Adenosine Triphosphate Molecules Research Articles

Mechanistic insights into the phosphoryl transfer reaction in cyclin-dependent kinase 2: A QM/MM study.

Cyclin-dependent kinase 2 (CDK2) is an important member of the CDK family exerting its most important function in the regulation of the cell cycle. It catalyzes the transfer of the gamma phosphate group from an ATP (adenosine triphosphate) molecule to a Serine/Threonine residue of a peptide substrate. Due to the importance of this enzyme, and protein kinases in general, a detailed understanding of the reaction mechanism is desired. Thus, in this work the phosphoryl transfer reaction catalyzed by CDK2 was revisited and studied by means of hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. Our results suggest that the base-assisted mechanism is preferred over the substrate-assisted pathway when one Mg2+ is present in the active site, in agreement with a previous theoretical study. The base-assisted mechanism resulted to be dissociative, with a potential energy barrier of 14.3 kcal/mol, very close to the experimental derived value. An interesting feature of the mechanism is the proton transfer from Lys129 to the phosphoryl group at the second transition state, event that could be helping in neutralizing the charge on the phosphoryl group upon the absence of a second Mg2+ ion. Furthermore, important insights into the mechanisms in terms of bond order and charge analysis were provided. These descriptors helped to characterize the synchronicity of bond forming and breaking events, and to characterize charge transfer effects. Local interactions at the active site are key to modulate the charge distribution on the phosphoryl group and therefore alter its reactivity.

Amplification of aptamer sensor signals by four orders of magnitude via interdigitated organic electrochemical transistors

Electrochemical aptamer receptor/transducer systems are key elements of emerging E-AB sensors (aptasensor) used for the detection of various kinds of targets. However, the performance of these amperometric sensors is often limited by the low density of receptors attached to the sensor surface and high background signals. In the present work, interdigitated organic electrochemical transistors (iOECT) were used as a transducer to enhance the sensitivity and dynamic detection range of aptasensors. Therefore, the electrode of an amperometric sensor was utilized as gate electrode to operate the iOECT. This device was used to detect the low weight target molecule adenosine triphosphate (ATP), a common biomarker, which plays an important role for cardiovascular, neurodegenerative, and immune deficiency diseases. The novel aptasensor can selectively detect ATP with ultrahigh sensitivity down to the concentration of 10 pM, which is four orders of magnitude lower than the detection limit of the same aptasensor using an amperometric transducer principle (limit-of-detection of 106 nM) and most other previously reported electrochemical sensors. Furthermore, sensor regeneration was demonstrated, which facilitates reusability of OECT aptasensors. The small device size in combination with high transconductances paves the way for the development of highly sensitive integrated micro-biosensors for point-of-care applications.

Consideration of gravity as a possible etiological factor in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with an unknown mechanism of onset that predominantly impairs the upper and lower motor neurons. Components of the sensory and autonomic nervous system were once thought to be spared in the disease, but more recently they have been identified to be impaired at various levels. However, some of the motor neurons such as oculomotor, abducens, or pudendal nerves are spared even in the later stages of ALS. The mechanism of such complex and heterogeneous neuronal loss in typical ALS is still unknown. In this study, the characteristics of the nervous system involved in the pathogenesis of ALS were comprehensively reviewed. As a result, the direction of the axon in the anatomical position, rather than the functional type or length of the axon, was suggested to contribute the most to the onset of ALS. This finding suggested that downward directed axons, represented by motor neurons, require extra energy to move waste or unnecessary substances from the synapse side to the neural cell body with retrograde fast axonal transport. Based on this theory, the extra energy that is required in vertically directed axons due to the effect of gravity was mathematically estimated. As a result, several percent more adenosine triphosphate molecules were suggested to be consumed in vertical axonal transport by gravity, compared to those consumed in transverse axonal transport. Because most of the motor neurons project downward in the anatomical position, unretrieved waste will gradually sediment in axon terminals by gravity, which could eventually result in motor neuron-dominant neuronal loss. Although the theory that gravity is one of the mechanisms responsible for ALS is still hypothetical, it is theoretically reasonable and compatible with the clinical manifestations of the disease. Further basic research studies with cultured neurons or animal models are necessary to confirm the association between gravity and the onset of ALS.

