scholarly journals The Nanopore-Tweezing-Based, Targeted Detection of Nucleobases on Short Functionalized Peptide Nucleic Acid Sequences

Polymers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1210
Author(s):  
Isabela S. Dragomir ◽  
Alina Asandei ◽  
Irina Schiopu ◽  
Ioana C. Bucataru ◽  
Loredana Mereuta ◽  
...  
Keyword(s):  
Single Molecule ◽  
Peptide Nucleic Acid ◽  
Conformational Dynamics ◽  
Ionic Current ◽  
Molecular Medicine ◽  
Single Molecule Sequencing ◽  
Stochastic Fluctuations ◽  
Wide Range ◽  
Targeted Detection ◽  
Recognition Capability

The implication of nanopores as versatile components in dedicated biosensors, nanoreactors, or miniaturized sequencers has considerably advanced single-molecule investigative science in a wide range of disciplines, ranging from molecular medicine and nanoscale chemistry to biophysics and ecology. Here, we employed the nanopore tweezing technique to capture amino acid-functionalized peptide nucleic acids (PNAs) with α-hemolysin-based nanopores and correlated the ensuing stochastic fluctuations of the ionic current through the nanopore with the composition and order of bases in the PNAs primary structure. We demonstrated that while the system enables the detection of distinct bases on homopolymeric PNA or triplet bases on heteropolymeric strands, it also reveals rich insights into the conformational dynamics of the entrapped PNA within the nanopore, relevant for perfecting the recognition capability of single-molecule sequencing.

scholarly journals The Nanopore-Tweezing-Based, Targeted Detection of Nucleobases on Short Functionalized Peptide-Nucleic Acid Sequences

2021 ◽  
Author(s):  
Isabela Dragomir ◽  
Alina Asandei ◽  
Irina Schiopu ◽  
Ioana Bucataru ◽  
Loredana Mereuta ◽  
...  
Keyword(s):  
Single Molecule ◽  
Peptide Nucleic Acid ◽  
Peptide Nucleic Acids ◽  
Conformational Dynamics ◽  
Ionic Current ◽  
Single Molecule Sequencing ◽  
The Past ◽  
Stochastic Fluctuations ◽  
Targeted Detection ◽  
Recognition Capability

Quantum leaps advances in the single-molecule investigative science have been made possible over the past decades through the implication of nanopores, as versatile components on dedicated biosensors. Here, we employed the nanopore-tweezing technique to capture amino acid-functionalized, peptide-nucleic acids (PNA) with -hemolysin-based nanopores, and correlate the ensuing stochastic fluctuations of the ionic current through the nanopore with the composition and order of bases in the PNAs primary structure. We demonstrate that while the system enables detection of distinct bases on homopolymeric PNA or triplet bases on heteropolymeric strands, it also reveals rich insights into the conformational dynamics of the entrapped PNA within the nanopore, relevant for perfecting the recognition capability single-molecule sequencing.

scholarly journals Protein dynamics enables phosphorylation of buried residues in Cdk2/Cyclin A-bound p27

2020 ◽  
Author(s):  
João Henriques ◽  
Kresten Lindorff-Larsen
Keyword(s):  
Protein Phosphorylation ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Protein Function ◽  
Cell Cycle Progression ◽  
Conformational Dynamics ◽  
Protein Structures ◽  
Cyclin A ◽  
General Role ◽  
Wide Range

AbstractProteins carry out a wide range of functions that are tightly regulated in space and time. Protein phosphorylation is the most common post-translation modification of proteins and plays key roles in the regulation of many biological processes. The finding that many phosphorylated residues are not solvent exposed in the unphosphorylated state opens several questions for understanding the mechanism that underlies phosphorylation and how phosphorylation may affect protein structures. First, since kinases need access to the phosphorylated residue, how do such buried residues become modified? Second, once phosphorylated, what are the structural effects of phosphorylation of buried residues and do they lead to changed conformational dynamics. We have used the ternary complex between p27, Cdk2 and Cyclin A to study these questions using enhanced sampling molecular dynamics simulations. In line with previous NMR and single-molecule fluorescence experiments we observe transient exposure of Tyr88 in p27, even in its unphosphorylated state. Once Tyr88 is phosphorylated, we observe a coupling to a second site, thus making Tyr74 more easily exposed, and thereby the target for a second phosphorylation step. Our observations provide atomic details on how protein dynamics plays a role in modulating multi-site phosphorylation in p27, thus supplementing previous experimental observations. More generally, we discuss how the observed phenomenon of transient exposure of buried residues may play a more general role in regulating protein function.Significance StatementProtein phosphorylation is a common post-translation modification and is carried out by kinases. While many phosphorylation sites are located in disordered regions of proteins or in loops, a surprisingly large number of modification sites are buried inside folded domains. This observation led us to ask the question of how kinases gain access to such buried residues. We used the complex between p27, a regulator of cell cycle progression, and Cyclin-dependent kinase 2/Cyclin A to study this problem. We hypothesized that transient exposure of buried tyrosines in p27 to the solvent would make them accessible to kinases, explaining how buried residues get modified. We provide an atomic-level description of these dynamic processes revealing how protein dynamics plays a role in regulation.

scholarly journals Molecular fluctuations as a ruler of force-induced protein conformations

