Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular animals, making them a viable target for drug design against protozoan parasites such as Plasmodium falciparum. An excellent understanding of many of the catalytic states throughout the enzymatic cycle has already been afforded by crystallography. However, the dynamics and kinetics of the catalytic cycle between these static snapshots remain to be elucidated. Here, we employ single-molecule Förster resonance energy transfer (FRET) measurements to determine the dynamic range and frequency of conformations available to the enzyme in a lipid bilayer during the catalytic cycle. First, we explore issues related to the introduction of fluorescent dyes by cysteine mutagenesis; we discuss the importance of residue selection for dye attachment, and the balance between mutating areas of the protein that will provide useful dynamics while not altering highly conserved residues that could disrupt protein function. To complement and guide the experiments, we used all-atom molecular dynamics simulations and computational methods to estimate FRET efficiency distributions for dye pairs at different sites in different protein conformational states. We present preliminary single-molecule FRET data that points to insights about the binding modes of different membrane-bound pyrophosphatase substrates and inhibitors.

• MeSH
• bakteriální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• enzymatické testy přístrojové vybavení   metody   MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční mikroskopie přístrojové vybavení   metody   MeSH
• mutageneze MeSH
• protozoální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• pyrofosfatasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• racionální návrh léčiv MeSH
• rekombinantní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• rezonanční přenos fluorescenční energie přístrojové vybavení   metody   MeSH
• Saccharomyces cerevisiae MeSH
• sekvenční seřazení MeSH
• simulace molekulární dynamiky * MeSH
• software MeSH
• zobrazení jednotlivé molekuly přístrojové vybavení   metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Fluorescence resonance energy transfer (FRET) under in vivo conditions is a well-established technique for the evaluation of populations of protein bound/unbound nucleic acid (NA) molecules or NA hybridization kinetics. However, in vivo FRET has not been applied to in vivo quantitative conformational analysis of NA thus far. Here we explored parameters critical for characterization of NA structure using single-pair (sp)FRET in the complex cellular environment of a living Escherichia coli cell. Our measurements showed that the fluorophore properties in the cellular environment differed from those acquired under in vitro conditions. The precision for the interprobe distance determination from FRET efficiency values acquired in vivo was found lower (≈ 31%) compared to that acquired in diluted buffers (13%). Our numerical simulations suggest that despite its low precision, the in-cell FRET measurements can be successfully applied to discriminate among various structural models. The main advantage of the in-cell spFRET setup presented here over other established techniques allowing conformational analysis in vivo is that it allows investigation of NA structure in various cell types and in a native cellular environment, which is not disturbed by either introduced bulk NA or by the use of chemical transfectants.

• MeSH
• DNA chemie   MeSH
• Escherichia coli genetika   MeSH
• fluorescenční barviva MeSH
• konformace nukleové kyseliny MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Several small-molecule ligands specifically bind and stabilize G-quadruplex (G4) nucleic acid structures, which are considered to be promising therapeutic targets. G4s are polymorphic structures of varying stability, and their formation is dynamic. Here, we investigate the mechanisms of ligand binding to dynamically populated human telomere G4 DNA by using the bisquinolinium based ligand Phen-DC3 and a combination of single-molecule FRET microscopy, ensemble FRET and CD spectroscopies. Different cations are used to tune G4 polymorphism and folding dynamics. We find that ligand binding occurs to pre-folded G4 structures and that Phen-DC3 also induces G4 formation in unfolded single strands. Following ligand binding to dynamically populated G4s, the DNA undergoes pronounced conformational redistributions that do not involve direct ligand-induced G4 conformational interconversion. On the contrary, the redistribution is driven by ligand-induced G4 folding and trapping of dynamically populated short-lived conformation states. Thus, ligand-induced stabilization does not necessarily require the initial presence of stably folded G4s.

• MeSH
• chinoliny chemie   metabolismus   MeSH
• G-kvadruplexy * MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• ligandy * MeSH
• rezonanční přenos fluorescenční energie MeSH
• simulace molekulární dynamiky MeSH
• telomery chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

With the goal to investigate biological phenomena at a single-cell level, we designed, synthesized and tested a molecular probe based on Förster resonance energy transfer (FRET) between a highly luminescent quantum dot (QD) as a donor and a fluorophore or fluorescence quencher as an acceptor linked by a specific peptide. In principle, QD luminescence, effectively dissipated in the probe, is switched on after the cleavage of the peptide by a protease and the release of the quencher. We proposed a novel synthesis strategy of a probe. A two-step synthesis consists of: (i) Conjugation of CdTe QDs functionalized by -COOH groups of succinic acid on the nanoparticle surface with the designed specific peptide (GTADVEDTSC) using a ligand-exchange approach; (ii) A fast, high-yield reaction of amine-reactive succinimidyl group on the BHQ-2 quencher with N-terminal of the peptide. This way, any crosslinking between individual nanoparticles and any nonspecific conjugation bonds are excluded. The analysis of the product after the first step proved a high reaction yield and nearly no occurrence of unreacted QDs, a prerequisite of the specificity of our luminescent probe. Its parameters evaluated as Michaelis-Menten description of enzymatic kinetics are similar to products published by other groups. Our research is focused on the fluorescence microscopy analyses of biologically active molecules, such as proteolytic active caspases, playing important roles in cell signaling regulations in normal and diseased states. Consequently, they are attractive targets for clinical diagnosis and medical therapy. The ultimate goal of our work was to synthesize a new QD luminescent probe for a long-time quantitative monitoring of active caspase-3/7 distribution in apoptotic osteoblastic MC3T3-E1 cells treated with camptothecin. As a result of comparison, our synthetized luminescent probe provides longer imaging times of caspases than commercial products. The probe proved the stability of the luminescence signal inside cells for more than 14 days.