Lambda-cyhalothrin exposure alters purine nucleotide hydrolysis and nucleotidase gene expression pattern in platelets and liver of rats

Lambda-cyhalothrin (LCT) is a broad-spectrum pesticide widely used in agriculture throughout the world. This pesticide is considered a potential contaminant of surface and underground water as well as food, posing a risk to ecosystems and humans. In this sense, we decided to evaluate the activity of enzymes belonging to the purinergic system, which is linked with regulation of extracellular nucleotides and nucleosides, such as adenosine triphosphate (ATP) and adenosine (Ado) molecules involved in the regulation of inflammatory response. However, there are no data concerning the effects of LCT exposure on the purinergic system, where extracellular nucleotides act as signaling molecules. The aim of this study was to evaluate nucleotide hydrolysis by E-NTPDase (ecto-nucleoside triphosphate diphosphohydrolase), Ecto-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase), ecto-5′-nucleotidase and ecto-adenosine deaminase (E-ADA) in platelets and liver of adult rats on days 7, 30, 45 and 60 after daily gavage with 6.2 and 31.1 mg/kg bw of LCT. Gene expression patterns of NTPDases1–3 and 5′-nucleotidase were also determined in those tissues. In parallel, lambda-cyhalothrin metabolites [3-(2-chloro-3,3,3- trifluoroprop-1-enyl)-2,2-dimethyl-cyclopropane carboxylic acid (CFMP), 4-hydroxyphenoxybenzoic acid (4-OH-3-PBA), and 3-phenoxybenzoic acid (3-PBA)] were measured in plasma. Results showed that exposure rats to LCT caused a significant increase in the assessed enzymes activities. Gene expression pattern of ectonucleotidases further revealed a significant increase in E-NTPDase1, E-NTPDase2, and E-NTPDase3 mRNA levels after LCT administration at all times. A dose-dependent increase in LCT metabolite levels was also observed but there no significant variations in levels from weeks to week, suggesting steady-steady equilibrium. Correlation analyses revealed that LCT metabolites in the liver and plasma were positively correlated with the adenine nucleotides hydrolyzing enzyme, E-ADA and E-NPP activities in platelets and liver of rats exposed to lambda-cyhalothin.Our results show that LCT and its metabolites may affect purinergic enzymatic cascade and cause alterations in energy metabolism.

Hypertension-induced alarm signal

Immunology Human hypertension is a highly prevalent disease known to be associated with chronic low-grade inflammation. Zhao et al. used mouse models to look for hypertension-induced proinflammatory molecules that contribute to T cell activation and inflammation. They found consistent elevations in plasma levels of the alarmin molecule adenosine triphosphate in hypertensive mice. Increased adenosine triphosphate (ATP) concentrations promoted T cell responses by enhancing expression of the CD86 costimulatory molecule on antigen-presenting cells, an effect mediated through the P2X7 purinergic receptor. Elevations of plasma ATP were also detected in a cohort of hypertensive human patients when compared with normotensive controls. Thus, ATP release and the ATP-P2X7 signaling axis represent potential targets to help rein in the proinflammatory sequelae associated with chronic hypertension. Sci. Immunol. 4 , eaau6426 (2019).

