diphenyliodonium salt. Real-time FTIR and photo-DSC photopolymerization experiments with a cycloaliphatic epoxide and vinyl monomers showed surprisingly good reactivity of the bimolecular photoinitiating systems under UV-A, as well as under visible light sources. Steady-state photolysis, fluorescence experiments, theoretical calculations of molecular orbitals, and electrochemical analysis demonstrated photo-redox behavior as well as the ability to form initiating species via photo-reduction or photo-oxidation pathways, respectively. Therefore, the 2-amino-4,6-diphenylpyridine-3-carbonitrile derivatives were also investigated as a type II free-radical photoinitiator with amine. It was confirmed that the 2-amino-4,6-diphenylpyridine-3-carbonitrile derivatives, in combination with different types of additives, e.g., amine as a co-initiator or the presence of onium salt, can act as bimolecular photoinitiating systems for cationic, free-radical, and thiol-ene photopolymerization processes by hydrogen abstraction and/or electron transfer reactions stimulated by either near-UV or visible light irradiation. Finally, the 2-amino-4,6-diphenylpyridine-3-carbonitrile derivatives were selected for 3D printing rapid prototyping experiments. Test objects were successfully printed using purely cationic photosensitive resin, created on a 3D printer with a visible LED light source.

Interaction of the synthesized anticancer compound of the methyl-glycine 1,10-phenanthroline platinum nitrate with human serum albumin and human hemoglobin proteins by spectroscopy methods and molecular docking

In the present study, the interaction of the novel synthesized platinum complex named methyl-glycine 1,10-phenanthroline platinum nitrate product with two most crucial blood carrier proteins of hemoglobin (Hb) and human serum albumin (HSA) has been investigated. Notably, structural alternation ability of the above anticancer compound was assessed at various temperatures through multispectroscopic techniques such as far-UV circular dichroism (CD) and fluorescence with the participation of the molecular docking targeting Hb and HSA in the systems of the medicine delivery. Fluctuations in the inherent fluorescence intensities of the proteins in consequence of the Pt(II) complex binding represented statics of the quenching system. It should be mentioned that thermodynamic and binding parameters have been calculated through the analysis of the outcomes of the quenching and outcomes of van’t Hoff equation for the two proteins. Theoretical and experimental findings pointed out that the leading force for the Pt(II) complex interaction was electrostatic for HSA and hydrophobic interactions for Hb. Moreover, fluorescence experiments discovered that one binding site would be available because of the Pt(II) complex binding in the two proteins accompanied by a negative Gibbs free energy value. Additionally, the far-UV CD outputs suggested that the Pt(II) complex would cause some variations in the regular secondary and tertiary structures of the HSA as the remarkable going down in the α helical contents of proteins structures at different temperatures equal to 25 °C and 37 °C; however, any noteworthy modifications have not been seen in the respective structures of Hb. However, the interaction between the newly made-up medicine (Pt(II) complex) and the two most crucial blood carrier proteins of the Hb and HSA made a notable shifting in the structures and conformations of the proteins via many fluctuations in the secondary and tertiary structures of the two proteins. Hence, such researches of designing the novel metal anticancer medicines may gather useful data for exploring the more suitable path in the face-off with severe disorders like various cancers through inventing and developing metal medical compounds that are more advantageous and have fewer consequences.

The Effect of Temperature and Magnetic Field on the Precipitation of Insoluble Salts of Alkaline Earth Metals

A study of the effects induced by a weak magnetic field of about 0.4 T on the precipitation of insoluble alkaline earth carbonates is reported. Optical microscopy, X-ray diffraction and fluorescence experiments are employed at 25 °C and 60 °C in either H2O or D2O solutions to explore the role played by temperature and solvent structure on the magnetic effect. The results reveal that the combination of an external magnetic field and high temperature limits the precipitation of scarcely soluble salts with the strongest effect on calcium carbonate. Furthermore, it is found that the magnetic field affects the structure of the solvent molecules in the hydration shells of the ions. The current work represents a step forward in our understanding of the curious effects of magnetic fields on salt precipitation. It may have application in various areas of chemical science, such as water treatment, shale gas extraction, colloid science, biology, and chirality.