• MeSH
• kaspasy MeSH
• kvantové tečky * MeSH
• peptidy chemie   MeSH
• proteasy MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• sloučeniny kadmia * chemie   MeSH
• telur MeSH
• Publikační typ
• časopisecké články MeSH

The implementation of quantum dots in analytical chemistry has already advanced from basic research activities to routine applications of commercially available fluorescent agents present in sophisticated assays kits. Nevertheless, a further development of new preparation and characterization methods of nanoparticles is still required to increase the sensitivity of analytical methods substantially. Thus, in many bioanalytical applications, important molecules such as DNA, proteins, and antibodies are routinely conjugated with fluorescent tags to reach even the absolute sensitivity, that is, the capability to detect a single molecule in complex matrices. Semiconductor quantum dots have already proved to be suitable components of highly luminescent tags, probes, and sensors with broad applicability in analytical chemistry. Quantum dots provide high extinction coefficients together with wide ranges of excitation wavelengths, size- and composition-tunable emissions, narrow and symmetric emission spectra, good quantum yields, relatively long size-dependent luminescence lifetime, and low photobleaching. Most of these properties are superior when compared with conventional organic fluorescent dyes. In this chapter, optimized procedures for the preparation of water-dispersed CdTe quantum dots; their coatings and conjugation reactions with antibodies, DNA, and macrocycles; and their analyses by capillary electrophoresis are described. The potential of capillary electrophoresis for fast analyses of nanoparticles, their conjugates with antibodies and immunocomplexes with targeted antigens, is demonstrated as an example.

• MeSH
• elektroforéza kapilární metody   MeSH
• fluorescenční barviva chemie   MeSH
• kvantové tečky chemie   MeSH
• luminiscenční měření metody   MeSH
• nanočástice MeSH
• nanotechnologie MeSH
• proteiny chemie   MeSH
• protilátky chemie   MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• sloučeniny kadmia chemie   MeSH
• telur chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

DNA nanodevices have been developed as platforms for the manipulation of gene expression, delivery of molecular payloads, and detection of various molecular targets within cells and in other complex biological settings. Despite efforts to translate DNA nanodevices from the test tube (in vitro) to living cells, their intracellular trafficking and functionality remain poorly understood. Herein, quantitative and super-resolution microscopy approaches were employed to track and visualise, with nanometric resolution, the molecular interactions between a synthetic DNA nanosensor and transcription factors in intracellular compartments. Specifically, fluorescence resonance energy transfer microscopy, fluorescence correlation spectroscopy, fluorescence lifetime imaging microscopy and multicolour single-molecule localisation microscopy were employed to probe the specific binding of the DNA nanosensor to the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). We monitored the mobility, subcellular localisation and degradation of the DNA nanosensor inside living prostate cancer PC3 cells. Super-resolution imaging enabled the direct visualisation of the molecular interactions between the synthetic DNA nanosensors and the NF-κB molecules in cells. This study represents a significant advance in the effective detection as well as understanding of the intracellular dynamics of DNA nanosensors in a complex biological milieu.

• MeSH
• DNA vazebné proteiny MeSH
• DNA MeSH
• lidé MeSH
• NF-kappa B * genetika   metabolismus   MeSH
• rezonanční přenos fluorescenční energie MeSH
• signální transdukce * MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

N-Methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for synaptic plasticity and memory. Receptor activation involves glycine- and glutamate-stabilized closure of the GluN1 and GluN2 subunit ligand binding domains that is allosterically regulated by the amino-terminal domain (ATD). Using single molecule fluorescence resonance energy transfer (smFRET) to monitor subunit rearrangements in real-time, we observe a stable ATD inter-dimer distance in the Apo state and test the effects of agonists and antagonists. We find that GluN1 and GluN2 have distinct gating functions. Glutamate binding to GluN2 subunits elicits two identical, sequential steps of ATD dimer separation. Glycine binding to GluN1 has no detectable effect, but unlocks the receptor for activation so that glycine and glutamate together drive an altered activation trajectory that is consistent with ATD dimer separation and rotation. We find that protons exert allosteric inhibition by suppressing the glutamate-driven ATD separation steps, and that greater ATD separation translates into greater rotation and higher open probability.