Near-infrared squaraine fluorescent probe for imaging adenosine 5′-triphosphate in live cells

Adenosine 5′-triphosphate (ATP), as a “molecular currency” for intracellular energy transfer, not only participates in various physiological activities, but also involves in various pathological processes of living cells. Therefore, the establishment of a simple, rapid, sensitive and accurate ATP analysis method is of great significance for the study of its physiological functions. In this work, we rationally design a near-infrared phenylboronic acid group-functionalized dicyanovinyl squaraine (SQ-PBA3), with which a supramolecular assembly was constructed for fluorescent imaging of ATP. In PBS buffer, SQ-PBA3 changes self-assembly when adding cetyltrimethyl ammonium bromide (CTAB) deriving fluorescence response of SQ-PBA3 to ATP with the maximum emission at 700 nm, due to the specificity of phenylboronic acid to diols and the multiple electrostatic interactions between SQ-PBA3, ATP and CTAB molecules. Meanwhile, the probe was successfully applied to monitor intracellular ATP level in MCF-7 cells with high sensitivity and selectivity.

Nano‐bio interface study between Fe content TiO2 nanoparticles and adenosine triphosphate biomolecules

The advent of nano‐biotechnology has inspired the interface interaction study between engineered nanoparticles (NPs) and biomolecules. The interaction between Fe content titanium dioxide (TiO2) NPs and adenosine triphosphate (ATP) biomolecules has been envisioned. The effect of Fe content in TiO2 matrix was studied using X‐ray diffraction (XRD) and transmission electron microscopy (TEM). The increase in Fe content caused a decrease in particle size with change in morphology from spherical to one‐dimensional rod structure. The Fe incorporation in the TiO2 matrix reduced the transition temperature from anatase to rutile (A‐R) phase along with formation of haematite phase of iron oxide at 400°C. The interaction of Fe content TiO2 NPs with ATP molecule has been studied using spectroscopic method of Raman scattering and infrared vibration spectrum along with TEM. Fe content in TiO2 has enhanced the interaction efficiency of the NPs with ATP biomolecules. Raman spectroscopy confirms that the NPs interact strongly with nitrogen (N7) site in the adenine ring of ATP biomolecule. Engineering of Fe content TiO2 NP could successfully tune the coordination between metal oxide NPs with biomolecules, which could help in designing devices for biomedical applications.

Integration of demand and supply sides in the ATP energy economics of cells

The central aspects of the energy economics of living cells revolve around the synthesis and utilization of molecules of adenosine triphosphate (ATP). Current descriptions of cell metabolism and its regulation in most textbooks of biochemistry assume that enzymes and transporters behave in the same way in isolation and in a cell. Calculations of the mechanistic or maximal P/O ratios in oxidative phosphorylation by mammalian cells generally consider only the supply side of the problem without linking to ATP-demand processes. The purpose of this article is to calculate the mechanistic P/O ratio by integration of the supply and demand sides of ATP reactions. The mechanistic stoichiometry calculated from an integrated approach is compared with that obtained from the standard model that considers only ATP supply. After accounting for leaks, slips, and other losses, the actual or operative P/O calculated by the integrated method is found to be in good agreement with the experimental values of the P/O ratio determined in mitochondria for both succinate and NADH-linked respiratory substrates. The thermodynamic consequences of these results and the biological implications are discussed. An integrated model of oxidative phosphorylation that goes beyond the chemiosmotic theory is presented, and a solution to the longstanding fundamental problem of respiratory control is found.

