scholarly journals Modeling of Ion and Water Transport in the Biological Nanopore ClyA

2020 ◽  
Author(s):  
Kherim Willems ◽  
Dino Ruić ◽  
Florian Lucas ◽  
Ujjal Barman ◽  
Johan Hofkens ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Water Transport ◽  
Single Molecule ◽  
Computational Study ◽  
Molecular Transport ◽  
Navier Stokes ◽  
Ionic Conductance ◽  
Computationally Efficient ◽  
Single Molecule Level ◽  
Wide Range

AbstractIn recent years, the protein nanopore cytolysin A (ClyA) has become a valuable tool for the detection, characterization and quantification of biomarkers, proteins and nucleic acids at the single-molecule level. Despite this extensive experimental utilization, a comprehensive computational study of ion and water transport through ClyA is currently lacking. Such a study yields a wealth of information on the electrolytic conditions inside the pore and on the scale the electrophoretic forces that drive molecular transport. To this end we have built a computationally efficient continuum model of ClyA which, together with an extended version of Poison-Nernst-Planck-Navier-Stokes (ePNP-NS) equations, faithfully reproduces its ionic conductance over a wide range of salt concentrations. These ePNP-NS equations aim to tackle the shortcomings of the traditional PNP-NS models by self-consistently taking into account the influence of both the ionic strength and the nanoscopic scale of the pore on all relevant electrolyte properties. In this study, we give both a detailed description of our ePNP-NS model and apply it to the ClyA nanopore. This enabled us to gain a deeper insight into the influence of ionic strength and applied voltage on the ionic conductance through ClyA and a plethora of quantities difficult to assess experimentally. The latter includes the cation and anion concentrations inside the pore, the shape of the electrostatic potential landscape and the magnitude of the electro-osmotic flow. Our work shows that continuum models of biological nanopores—if the appropriate corrections are applied—can make both qualitatively and quantitatively meaningful predictions that could be valuable tool to aid in both the design and interpretation of nanopore experiments.

Quantitative single molecule FRET efficiencies using TIRF microscopy

2015 ◽  
Vol 184 ◽  
pp. 131-142 ◽  
Author(s):  
Lasse L. Hildebrandt ◽  
Søren Preus ◽  
Victoria Birkedal
Keyword(s):  
Single Molecule ◽  
Structural Information ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Complexes ◽  
Distance Information ◽  
Single Molecule Level ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Intermolecular Distances

Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2–10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (http://www.isms.au.dk), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

scholarly journals Broken force dispersal network in tip-links by the mutations at the Ca2+-binding residues induces hearing-loss

2019 ◽  
Vol 476 (16) ◽  
pp. 2411-2425 ◽  
Author(s):  
Jagadish P. Hazra ◽  
Amin Sagar ◽  
Nisha Arora ◽  
Debadutta Deb ◽  
Simerpreet Kaur ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Inner Ear ◽  
Single Molecule ◽  
Force Sensor ◽  
Force Sensors ◽  
Single Molecule Level ◽  
Conformational Variability ◽  
Atomic Force ◽  
Wide Range ◽  
And Function

Abstract Tip-link as force-sensor in hearing conveys the mechanical force originating from sound to ion-channels while maintaining the integrity of the entire sensory assembly in the inner ear. This delicate balance between structure and function of tip-links is regulated by Ca2+-ions present in endolymph. Mutations at the Ca2+-binding sites of tip-links often lead to congenital deafness, sometimes syndromic defects impairing vision along with hearing. Although such mutations are already identified, it is still not clear how the mutants alter the structure-function properties of the force-sensors associated with diseases. With an aim to decipher the differences in force-conveying properties of the force-sensors in molecular details, we identified the conformational variability of mutant and wild-type tip-links at the single-molecule level using FRET at the endolymphatic Ca2+ concentrations and subsequently measured the force-responsive behavior using single-molecule force spectroscopy with an Atomic Force Microscope (AFM). AFM allowed us to mimic the high and wide range of force ramps (103–106 pN s−1) as experienced in the inner ear. We performed in silico network analysis to learn that alterations in the conformations of the mutants interrupt the natural force-propagation paths through the sensors and make the mutant tip-links vulnerable to input forces from sound stimuli. We also demonstrated that a Ca2+ rich environment can restore the force-response of the mutant tip-links which may eventually facilitate the designing of better therapeutic strategies to the hearing loss.