Protein assembled nano-vehicle entrapping photosensitizer molecules for efficient lung carcinoma therapy.

The efficiency of drug depends not only on its potency but also on its ability to reach the target sites in preference to non-target sites. In this regard, protein assembled nanocarrier is the most promising strategy for intracellular anti-cancer drug delivery. The key motive of this study is to fabricate biocompatible protein assembled nanocarrier conjugated photosensitizer system for stimuli-responsive treatment of lung carcinoma. Here, we have synthesized a unique nanohybrid of protein assembled gold nanoparticles (AuNPs), attaching a model photosensitizer, Protoporphyrin IX (PpIX) to the protein shell of the nanoparticles (NPs) imparting an ideal drug-carrier nature. Photo-induced alteration in hydrodynamic diameter suggests structural perturbation of the nanohybrid which in terms signifies on-demand drug delivery. The drug release profile has been further confirmed by using steady-state fluorescence experiments. AuNP-PpIX showed excellent anti-cancer efficiency upon green light irradiation on lung adenocarcinoma cell line (A549) through intracellular reactive oxygen species (ROS) generation. The cellular morphological changes upon PDT and internalization of nanohybrid were monitored using confocal laser scanning microscope. This anti-cancer effect of nanohybrid was associated with apoptotic pathway which was confirmed in the flow cytometric platform. The developed nanomedicine is expected to find relevance in clinical anti-cancer PDT models in the near future.

What does fluorine do to a protein? Thermodynamic, and highly-resolved structural insights into fluorine-labelled variants of the cold shock protein

Fluorine labelling represents one promising approach to study proteins in their native environment due to efficient suppressing of background signals. Here, we systematically probe inherent thermodynamic and structural characteristics of the Cold shock protein B from Bacillus subtilis (BsCspB) upon fluorine labelling. A sophisticated combination of fluorescence and NMR experiments has been applied to elucidate potential perturbations due to insertion of fluorine into the protein. We show that single fluorine labelling of phenylalanine or tryptophan residues has neither significant impact on thermodynamic stability nor on folding kinetics compared to wild type BsCspB. Structure determination of fluorinated phenylalanine and tryptophan labelled BsCspB using X-ray crystallography reveals no displacements even for the orientation of fluorinated aromatic side chains in comparison to wild type BsCspB. Hence we propose that single fluorinated phenylalanine and tryptophan residues used for protein labelling may serve as ideal probes to reliably characterize inherent features of proteins that are present in a highly biological context like the cell.

Transmembrane and Juxtamembrane Interactions of EphA2 with Lipid Membranes in the Active and Inactive States

Aberrant signaling of the receptor tyrosine kinase EphA2 is linked to tumor aggressiveness in many types of cancers. Upon binding of an extracellular ephrin ligand EphA2 undergoes oligomerization and phosphorylation inducing an intracellular signaling cascade. Little else is known of the mechanisms by which the signal is conferred across the membrane to drive phosphorylation or how in the absence of ligand the receptor remains inactive. Evidence suggests that prior to activation EphA2 dimers exhibit a distinct 15° interhelical crossing angle between the transmembrane domains (TMD) and that upon activation a rotation of the TMDs occurs and it widens to 45°. It is hypothesized that positively charged residues on the intracellular juxtamembrane (JM) segment interact with negatively charged Phosphatidylinositol 4,5-bisphosphate (PIP2) lipids on the inner leaflet of the cell membrane inhibiting activation in the absence of bound ligand. Here we use a method of studying a peptide comprised of the TMD and first five JM residues of EphA2 in the putative active and inactive conformations by varying membrane thickness using 14:1 PC and 22:1 PC. We established that the TMD orientation is more tilted in thin bilayers and a less tilted in thick bilayers. We used this system to investigate the interaction of JM residues with membranes containing PIP2 in thin and thick bilayers. Fluorescence experiments indicate that JM residues experience different environments in thin vs. thick bilayers in the presence of PIP2. FRET results suggest that the JM is buried deeper into the membrane in the presence of PIP2 in 22:1 PC but not 14:1 PC. The extent of burial has been further examined by quenching experiments using brominated lipids. These results provide new mechanistic insights into the inhibition and activation of EphA2 signaling.