A Protonic Biotransducer for Controlling ATP Synthesis in Mitochondria

Controlling the flow of ions and electrons at the device/biological interface has become an important challenge in broad fields such as bioelectronics and medical biology. These devices provide a new way to translate bidirectionally between the ionic language of biology and the electronic language of circuitry. The current strategy to develop such ion and electron controlling devices is to use new functional materials at the device/biological interface. For example, silicon nanowire and carbon nanotube based transistors use ions to read out biological functions. Aluminum nanostraws and carbon nanotube porins can selectively deliver molecules such as cations, DNA, and nicotine into biological materials. Since conductive polymers allow the transport of both ions and electrons, there are many conductive polymer based examples including organic field effect transistors for biosensing and organic ion pumps for locally delivering ions and neurotransmitters into cells and the brain. Along with ions and small molecules, protons (H+) play an important role in biology. Examples include the homeostatic pH regulation in body and bacteria, acid sensing ion channels activating in neuron cells, proton activated bioluminescence in dinoflagellates, and pH responsive flagella in bacteria. Mitochondria in particular are a noteworthy organelle that utilize the transport of protons and electrons across a membrane to synthesize adenosine triphosphate (ATP) molecules. To translate H+ signals from biological environments into measureable electronic signals, Rolandi and co-workers developed a prototype bioprotonic device using a Pd/PdHx protode. In his group, the metallic protode was used to measure the protonic conductivity in biological materials such as chitosan and jelly from in the ampullae of Lorenzini of sharks, and to integrate electronic signals with an enzymatic flip flop circuit, ion channels, and light-sensitive bacteriorhodopsin. Gorodestky and co-workers have separately demonstrated high proton conductivity in reflectin squid proteins and Pd/PdHx based transistors. All attempts however focused on the interfacing between Pd-based metal and biological materials, even though the Pd protode may occur side reactions such as oxygen conversion (PdO, PdO2 and Pd(OH)x) at the potentials beginning at 0.9 V vs. NHE and toxic hydrogen peroxide production. Here, we develop an organic biotransducer using a high H+-coupling conductive polymer of sulfonated polyaniline (SPA) that monitors and modulates the pH in the vicinity of the SPA electrode, even in solutions with the high buffering capacity typical of mitochondrial environments (Fig.1). We integrate this biotransducer with mitochondria isolated from pig hearts to translate the H+ signal from the activated mitochondria into a measureable protonic current, and also to control the activity of ATP synthase through electrochemical pH modulation at the SPA surface. Figure 1

Molecular dynamics simulation of the hydration of adenosine phosphates

The hydration behavior of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) has been investigated with molecular dynamics simulations. The spatial distribution of water molecules around different hydration sites of the adenosine phosphates and the average times that water molecules spend in the vicinity of the hydration sites of the phosphate, ribose, and adenine groups have been examined in order to characterize the hydration extent of different regions of the ATP, ADP, and AMP molecules. The water molecules strongly hydrate the negatively-charged phosphate groups of the adenosine phosphates and the interaction between the oxygen and nitrogen atoms of the ribose and adenine rings with water molecules lead to the full hydration of these groups by the water molecules as well. The adenosine phosphates ATP, ADP, and AMP were found to be fully hydrated by the water molecules regardless of the magnitude of their negative charges. Results of this work provide atomic-level information about the hydration of ATP, ADP, and AMP were and are expected to help better understand the behavior of these molecules in biological systems.

The structure of the catalytic domain of the ATP synthase from Mycobacterium smegmatis is a target for developing antitubercular drugs

The crystal structure of the F1-catalytic domain of the adenosine triphosphate (ATP) synthase has been determined from Mycobacterium smegmatis which hydrolyzes ATP very poorly. The structure of the α3β3-component of the catalytic domain is similar to those in active F1-ATPases in Escherichia coli and Geobacillus stearothermophilus However, its ε-subunit differs from those in these two active bacterial F1-ATPases as an ATP molecule is not bound to the two α-helices forming its C-terminal domain, probably because they are shorter than those in active enzymes and they lack an amino acid that contributes to the ATP binding site in active enzymes. In E. coli and G. stearothermophilus, the α-helices adopt an "up" state where the α-helices enter the α3β3-domain and prevent the rotor from turning. The mycobacterial F1-ATPase is most similar to the F1-ATPase from Caldalkalibacillus thermarum, which also hydrolyzes ATP poorly. The βE-subunits in both enzymes are in the usual "open" conformation but appear to be occupied uniquely by the combination of an adenosine 5'-diphosphate molecule with no magnesium ion plus phosphate. This occupation is consistent with the finding that their rotors have been arrested at the same point in their rotary catalytic cycles. These bound hydrolytic products are probably the basis of the inhibition of ATP hydrolysis. It can be envisaged that specific as yet unidentified small molecules might bind to the F1 domain in Mycobacterium tuberculosis, prevent ATP synthesis, and inhibit the growth of the pathogen.

Design and Evaluation of Potent EGFR Inhibitors through the Incorporation of Macrocyclic Polyamine Moieties into the 4-Anilinoquinazoline Scaffold.

Adenosine triphosphate (ATP)-competitive inhibitors of the epidermal growth factor receptor (EGFR) have provided a significant improvement in the disease outcome of nonsmall cell lung cancer (NSCLC). Unfortunately, some marketed drugs affect a transient beneficial response in EGFR mutant NSCLC patients. We reported a series of potential EGFR inhibitors through incorporation of macrocyclic polyamine into 4-anilinoquinazoline scaffold. It is expected that anilinoquinazoline part effectively bind to EGFR domain, while ATP molecules are captured by a macrocyclic polyamine moiety. In vitro experiments exhibited that most of tested compounds suppressed tumor cell proliferation more strongly than Gefitinib and Lapatinib (dual inhibitor of EGFR/HER2) as controls. In kinase assays, the compound 1f showed excellent dual inhibition activity toward EGFRWT (IC50 = 1.4 nM) and HER2 (IC50 = 2.1 nM). In vivo pharmacology evaluation of 1f showed significant antitumor activity (TGI = 44.2%) in A549 xenografts mice. The current work provided a feasible solution to optimize anilinoquinazoline-based inhibitors.

Simultaneous Observation of Kinesin-Driven Microtubule Motility and Binding of Adenosine Triphosphate Using Linear Zero-Mode Waveguides.

Single-molecule fluorescence observation of adenosine triphosphate (ATP) is a powerful tool to elucidate the chemomechanical coupling of ATP with a motor protein. However, in total internal reflection fluorescence microscopy (TIRFM), available ATP concentration is much lower than that in the in vivo environment. To achieve single-molecule observation with a high signal-to-noise ratio, zero-mode waveguides (ZMWs) are utilized even at high fluorescent molecule concentrations in the micromolar range. Despite the advantages of ZMWs, the use of cytoskeletal filaments for single-molecule observation has not been reported because of difficulties in immobilization of cytoskeletal filaments in the cylindrical aperture of ZMWs. Here, we propose linear ZMWs (LZMWs) to visualize enzymatic reactions on cytoskeletal filaments, specifically kinesin-driven microtubule motility accompanied by ATP binding/unbinding. Finite element method simulation revealed excitation light confinement in a 100 nm wide slit of LZMWs. Single-molecule observation was then demonstrated with up to 1 μM labeled ATP, which was 10-fold higher than that available in TIRFM. Direct observation of binding/unbinding of ATP to kinesins that propel microtubules enabled us to find that a significant fraction of ATP molecules bound to kinesins were dissociated without hydrolysis. This highlights the advantages of LZMWs for single-molecule observation of proteins that interact with cytoskeletal filaments such as microtubules, actin filaments, or intermediate filaments.

Dynamic Plasmonic System That Responds to Thermal and Aptamer-Target Regulations.

The DNA origami technique has empowered a new paradigm in plasmonics for manipulating light and matter at the nanoscale. This interdisciplinary field has witnessed vigorous growth, outlining a viable route to construct advanced plasmonic architectures with tailored optical properties. However, so far plasmonic systems templated by DNA origami have been restricted to respond to only single stimuli. Despite broad interest and scientific importance, thermal and aptamer-target regulations have not yet been widely utilized to reconfigure three-dimensional plasmonic architectures. In this Letter, we demonstrate a chiral plasmonic nanosystem integrated with split aptamers, which can respond to both thermal and aptamer-target regulations. We show that our dual-responsive system can be noninvasively tuned in a wide range of temperatures, readily correlating thermal control with optical signal changes. Meanwhile, our system can detect specific targets including adenosine triphosphate and cocaine molecules, which further enhance the optical response modulations and in turn influence the thermal tunability.