scholarly journals A Computational Study of a Data Assimilation Algorithm for the Two-dimensional Navier-Stokes Equations

2016 ◽  
Vol 19 (4) ◽  
pp. 1094-1110 ◽  
Author(s):  
Masakazu Gesho ◽  
Eric Olson ◽  
Edriss S. Titi
Keyword(s):  
Data Assimilation ◽  
Stokes Equations ◽  
Computational Study ◽  
Nodal Point ◽  
Navier Stokes ◽  
Reference Solution ◽  
Two Dimensional ◽  
Computationally Efficient ◽  
Navier Stokes Equations ◽  
Feedback Control Theory

AbstractWe study the numerical performance of a continuous data assimilation (downscaling) algorithm, based on ideas from feedback control theory, in the context of the two-dimensional incompressible Navier-Stokes equations. Our model problem is to recover an unknown reference solution, asymptotically in time, by using continuous-in-time coarse-mesh nodal-point observational measurements of the velocity field of this reference solution (subsampling), as might be measured by an array of weather vane anemometers. Our calculations show that the required nodal observation density is remarkably less than what is suggested by the analytical study; and is in fact comparable to the number of numerically determining Fourier modes, which was reported in an earlier computational study by the authors. Thus, this method is computationally efficient and performs far better than the analytical estimates suggest.

scholarly journals Nanometer-accuracy distance measurements between fluorophores at the single-molecule level

2019 ◽  
Vol 116 (10) ◽  
pp. 4275-4284 ◽  
Author(s):  
Stefan Niekamp ◽  
Jongmin Sung ◽  
Walter Huynh ◽  
Gira Bhabha ◽  
Ronald D. Vale ◽  
...  
Keyword(s):  
Single Molecule ◽  
Open Source Software ◽  
Single Molecules ◽  
Coiled Coil ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Distance Measurements ◽  
Single Molecule Level ◽  
Wide Range ◽  
Different Color

Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil “stalk” of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.

Stokes Layers in Horizontal-Wave Outer Flows

1996 ◽  
Vol 118 (3) ◽  
pp. 537-545 ◽  
Author(s):  
J. E. Choi ◽  
M. K. Sreedhar ◽  
F. Stern
Keyword(s):  
Boundary Layer ◽  
Computational Study ◽  
Perturbation Expansion ◽  
Plate Boundary ◽  
Navier Stokes ◽  
Additional Parameter ◽  
Small Disturbance ◽  
Wave Speeds ◽  
Wide Range ◽  
Horizontal Wave

Results are reported of a computational study investigating the responses of flat plate boundary layers and wakes to horizontal wave outer flows. Solutions are obtained for temporal, spatial, and traveling waves using Navier Stokes, boundary layer, and perturbation expansion equations. A wide range of parameters are considered for all the three waves. The results are presented in terms of Stokes-layer overshoots, phase leads (lags), and streaming. The response to the temporal wave showed all the previously reported features. The magnitude and nature of the response are small and simple such that it is essentially a small disturbance on the steady solution. Results are explainable in terms of one parameter ξ (the frequency of oscillation). For the spatial wave, the magnitude and the nature of the response are significantly increased and complex such that it cannot be considered simply a small disturbance on the without-wave solution. The results are explainable in terms of the two parameters λ−1 and x/λ (where λ is the wavelength). A clear asymmetry is observed in the wake response for the spatial wave. An examination of components of the perturbation expansion equations indicates that the asymmetry is a first-order effect due to nonlinear interaction between the steady and first-harmonic velocity components. For the traveling wave, the responses are more complex and an additional parameter, c (the wave speed), is required to explain the results. In general, for small wave speeds the results are similar to a spatial wave, whereas for higher wave speeds the response approaches the temporal wave response. The boundary layer and perturbation expansion solutions compares well with the Navier Stokes solution in their range of validity.

A Comprehensive Through-Flow Solver Method for Modern Turbomachinery Design

2015 ◽  
Author(s):  
Mark R. Anderson ◽  
Daryl L. Bonhaus
Keyword(s):  
High Speed ◽  
Three Dimensional ◽  
Optimization Techniques ◽  
Navier Stokes ◽  
Computationally Efficient ◽  
Dimensional Solution ◽  
Loss Model ◽  
Flow Solver ◽  
Through Flow ◽  
Wide Range

Through-flow solvers have historically played a very prominent role in the design and analysis of axial turbomachinery. While three-dimensional, Full Navier-Stokes (FNS) CFD is taking an increasing larger role, quasi-3D through-flow methods are still widely used. Automated optimization techniques that search over a wide design space, involving many possible variables, are particularly suitable for the computationally efficient through-flow solver. Pressure-based methods derived from CFD solution techniques have gradually replaced older streamline curvature methods, due to their ability to capture flow across a wide range of Mach numbers, particularly the transonic and supersonic regimes. The through-flow approach allows for the solution of the three-dimensional problem with the computational efficiency of a two-dimensional solution. Since the losses are explicitly calculated through empirically based models, the need for detailed grid resolution to capture tiny flow entities (such as wakes and boundary layers) is also greatly reduced. The combined savings can result in computational costs as much as two orders of magnitude lower than full 3D CFD methods. A state-of-the-art through-flow solver has several features that are crucial in the design process. One of these is the ability to run in both a design and an analysis mode. Also important, is the ability to generate solutions where critical components are solved using 3D FNS, while others are run using a through-flow method. Other desirable features in a through-flow solver are: an advanced equation of state, injection and extraction ability, the handling of arbitrary (non-axial) shapes, and a link to a capable geometry generation engine. Through-flow solvers represent a unique mix of higher order numerical methods (increasingly CFD-based) coupled with empirically derived models (generally meanline based). The combination of these two methods in one solver creates a particularly challenging programming problem. This paper details the techniques required to effectively generate through-flow solutions. Special attention is given to an improved off-design loss model for compressors, as well as a transonic loss model needed for high-speed compressor and turbine flows. Validation with recognized test data along with corresponding 3D FNS CFD results are presented.

scholarly journals A flow extension tethered particle motion assay for single-molecule proteolysis

2019 ◽  
Author(s):  
Andrew A. Drabek ◽  
Joseph J. Loparo ◽  
Stephen C. Blacklow
Keyword(s):  
Single Molecule ◽  
Magnetic Force ◽  
Magnetic Tweezers ◽  
Signaling Proteins ◽  
Mechanical Tension ◽  
Tobacco Etch Virus Protease ◽  
Single Molecule Level ◽  
Wide Range ◽  
Tethered Particle Motion

AbstractRegulated proteolysis of signaling proteins under mechanical tension enables cells to communicate with their environment in a variety of developmental and physiologic contexts. The role of force in inducing proteolytic sensitivity has been explored using magnetic tweezers at the single-molecule level with bead-tethered assays, but such efforts have been limited by challenges in ensuring that beads are not restrained by multiple tethers. Here, we describe a multiplexed assay for single-molecule proteolysis that overcomes the multiple-tether problem using a flow extension (FLEX) strategy on a microscope equipped with magnetic tweezers. Particle tracking and computational sorting of flow-induced displacements allows assignment of tethered substrates into singly-captured and multiply-tethered bins, with the fraction of fully mobile, single-tethered substrates depending inversely on the concentration of substrate loaded on the coverslip. Computational exclusion of multiply-tethered beads enables robust assessment of on-target proteolysis by the highly specific tobacco etch virus protease and the more promiscuous metalloprotease ADAM17. This method should be generally applicable to a wide range of proteases and readily extensible to robust evaluation of proteolytic sensitivity as a function of applied magnetic force.

scholarly journals Toward Sub-Diffraction Imaging of Single-DNA Molecule Sensors Based on Stochastic Switching Localization Microscopy

Sensors ◽  
2020 ◽  
Vol 20 (22) ◽  
pp. 6667
Author(s):  
Seungah Lee ◽  
Indra Batjikh ◽  
Seong Ho Kang
Keyword(s):  
Single Molecule ◽  
Specific Binding ◽  
Super Resolution ◽  
Localization Microscopy ◽  
Single Molecule Level ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Wide Range ◽  
Nanoscale Topography ◽  
Diffraction Imaging ◽  
Optical Reconstruction

