scholarly journals Displacement statistics of unhindered single molecules show no enhanced diffusion in enzymatic reactions

2021 ◽  
Author(s):  
Alexander Choi ◽  
Ha Park ◽  
Kun Chen ◽  
Rui Yan ◽  
Wan Li ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Single Molecule ◽  
Confidence Level ◽  
Diffusion Coefficients ◽  
Single Molecules ◽  
Enzymatic Reactions ◽  
Enhanced Diffusion ◽  
Heated Debate ◽  
Better Than

Recent studies have sparked heated debate over whether catalytical reactions would enhance the diffusion coefficients D of enzymes. Through high statistics of the transient (600 μs) displacements of unhindered single molecules freely diffusing in common buffers, we here quantify D for four highly contested enzymes under catalytic turnovers. We thus formulate how precisions of better than ±1% may be achieved for D at the 95% confidence level, and show no changes in diffusivity for catalase, urease, aldolase, and alkaline phosphatase under the application of wide concentration ranges of substrates. Our single-molecule approach thus overcomes potential limitations and artifacts underscored by recent studies to show no enhanced diffusion in enzymatic reactions.

scholarly journals Biological Nanopores: Engineering on Demand

Life ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 27
Author(s):  
Ana Crnković ◽  
Marija Srnko ◽  
Gregor Anderluh
Keyword(s):  
Molecular Biology ◽  
Single Molecule ◽  
Enzymatic Reactions ◽  
Label Free ◽  
Natural Sources ◽  
On Demand ◽  
Label Free Detection ◽  
Long Read ◽  
Inorganic Molecules ◽  
Standard Molecular Biology

Nanopore-based sensing is a powerful technique for the detection of diverse organic and inorganic molecules, long-read sequencing of nucleic acids, and single-molecule analyses of enzymatic reactions. Selected from natural sources, protein-based nanopores enable rapid, label-free detection of analytes. Furthermore, these proteins are easy to produce, form pores with defined sizes, and can be easily manipulated with standard molecular biology techniques. The range of possible analytes can be extended by using externally added adapter molecules. Here, we provide an overview of current nanopore applications with a focus on engineering strategies and solutions.

scholarly journals Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alan M. Szalai ◽  
Bruno Siarry ◽  
Jerónimo Lukin ◽  
David J. Williamson ◽  
Nicolás Unsain ◽  
...  
Keyword(s):  
Single Molecule ◽  
Total Internal Reflection ◽  
Single Molecules ◽  
Fluorescence Microscope ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Axial Position ◽  
Localization Microscopy ◽  
Localization Precision ◽  
Single Molecule Localization Microscopy

AbstractSingle-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule’s image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

Interfacial charge transfer events of BODIPY molecules: single molecule spectroelectrochemistry and substrate effects

2014 ◽  
Vol 16 (42) ◽  
pp. 23150-23156 ◽  
Author(s):  
Jia Liu ◽  
Caleb M. Hill ◽  
Shanlin Pan ◽  
Haiying Liu
Keyword(s):  
Charge Transfer ◽  
Single Molecule ◽  
Single Molecules ◽  
Transfer Mechanism ◽  
Substrate Effects ◽  
Interfacial Charge Transfer ◽  
Charge Transfer Mechanism ◽  
Nanostructured Substrates ◽  
Interfacial Charge

BODIPY dye single molecules on nanostructured substrates are studied with a single molecule spectroelectrochemistry technique to reveal the heterogeneous charge transfer mechanism.

scholarly journals Conformational entropy of a single peptide controlled under force governs protease recognition and catalysis

2018 ◽  
Vol 115 (45) ◽  
pp. 11525-11530 ◽  
Author(s):  
Marcelo E. Guerin ◽  
Guillaume Stirnemann ◽  
David Giganti
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Conformational Sampling ◽  
Enzymatic Reactions ◽  
Sequence Specificity ◽  
Proteomics Data ◽  
Conformational Entropy ◽  
Substrate Peptide ◽  
The Impact

An immense repertoire of protein chemical modifications catalyzed by enzymes is available as proteomics data. Quantifying the impact of the conformational dynamics of the modified peptide remains challenging to understand the decisive kinetics and amino acid sequence specificity of these enzymatic reactions in vivo, because the target peptide must be disordered to accommodate the specific enzyme-binding site. Here, we were able to control the conformation of a single-molecule peptide chain by applying mechanical force to activate and monitor its specific cleavage by a model protease. We found that the conformational entropy impacts the reaction in two distinct ways. First, the flexibility and accessibility of the substrate peptide greatly increase upon mechanical unfolding. Second, the conformational sampling of the disordered peptide drives the specific recognition, revealing force-dependent reaction kinetics. These results support a mechanism of peptide recognition based on conformational selection from an ensemble that we were able to quantify with a torsional free-energy model. Our approach can be used to predict how entropy affects site-specific modifications of proteins and prompts conformational and mechanical selectivity.