Absolute proteome quantification of highly purified populations of circulating reticulocytes and mature erythrocytes

AbstractReticulocytes produced in the bone marrow undergo maturation in the bloodstream to give rise to erythrocytes. Although the proteome of circulating red cells has been the subject of several reports, the cellular populations used for these studies were never completely devoid of reticulocytes. In our current study, we used highly purified erythrocyte and reticulocyte populations to quantify the absolute expression levels of the proteins in each cell population. Erythrocytes and reticulocytes were purified in a multistep process involving cellulose chromatography, Percoll gradient centrifugation, and fluorescence cell sorting after thiazole orange labeling. Proteins were analyzed by mass spectrometry from whole cells and erythrocyte plasma membrane (ghosts), leading to the identification and quantification of 2077 proteins, including 654 that were reticulocyte-specific. Absolute quantifications of these proteins were made using the mean corpuscular hemoglobin content of the cells as a standard. For each protein, we calculated the percentage loss during the terminal stages of reticulocyte maturation and the percentage of association with the plasma membrane. In addition, we used modified adenosine triphosphate and adenosine diphosphate molecules that enable the transfer of a biotin molecule to the catalytic sites of kinases to isolate active kinases in the erythrocytes and determined the absolute expression of 75 protein kinases and the modification of their expression during reticulocyte maturation. Our findings represent the first absolute quantification of proteins that are specifically expressed in normal erythrocytes with no detectable contamination by reticulocytes. Our findings thus represent a reference database for the future proteomic analysis of pathological erythrocytes.

A Type III-B Cmr effector complex catalyzes the synthesis of cyclic oligoadenylate second messengers by cooperative substrate binding.

Recently, Type III-A CRISPR-Cas systems were found to catalyze the synthesis of cyclic oligoadenylates (cOAs), a second messenger that specifically activates Csm6, a Cas accessory RNase and confers antiviral defense in bacteria. To test if III-B CRISPR-Cas systems could mediate a similar CRISPR signaling pathway, the Sulfolobus islandicus Cmr-α ribonucleoprotein complex (Cmr-α–RNP) was purified from the native host and tested for cOA synthesis. We found that the system showed a robust production of cyclic tetra-adenylate (c-A4), and that c-A4 functions as a second messenger to activate the III-B-associated RNase Csx1 by binding to its CRISPR-associated Rossmann Fold domain. Investigation of the kinetics of cOA synthesis revealed that Cmr-α–RNP displayed positively cooperative binding to the adenosine triphosphate (ATP) substrate. Furthermore, mutagenesis of conserved domains in Cmr2α confirmed that, while Palm 2 hosts the active site of cOA synthesis, Palm 1 domain serves as the primary site in the enzyme–substrate interaction. Together, our data suggest that the two Palm domains cooperatively interact with ATP molecules to achieve a robust cOA synthesis by the III-B CRISPR-Cas system.

Interactions of bioactive molecules with thin dendritic glycopolymer layers.

The authors report on highly swellable, stable layers of spherical dendritic glycopolymers, composed of hyperbranched poly(ethylene imine) (PEI) as core and two different maltose shells (A = dense shell and B = open shell). These glycopolymers are cross-linked and attached with poly(ethylene-alt-maleic anhydride) (PEMA) or citric acid on SiOx substrates. The swelling and adsorption of biomolecules were analyzed by spectroscopic ellipsometry and quartz crystal microbalance with dissipation. The swelling degree and complexation with the drug molecule adenosine triphosphate (ATP) were found to be up to 10 times higher for dendritic glycopolymer layers cross-linked with PEMA than for layers cross-linked with citric acid. ATP complexation by electrostatic interaction with the PEI cores was confirmed by x-ray photoelectron spectroscopy analysis. Complexation led to partial collapsing, stiffening, and increase of polymer layer viscosity of the PEMA cross-linked layers. From modeling of ellipsometric data, it was deduced that ATP complexation preferably takes place at the polymer layer-solution interface. The size effect of the adsorbates was investigated by comparing ATP complexation with the adsorption of larger vitamin B12 and human serum albumin (HSA) protein. PEI-Mal A cross-linked with PEMA was found to be resistant toward B12 and HSA adsorption due to the diffusion barrier of the polymer layer. Thus, the authors present potentially biocompatible polymer surfaces for drug loading and their surface supported release.