The natural characteristics of deoxyribonucleic acid (DNA) enable its advanced applications in nanotechnology as a special tool that can be detected by high-resolution imaging with precise localization. Super-resolution (SR) microscopy enables the examination of nanoscale molecules beyond the diffraction limit. With the development of SR microscopy methods, DNA nanostructures can now be optically assessed. Using the specific binding of fluorophores with their target molecules, advanced single-molecule localization microscopy (SMLM) has been expanded into different fields, allowing wide-range detection at the single-molecule level. This review discusses the recent progress in the SR imaging of DNA nano-objects using SMLM techniques, such as direct stochastic optical reconstruction microscopy, binding-activated localization microscopy, and point accumulation for imaging nanoscale topography. Furthermore, we discuss their advantages and limitations, present applications, and future perspectives.

scholarly journals Supercoiling DNA optically

2019 ◽  
Vol 116 (52) ◽  
pp. 26534-26539
Author(s):  
Graeme A. King ◽  
Federica Burla ◽  
Erwin J. G. Peterman ◽  
Gijs J. L. Wuite
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Dna Supercoiling ◽  
Biological Interactions ◽  
Mitochondrial Transcription ◽  
Supercoiled Dna ◽  
Single Molecule Level ◽  
Mitochondrial Transcription Factor ◽  
Wide Range

Cellular DNA is regularly subject to torsional stress during genomic processes, such as transcription and replication, resulting in a range of supercoiled DNA structures. For this reason, methods to prepare and study supercoiled DNA at the single-molecule level are widely used, including magnetic, angular-optical, micropipette, and magneto-optical tweezers. However, it is currently challenging to combine DNA supercoiling control with spatial manipulation and fluorescence microscopy. This limits the ability to study complex and dynamic interactions of supercoiled DNA. Here we present a single-molecule assay that can rapidly and controllably generate negatively supercoiled DNA using a standard dual-trap optical tweezers instrument. This method, termed Optical DNA Supercoiling (ODS), uniquely combines the ability to study supercoiled DNA using force spectroscopy, fluorescence imaging of the whole DNA, and rapid buffer exchange. The technique can be used to generate a wide range of supercoiled states, with between <5 and 70% lower helical twist than nonsupercoiled DNA. Highlighting the versatility of ODS, we reveal previously unobserved effects of ionic strength and sequence on the structural state of underwound DNA. Next, we demonstrate that ODS can be used to directly visualize and quantify protein dynamics on supercoiled DNA. We show that the diffusion of the mitochondrial transcription factor TFAM can be significantly hindered by local regions of underwound DNA. This finding suggests a mechanism by which supercoiling could regulate mitochondrial transcription in vivo. Taken together, we propose that ODS represents a powerful method to study both the biophysical properties and biological interactions of negatively supercoiled DNA.

scholarly journals Finite-Temperature Single Molecule Vibrational Dynamics from Combined Density Functional Tight Binding Extended Lagrangian Dynamics Simulations and Time Series Analysis

2020 ◽  
Vol 20 (6) ◽  
pp. 201-212
Author(s):  
Bojana Koteska ◽  
Anastas Mishev ◽  
Ljupco Pejov
Keyword(s):  
Molecular Dynamics ◽  
Time Series ◽  
Single Molecule ◽  
Density Functional ◽  
Tight Binding ◽  
Range Data ◽  
Computationally Efficient ◽  
Vibrational Dynamics ◽  
Wide Range ◽  
Dynamics Simulations

AbstractCombining a computationally efficient and affordable molecular dynamics approach, based on atom-centered density matrix propagation scheme, with the density functional tight binding semiempirical quantum mechanics, we study the vibrational dynamics of a single molecule at series of finite temperatures, spanning quite wide range. Data generated by molecular dynamics simulations are further analyzed and processed using time series analytic methods, based on correlation functions formalism, leading to both vibrational density of states spectra and infrared absorption spectra at finite temperatures. The temperature-induced dynamics in structural intramolecular parameters is correlated to the observed changes in the spectral regions relevant to molecular detection. In particular, we consider a case when an intramolecular X-H stretching vibrational states are notably dependent on the intramolecular torsional degree of freedom, the dynamics of which is, on the other hand, strongly temperature-dependent.

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