• MeSH
• alosterická regulace MeSH
• glycin chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• kinetika MeSH
• konfokální mikroskopie MeSH
• konformace proteinů * MeSH
• kyselina glutamová chemie   metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• multimerizace proteinu * MeSH
• receptory N-methyl-D-aspartátu chemie   genetika   metabolismus   MeSH
• rezonanční přenos fluorescenční energie metody   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

STIM1 (stromal interaction molecule 1) and Orai proteins are the essential components of Ca(2+) release-activated Ca(2+) (CRAC) channels. We focused on the role of cholesterol in the regulation of STIM1-mediated Orai1 currents. Chemically induced cholesterol depletion enhanced store-operated Ca(2+) entry (SOCE) and Orai1 currents. Furthermore, cholesterol depletion in mucosal-type mast cells augmented endogenous CRAC currents, which were associated with increased degranulation, a process that requires calcium influx. Single point mutations in the Orai1 amino terminus that would be expected to abolish cholesterol binding enhanced SOCE to a similar extent as did cholesterol depletion. The increase in Orai1 activity in cells expressing these cholesterol-binding-deficient mutants occurred without affecting the amount in the plasma membrane or the coupling of STIM1 to Orai1. We detected cholesterol binding to an Orai1 amino-terminal fragment in vitro and to full-length Orai1 in cells. Thus, our data showed that Orai1 senses the amount of cholesterol in the plasma membrane and that the interaction of Orai1 with cholesterol inhibits its activity, thereby limiting SOCE.

• MeSH
• biotinylace MeSH
• bodová mutace MeSH
• buněčná membrána metabolismus   MeSH
• buněčné linie MeSH
• cholesterol oxidasa metabolismus   MeSH
• cholesterol metabolismus   MeSH
• cirkulární dichroismus MeSH
• elektrofyziologické jevy MeSH
• fluorescenční spektrometrie MeSH
• HEK293 buňky MeSH
• histamin metabolismus   MeSH
• lidé MeSH
• mastocyty metabolismus   MeSH
• mutace MeSH
• peptidy metabolismus   MeSH
• rezonanční přenos fluorescenční energie MeSH
• signální transdukce MeSH
• terciární struktura proteinů MeSH
• vápník metabolismus   MeSH
• vápníkové kanály metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

V oblasti molekulární genetiky lze od nástupu nového milénia pozorovat velké pokroky ve vyšetřovacích metodách. Stěžejními momenty tohoto rozvoje byly: dokončení projektu Lidský genom, aplikace čipové technologie a sekvenování nové generace. Úloha bioanalytika při genetickém testování spočívá zejména v provedení spolehlivého laboratorního vyšetření a ve správném hodnocení získaných dat. Z těchto důvodů je v textu kladen důraz na principy používaných analýz. U čtenáře se předpokládají znalosti obecné a molekulární biologie týkající se procesů probíhajících v živých buňkách: replikace, transkripce, proteosyntézy a mechanismů jejich regulace.; Učebnice pro studenty magisterského studia bioanalytiky na Farmaceutické fakultě UK v Hradci Králové.

• Klíčová slova
• Přírodní vědy,
• MeSH
• genetika MeSH
• molekulární biologie MeSH

• NLK Obory
• biologie
• genetika, lékařská genetika

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) for the alpha-subunit of heterotrimeric G proteins. Several RGS proteins have been found to interact with 14-3-3 proteins. The 14-3-3 protein binding inhibits the GAP function of RGS proteins presumably by blocking their interaction with G(alpha) subunit. Since RGS proteins interact with G(alpha) subunits through their RGS domains, it is reasonable to assume that the 14-3-3 protein can either sterically occlude the G(alpha) interaction surface of RGS domain and/or change its structure. In this work, we investigated whether the 14-3-3 protein binding affects the structure of RGS3 using the time-resolved tryptophan fluorescence spectroscopy. Two single-tryptophan mutants of RGS3 were used to study conformational changes of RGS3 molecule. Our measurements revealed that the 14-3-3 protein binding induces structural changes in both the N-terminal part and the C-terminal RGS domain of phosphorylated RGS3 molecule. Experiments with the isolated RGS domain of RGS3 suggest that this domain alone can, to some extent, interact with the 14-3-3 protein in a phosphorylation-independent manner. In addition, a crystal structure of the RGS domain of RGS3 was solved at 2.3A resolution. The data obtained from the resolution of the structure of the RGS domain suggest that the 14-3-3 protein-induced conformational change affects the region within the G(alpha)-interacting portion of the RGS domain. This can explain the inhibitory effect of the 14-3-3 protein on GAP activity of RGS3.

• MeSH
• fluorescenční spektrometrie MeSH
• fosforylace MeSH
• interakční proteinové domény a motivy MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• multiproteinové komplexy MeSH
• mutageneze cílená MeSH
• podjednotky proteinů MeSH
• proteiny 14-3-3 chemie   metabolismus   MeSH
• proteiny aktivující GTPasu chemie   genetika   metabolismus   MeSH
• proteiny vázající GTP chemie   genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• rezonanční přenos fluorescenční energie MeSH
• sekvence aminokyselin MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• stabilita proteinů MeSH
• substituce aminokyselin MeSH
• techniky in vitro MeSH
• terciární struktura proteinů MeSH
• tryptofan chemie   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH