Multiplexed single-molecule assay for enzymatic activity on flow-stretched DNA

2007 ◽  
Vol 4 (5) ◽  
pp. 397-399 ◽  
Author(s):  
Sangjin Kim ◽  
Paul C Blainey ◽  
Charles M Schroeder ◽  
X Sunney Xie
Keyword(s):  
Enzymatic Activity ◽  
Single Molecule

scholarly journals Ultrasensitive Detection of Enzymatic Activity Using Single Molecule Arrays

2020 ◽  
Vol 142 (35) ◽  
pp. 15098-15106
Author(s):  
Xu Wang ◽  
Alana F. Ogata ◽  
David R. Walt
Keyword(s):  
Enzymatic Activity ◽  
Single Molecule ◽  
Ultrasensitive Detection

scholarly journals 1PT236 Monitoring the single-molecule enzymatic activity of β-glucosidase in a microchamber array chip(The 50th Annual Meeting of the Biophysical Society of Japan)

2012 ◽  
Vol 52 (supplement) ◽  
pp. S108-S109
Author(s):  
Kentaro Tahara ◽  
Ryo Iizuka ◽  
Takao Ono ◽  
Kiyohiko Igarashi ◽  
Masahiro Samejima ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Annual Meeting ◽  
Single Molecule ◽  
Biophysical Society

scholarly journals A versatile platform for single-molecule enzymology of restriction endonuclease

2019 ◽  
Vol 12 (01) ◽  
pp. 1841002 ◽  
Author(s):  
Xin Wang ◽  
Jingyuan Nie ◽  
Yi Li ◽  
Hai Pan ◽  
Peng Zheng ◽  
...  
Keyword(s):  
Experimental Design ◽  
Enzymatic Activity ◽  
Single Molecule ◽  
Holliday Junction ◽  
Biological Processes ◽  
Surface Anchoring ◽  
Single Molecule Level ◽  
Enzyme Binding ◽  
Single Molecule Fret ◽  
Single Molecule Enzymology

Enzymes are the major players for many biological processes. Fundamental studies of the enzymatic activity at the single-molecule level provides important information that is otherwise inaccessible at the ensemble level. Yet, these single-molecule experiments are technically difficult and generally require complicated experimental design. Here, we develop a Holliday junction (HJ)-based platform to study the activity of restriction endonucleases at the single-molecule level using single-molecule FRET (sm-FRET). We show that the intrinsic dynamics of HJ can be used as the reporter for both the enzyme-binding and the substrate-release events. Thanks to the multiple-arms structure of HJ, the fluorophore-labeled arms can be different from the surface anchoring arm and the substrate arm. Therefore, it is possible to independently change the substrate arm to study different enzymes with similar functions. Such a design is extremely useful for the systematic study of enzymes from the same family or enzymes bearing different pathologic mutations. Moreover, this method can be easily extended to study other types of DNA-binding enzymes without too much modification of the design. We anticipate it can find broad applications in single-molecule enzymology.

scholarly journals Dynamic disorder in simple enzymatic reactions induces stochastic amplification of substrate

2017 ◽  
Vol 14 (132) ◽  
pp. 20170311 ◽  
Author(s):  
Ankit Gupta ◽  
Andreas Milias-Argeitis ◽  
Mustafa Khammash
Keyword(s):  
Catalytic Activity ◽  
Enzymatic Activity ◽  
Single Molecule ◽  
Measurement Techniques ◽  
Reaction System ◽  
Enzymatic Reactions ◽  
Relative Speed ◽  
Dynamic Disorder ◽  
The Mean ◽  
Single Molecule Enzymology

A growing amount of evidence over the last two decades points to the fact that many enzymes exhibit fluctuations in their catalytic activity, which are associated with conformational changes on a broad range of timescales. The experimental study of this phenomenon, termed dynamic disorder, has become possible thanks to advances in single-molecule enzymology measurement techniques, through which the catalytic activity of individual enzyme molecules can be tracked in time. The biological role and importance of these fluctuations in a system with a small number of enzymes, such as a living cell, have only recently started being explored. In this work, we examine a simple stochastic reaction system consisting of an inflowing substrate and an enzyme with a randomly fluctuating catalytic reaction rate that converts the substrate into an outflowing product. To describe analytically the effect of rate fluctuations on the average substrate abundance at steady state, we derive an explicit formula that connects the relative speed of enzymatic fluctuations with the mean substrate level. Under fairly general modelling assumptions, we demonstrate that the relative speed of rate fluctuations can have a dramatic effect on the mean substrate, and lead to large positive deviations from predictions based on the assumption of deterministic enzyme activity. Our results also establish an interesting connection between the amplification effect and the mixing properties of the Markov process describing the enzymatic activity fluctuations, which can be used to easily predict the fluctuation speed above which such deviations become negligible. As the techniques of single-molecule enzymology continuously evolve, it may soon be possible to study the stochastic phenomena due to enzymatic activity fluctuations within living cells. Our work can be used to formulate experimentally testable hypotheses regarding the nature and magnitude of these fluctuations, as well as their phenotypic consequences.

scholarly journals Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy

2015 ◽  
Vol 112 (45) ◽  
pp. 13904-13909 ◽  
Author(s):  
Qing Guo ◽  
Yufan He ◽  
H. Peter Lu
Keyword(s):  
Enzymatic Activity ◽  
Single Molecule ◽  
Total Internal Reflection ◽  
Enzymatic Reaction ◽  
Magnetic Tweezers ◽  
Amplex Red ◽  
Enzyme Substrate ◽  
Conformational Fluctuation ◽  
Function Relationship ◽  
The Impact

Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure–function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H2O2–amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzyme–substrate interactions, enzyme–substrate active complex formation, and protein folding–binding interactions.

The method of replication affects metal film granularity and resolution

1989 ◽  
Vol 47 ◽  
pp. 708-709
Author(s):  
George C. Ruben
Keyword(s):  
Electron Beam ◽  
High Resolution ◽  
Film Thickness ◽  
Single Molecule ◽  
Metal Film ◽  
Vertical Surface ◽  
High Resolution Imaging ◽  
Controllable Factors ◽  
Resolution Imaging

Single molecule resolution in electron beam sensitive, uncoated, noncrystalline materials has been impossible except in thin Pt-C replicas ≤ 150Å) which are resistant to the electron beam destruction. Previously the granularity of metal film replicas limited their resolution to ≥ 20Å. This paper demonstrates that Pt-C film granularity and resolution are a function of the method of replication and other controllable factors. Low angle 20° rotary , 45° unidirectional and vertical 9.7±1 Å Pt-C films deposited on mica under the same conditions were compared in Fig. 1. Vertical replication had a 5A granularity (Fig. 1c), the highest resolution (table), and coated the whole surface. 45° replication had a 9Å granulartiy (Fig. 1b), a slightly poorer resolution (table) and did not coat the whole surface. 20° rotary replication was unsuitable for high resolution imaging with 20-25Å granularity (Fig. 1a) and resolution 2-3 times poorer (table). Resolution is defined here as the greatest distance for which the metal coat on two opposing faces just grow together, that is, two times the apparent film thickness on a single vertical surface.

Single molecule optical diffraction: A tool for understanding the structure of triple stranded left-hand helical cellulose

1989 ◽  
Vol 47 ◽  
pp. 1024-1025
Author(s):  
George C. Ruben ◽  
William Krakow
Keyword(s):  
Single Molecule ◽  
Helical Structure ◽  
Optical Diffraction ◽  
Maximum Diameter ◽  
Primary Cell Wall ◽  
Cellulose Synthesis ◽  
Left Hand ◽  
Left Handed ◽  
Freeze Dried ◽  
Diffraction Patterns

Tobacco primary cell wall and normal bacterial Acetobacter xylinum cellulose formation produced a 36.8±3Å triple-stranded left-hand helical microfibril in freeze-dried Pt-C replicas and in negatively stained preparations for TEM. As three submicrofibril strands exit the wall of Axylinum , they twist together to form a left-hand helical microfibril. This process is driven by the left-hand helical structure of the submicrofibril and by cellulose synthesis. That is, as the submicrofibril is elongating at the wall, it is also being left-hand twisted and twisted together with two other submicrofibrils. The submicrofibril appears to have the dimensions of a nine (l-4)-ß-D-glucan parallel chain crystalline unit whose long, 23Å, and short, 19Å, diagonals form major and minor left-handed axial surface ridges every 36Å.The computer generated optical diffraction of this model and its corresponding image have been compared. The submicrofibril model was used to construct a microfibril model. This model and corresponding microfibril images have also been optically diffracted and comparedIn this paper we compare two less complex microfibril models. The first model (Fig. 1a) is constructed with cylindrical submicrofibrils. The second model (Fig. 2a) is also constructed with three submicrofibrils but with a single 23 Å diagonal, projecting from a rounded cross section and left-hand helically twisted, with a 36Å repeat, similar to the original model (45°±10° crossover angle). The submicrofibrils cross the microfibril axis at roughly a 45°±10° angle, the same crossover angle observed in microflbril TEM images. These models were constructed so that the maximum diameter of the submicrofibrils was 23Å and the overall microfibril diameters were similar to Pt-C coated image diameters of ∼50Å and not the actual diameter of 36.5Å. The methods for computing optical diffraction patterns have been published before.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

2020 ◽  
Author(s):  
Nikolas Hundt
Keyword(s):  
Single Molecule ◽  
Cellular Systems ◽  
Single Molecule Imaging ◽  
Label Free ◽  
Measurement Sensitivity ◽  
Mass Distributions ◽  
Quantitative Measurements ◽  
Contrast Mechanism ◽  
Cellular Components ◽  
Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

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