A Novel Fluorescent Biosensor for Adenosine Triphosphate Detection Based on a Metal⁻Organic Framework Coating Polydopamine Layer.

In this work, a novel and sensitive fluorescent biosensor based on polydopamine coated Zr-based metal–organic framework (PDA/UiO-66) is presented for adenosine triphosphate (ATP) detection. This PDA/UiO-66 nanoparticle which holds a great potential to be excellent fluorescence quencher can protect the 6-carboxyfluorescein (FAM)-labeled probe from cleaved by DNase I dispersed in solution and the flurescence of labeled FAM is quenched. When ATP molecules exist, aptamers on the PDA/UiO-66 nanoparticles can hybridize with ATP molecule to form complex structure that will be desorbed from the PDA/UiO-66 and digested by DNase I. After that, the released ATP molecule can react with another aptamer on the PDA/UiO-66 complexes, then restarts a new cycle. Herein, the excellent strong fluorescence quenching ability and uploading more amount of aptamer probes of PDA/UiO-66 composites make them efficient biosensors, leading to a high sensitivity with detection limit of 35 nM. Compared with ATP detection directly by UiO-66-based method, the LOD is about 5.7 times higher with PDA/UiO-66 nanoparticle. Moreover, the enhanced biocompatibility and bioactivity with PDA layer of the composites render a proposed strategy for clinical diagnosis field of detecting small biological molecules in vivo in the future.

On DNA Motions under Action of Enzymes of Different Types. I

Different types of enzymes in rotational macromolecular motors of living cells are presented based on the data in the literature. Particular attention has been given to the restriction–modification enzymes and helicases, in which conformational changes in sites are physical reasons of linear motions and rotation of DNA molecules (caused by polar forces). It is shown that the structure and functioning of enzymes, similar to those in chiral smectic C-type liquid crystals, can be described within the formalism of generalized forces and currents. These forces include the membrane electrochemical gradient, the velocity of rotor mechanical motion, and the measure of change in free energy in the “fuel” adenosine triphosphate molecule.

Single Molecule Fluorescent Colocalization of Split Aptamers for Ultrasensitive Detection of Biomolecules.

Single-molecule fluorescence imaging is a promising strategy for biomolecule detection. However, the accuracy of single-molecule method is often compromised by the false-positive events at the ultralow sample levels that are caused by the nonspecific adsorption of the fluorescent labeled probe and other fluorescent impurities on the imaging surface. Here, we demonstrate an ultrasensitive single molecule detection assay based on dual-color fluorescent colocalization of spilt aptamers that was implemented to the measurement of adenosine triphosphate (ATP). The ATP aptamer was split into two fragments and labeled with green and red dye molecules, respectively. When the two probes of split aptamers were brought together by the target ATP molecule, the two colors of fluorescence of two probes were simultaneously detected through two channels and projected to the correlated locations in the two halves of image. The colocalizaiton imaging of two split apatamer probes greatly excluded the false detection of biomolecules that was usually caused by the fluorescent noise of single nonbound aptamer probes and impurities, and further improved the accuracy of measurement. The assay showed excellent selectivity and high sensitivity for ATP detection with linear range of 1 pM to 5 nM and a detection limit of 100 fM. This versatile protocol of single molecule colocalization of split apatamer can be widely applied to the ultrasensitive and highly accurate detection of many types of biomolecules in basic research and biomedical applications.