FRET-Based Integrative Structural Models and Their Database Deposition

Förster Resonance Energy Transfer (FRET) experiments provide information on the dynamics and the tertiary and super-tertiary structure of dynamic biomolecules. Combining FRET experiments with computational methods in integrative approaches allows to generate structural models of biomolecules with high accuracy and precision. We used single-molecule FRET measurements together with FRET-restrained structural modeling, molecular simulations and SAXS measurements in a hybrid approach to obtain structural models for a RNA four-way junction. This integrative approach allowed us to obtain structural models for three co-existing conformers with a precision between 1.3 and 3.0 Å. Software used in this workflow is available on our webpage (http://www.mpc.hhu.de/software.html). PDB-Dev is a prototype archiving system for deposition of integrative and hybrid structural models obtained from various experimental sources (https://pdb-dev.wwpdb.org/). In order to be able to deposit integrative structural models using data from FRET measurements, we worked with the PDB-Dev developers to extend the integrative/hybrid modeling (IHM) dictionary for fluorescence experiments. This extension (FLR dictionary: https://github.com/ihmwg/FLR-dictionary) includes the description of samples, fluorescent probes, analyses, structural modeling procedures and the resulting structural models. In addition to this, we developed a workflow to prepare fluorescence-based structural models and the respective metadata from fluorescence experiments to facilitate their deposition in PDB-Dev.

Characterizing the Interplay between Dynamics and Regulation in the Trypsinogen/Trypsin Protease System

Trypsin (Tn) is a digestive serine protease found in eukaryotes and prokaryotes. Tn is manufactured by the vertebrate pancreas as its proenzyme trypsinogen (Tgn) which includes an N-terminal six amino acid zymogen region. The zymogen form, Tgn, is disabled against cleavage of substrate until Tgn reaches the small intestine. Within the small intestine, enterokinase cleaves the inhibiting peptide from folded Tgn to release active Tn protease. Fluorescence experiments with bovine Tn demonstrate remarkably slow global unfolding, with a kinetic barrier to unfolding as large as kinetically stabilized bacterial homologues. Whereas bacterial homologues have evolved to resist proteolysis, thereby prolonging extracellular activity, mammalian Tgn/Tn is tightly regulated through a series of auto-cleavage events. We propose that Tgn/Tn experiences subglobal dynamics, and that sampling of these semi-unfolded states under physiological conditions causes this rapid autolysis. To explore this, we will perform native-state hydrogen exchange mass spectrometry of a purified inactive mutant. Exploring the Tgn/Tn subglobal dynamics which expose the native-state to autolysis will grant understanding of the link between Tn dynamics and the regulation of its function through its specific sequence of auto-cleavage events. By comprehensively characterizing the sampling of subglobally and globally unfolded states of Tgn/Tn, more can be inferred about the occurrence of non-native state protein aggregation and susceptibility to proteolytic attack. Studying Tgn/Tn from the inactive Tgn/Tn mutant allows direct assessment of the role of subglobal protein dynamics and the regulation of these dynamics in Tn folding disorders without the complication of autoproteolysis. The Tgn/Tn system will allow investigation of diseases characterized by abnormal protein aggregation and vulnerability to proteolytic attack, in addition to diseases directly related to Tn function such as pancreatitis.

Characterization of Small Objects in Homogenates of the Squid Optic Lobe

The squid (Loligo pealei) optic lobe is unique among sources of CNS synaptosomes via differential centrifugation, both in its ease and speed of dissection and preparation, and in the purity of the final result (Pekkurnaz et al. Biol. Bull. 2011). Despite the high purity of ∼1 µm-diameter synaptosomes in the synaptosome fraction, another class of 2-10 μm diameter objects were detected by one us (GP) in DIC microscopy of thin layers of this fraction. Recently, a publication assumed that these inclusions were large synaptosomes (Cefaliello et al. Molec. Neurobio. 2019). To test that assumption, and to further characterize these inclusions, we carried out a series of fluorescent labeling and dye exclusion experiments by direct imaging for size classification. Two major classes of objects were found, a larger class (L) 5-10 μm in diameter and a smaller class (S) that appeared more refractile, perfectly spherical, and averaged 2 μm in diameter. To identify some of the macromolecular content of these objects, we labeled them with fluorescent dyes for DNA, lipid, and protein aggregates. ‘L’ stained positive for the membrane dye BODIPY-sphingomyelin and the DNA marker Hoechst. The exclusion of rhodamine-dextran (10kDa) further corroborated the membrane-enclosed nature of the ‘L’ object. Electron microscopy showed a large ∼10 μm spheroid with a double membrane and dark material draping the internal membrane, suggesting that the ‘L’ inclusions were nuclei. The majority of the inclusions in the ‘S’ class did not exclude dyes, did not stain with the DNA dye Hoechst, and were positive for a rotamer fluorescent marker for intracellular aggregated misfolded proteins. Currently, efforts are underway to identify the protein composition of the ‘S’ droplets.

Altered Thick and Thin Filament Structural Dynamics in Mouse Myocardium Due to Ablation and Phosphorylation of Myosin Binding Protein-C

Cardiac Myosin Binding Protein-C (cMyBP-C) is an important regulator of myocardial contraction and cross-bridge disposition; however, its contributions to thick and thin filament structural dynamics is unknown. Therefore, we performed time-resolved fluorescence (TR-F) experiments to quantify lifetime changes of environmentally-sensitive probes introduced in myofibrils isolated from ventricles of wild-type (WT) and cMyBP-C null (cMyBP-C-/-) mice. Mutant ventricular regulatory light chain (RLC V105C) labeled with CPM or cardiac troponin C (cTnC T53C) labeled with IANBD were exchanged in skinned myofibrils to replace the endogenous proteins. ATPase measurements demonstrated that the exchanges did not affect contractile function. TR-F measurements revealed differences in lifetimes of both RLC and cTnC between WT and cMyBP-C-/- myofibrils. PKA-mediated phosphorylation of myofibrils also affected lifetimes with greater effects observed in WT as compared to cMyBP-C-/-. For the RLC probe, the differences in lifetimes due to PKA phosphorylation were greatest with addition of myosin inhibitors: blebbistatin or vanadate. These results are consistent with the idea that cMyBP-C plays a role in stabilizing myosin on the thick filament and promoting the super-relaxed state, whereas its absence or phosphorylation leads to increased molecular disorder. For the cTnC probe, PKA treatment of myofibrils reduced the Ca2+-sensitivity of lifetime in both WT and cMyBP-C-/-. These results suggest unique phosphorylation-dependent roles for cMyBP-C and cardiac troponin I (cTnI) on thin filament structural dynamics. Our findings demonstrate that cMyBP-C and cTnI contribute to phosphorylation-dependent effects on thick and thin filament structural dynamics to influence force development. These TR-F probes can also be useful for screening of compounds that alter cMyBP-C interactions with the myofilaments.

Synthesis, structure, and properties of complexes based on 3, 5-di-(Triazole-1-yl)-Benzoic acid ligands

Two kinds of new complexes based on 3, 5-bis (triazol-1-yl) benzoic acid (HL) and 1, 3, 5-benzenetricarboxylic acid (H3bta), {[Zn2(μ4-L)(μ3-bta(3H2O)]∙H2O}n(1) and {[Cd2(μ4-L)(μ3-bta(3H2O)]∙4H2O}n(2), were prepared by solvothermal reactions and the structures were characterized by single-crystal X-ray diffraction. The experimental results showed that complexes 1 and 2 were isomorphic, in which metal ions were separately chelated with the ligands to form one mononuclear Cd1 and another binuclear Cd2O4 structure. Each metal center (M) can be considered as a three-connection, each M2O4 as a six-connection, and the ligands of HL and H3bta can be used as three-junctions. Finally, complex 1 and 2 showed a (3, 6)-connection of {63}6{66;86;102;12} topology. Solid-state fluorescence experiments showed that only complex 1 could selectively identify Fe3+ (detection limit: 0.11 ​mM) and Cr6+ (detection limit: 0.1 ​mM) in aqueous solution by a fluorescence quenching mechanism and the fluorescence quenching constant of Fe3+ reached 104. Dye adsorption experiments showed that the complexes could absorb different organic dyes and the adsorption capacity was 1 ​> ​2. Furthermore, the adsorption efficiency of 1 for methylene blue was as high as 97.64%.