scholarly journals A method for imaging single molecules at the plasma membrane of live cells within tissue slices

2020 ◽  
Vol 153 (1) ◽  
Author(s):  
Gregory I. Mashanov ◽  
Tatiana A. Nenasheva ◽  
Tatiana Mashanova ◽  
Catherine Maclachlan ◽  
Nigel J.M. Birdsall ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Lines ◽  
Single Molecule ◽  
Cardiac Tissue ◽  
Single Molecules ◽  
Live Cells ◽  
Biological Macromolecules ◽  
Tissue Slices ◽  
Tissue Samples ◽  
Mammalian Cell Lines

Recent advances in light microscopy allow individual biological macromolecules to be visualized in the plasma membrane and cytosol of live cells with nanometer precision and ∼10-ms time resolution. This allows new discoveries to be made because the location and kinetics of molecular interactions can be directly observed in situ without the inherent averaging of bulk measurements. To date, the majority of single-molecule imaging studies have been performed in either unicellular organisms or cultured, and often chemically fixed, mammalian cell lines. However, primary cell cultures and cell lines derived from multi-cellular organisms might exhibit different properties from cells in their native tissue environment, in particular regarding the structure and organization of the plasma membrane. Here, we describe a simple approach to image, localize, and track single fluorescently tagged membrane proteins in freshly prepared live tissue slices and demonstrate how this method can give information about the movement and localization of a G protein–coupled receptor in cardiac tissue slices. In principle, this experimental approach can be used to image the dynamics of single molecules at the plasma membrane of many different soft tissue samples and may be combined with other experimental techniques.

scholarly journals Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

2020 ◽  
Vol 117 (35) ◽  
pp. 21328-21335
Author(s):  
Zhijie Chen ◽  
Alan Shaw ◽  
Hugh Wilson ◽  
Maxime Woringer ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Theoretical Models ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Single Molecule Level ◽  
Diffusion Phenomena ◽  
Diffusion Enhancement

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments usingp-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish thatpNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models––including those of our own––in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

scholarly journals Effect of Various Diluents on the Activity of Several Enzymes Present in Serum

1973 ◽  
Vol 19 (9) ◽  
pp. 1079-1080
Author(s):  
Ted W Fendley ◽  
Jane M Hochholzer ◽  
Christopher S Frings
Keyword(s):  
Alkaline Phosphatase ◽  
Creatine Kinase ◽  
Enzyme Activity ◽  
Lactate Dehydrogenase ◽  
Confidence Level ◽  
Aspartate Aminotransferase ◽  
Nacl Solution ◽  
Manual Method ◽  
Accuracy And Precision ◽  
Significant Difference

Abstract We have evaluated the effect of diluting serum with water or NaCl solution (8.5 or 9.0 g/liter) before assaying by a manual method for creatine kinase (EC 2.7.3.2), alkaline phosphatase (EC 3.1.3.1), lactate dehydrogenase (EC 1.1.1.27), and aspartate aminotransferase (EC 2.6.1.1) activity. The t test and the F test show no significant difference in the accuracy and precision of the assays at the 95% confidence level when 100 different samples were compared for each enzyme activity after use of the three diluents.

Use of monoclonal antibodies to detect human placental alkaline phosphatase.

1983 ◽  
Vol 29 (1) ◽  
pp. 115-119 ◽  
Author(s):  
G De Groote ◽  
P De Waele ◽  
A Van de Voorde ◽  
M De Broe ◽  
W Fiers
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Solid Phase ◽  
Enzymatic Reactions ◽  
Placental Alkaline Phosphatase ◽  
Human Placental Alkaline Phosphatase ◽  
Human Sera ◽  
Starch Gel ◽  
Alkaline Phosphatase Isoenzyme

Abstract Convenient, sensitive, and specific solid-phase immunoassays involving monoclonal antibody are described for the determination of human placental alkaline phosphatase (hPLAP). An endogenous enzyme immunoassay combined the specificity of the immunological and the enzymatic reactions. Alternatively, a solid-phase "sandwich" radioimmunoassay involving immobilized polyclonal rabbit anti-hPLAP in combination with iodinated monoclonal antibody provided some additional advantages. Both tests can be used to detect hPLAP from various sources, e.g., in human sera during pregnancy or as a tumor marker. The radioimmunoassay detected an increase in hPLAP at nine weeks of gestation. We discuss the use of monoclonal antibodies for the differentiation of different alkaline phosphatase isoenzyme types by electrophoresis on starch gel.

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