2020 ◽  
Author(s):  
Andrew Stannard ◽  
Marc Mora ◽  
Amy E.M. Beedle ◽  
Marta Castro-Lopez ◽  
Stephanie Board ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dynamic Equilibrium ◽  
Conformational Dynamics ◽  
Magnetic Tweezers ◽  
Polymer Physics ◽  
Variance Analysis ◽  
Contour Length ◽  
Stable States ◽  
Wide Range ◽  
Dynamics Simulations

Molecular fluctuations directly reflect the underlying energy landscape. Variance analysis can probe protein dynamics in several biochemistry-driven approaches, yet measurement of probe-independent fluctuations in proteins exposed to mechanical forces remains only accessible through steered molecular dynamics simulations. Using single molecule magnetic tweezers, here we conduct variance analysis to show that individual unfolding and refolding transitions occurring in dynamic equilibrium in a single protein under force are hallmarked by a change in the protein's end-to-end fluctuations, revealing a change in protein stiffness. By unfolding and refolding three structurally distinct proteins under a wide range of constant forces, we demonstrate that the associated change in protein compliance to reach force-induced thermodynamically-stable states scales with the protein's contour length, in agreement with the sequence-independent FJC model of polymer physics. Our findings will help probe the conformational dynamics of proteins exposed to mechanical force at high resolution, of central importance in mechanosensing and mechanotransduction.

Segmentation of Noisy Signals Generated By a Nanopore

2015 ◽  
Author(s):  
Jacob Matthew Schreiber ◽  
Kevin Karplus
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Ionic Current ◽  
Divide And Conquer ◽  
Single Molecule Sequencing ◽  
Automated Method ◽  
Noisy Signals ◽  
Low Pass ◽  
Current Steps ◽  
Pass Through

Nanopore-based single-molecule sequencing techniques exploit ionic current steps produced as biomolecules pass through a pore to reconstruct properties of the sequence. A key task in analyzing complex nanopore data is discovering the boundaries between these steps, which has traditionally been done in research labs by hand. We present an automated method of analyzing nanopore data, by detecting regions of ionic current corresponding to the translocation of a biomolecule, and then segmenting the region. The segmenter uses a divide-and-conquer method to recursively discover boundary points, with an implementation that works several times faster than real time and that can handle low-pass filtered signals.

scholarly journals PPDiffuse: A Quantitative Prediction Tool for Diffusion of Charged Polymers in a Nanopore

2020 ◽  
Vol 125 ◽  
Author(s):  
David P. Hoogerheide
Keyword(s):  
Single Molecule ◽  
Rational Design ◽  
First Passage Time ◽  
Ionic Current ◽  
Passage Time ◽  
Quantitative Prediction ◽  
Analyte Molecule ◽  
Wide Range ◽  
Electrochemical Electrodes ◽  
Set Up

Nanopore-based sensing of charged biopolymers is a powerful single-molecule method. In aconventional nanopore experiment, a single biological (proteinaceous) or solid-state nanopore perforates a thin membrane that is wetted by, and electrically isolates, two opposing reservoirs of electrolyte solution. A potential is applied across the membrane via external electronics coupled to the electrolyte reservoirs with electrochemical electrodes, actuating the system. The electric field set up by the applied potential in the nanopore and its immediate environment plays two roles: supporting an ionic current through the nanopore, which reports on the properties of the pore and its contents; and acting on analyte molecules to attract them to, and drive them into, the nanopore. The presence of a large biopolymer in the pore modulates the ionic current 𝐼(𝑡). The duration of the ionic current modulation corresponds to the length of time the polymer spends in the pore from capture to its ultimate escape, either by retraction to the reservoir from which it was captured, or by translocation to the opposite reservoir . The probabilities of retraction or translocation, or splitting probabilities, and the corresponding distributions of escape times (𝑡esc), are particularly sensitive to the size and charge of the analyte molecule and have been the focus of much theoretical, computational, and experimental effort. An underlying physical framework in which the distribution of escape times is modeled as a first-passage time from a one-dimensional potential is quantitatively predictive for a wide range of experiments. The complexity of this potential for the general case, however, requires calculations to guide experimental design that can be tedious to implement. PPDiffuse is intended to remove this burden from the nanopore research community and enable convenient, rational design of nanopore experiments with complex substrates such as polypeptides.

Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump

2017 ◽  
Author(s):  
Jana Shen ◽  
Zhi Yue ◽  
Helen Zgurskaya ◽  
Wei Chen
Keyword(s):  
Molecular Dynamics ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Conformational Transition ◽  
Conformational Dynamics ◽  
Proton Release ◽  
Intrinsic Resistance ◽  
Constant Ph ◽  
Wide Range ◽  
Constant Ph Molecular Dynamics

AcrB is the inner-membrane transporter of E. coli AcrAB-TolC tripartite efflux complex, which plays a major role in the intrinsic resistance to clinically important antibiotics. AcrB pumps a wide range of toxic substrates by utilizing the proton gradient between periplasm and cytoplasm. Crystal structures of AcrB revealed three distinct conformational states of the transport cycle, substrate access, binding and extrusion, or loose (L), tight (T) and open (O) states. However, the specific residue(s) responsible for proton binding/release and the mechanism of proton-coupled conformational cycling remain controversial. Here we use the newly developed membrane hybrid-solvent continuous constant pH molecular dynamics technique to explore the protonation states and conformational dynamics of the transmembrane domain of AcrB. Simulations show that both Asp407 and Asp408 are deprotonated in the L/T states, while only Asp408 is protonated in the O state. Remarkably, release of a proton from Asp408 in the O state results in large conformational changes, such as the lateral and vertical movement of transmembrane helices as well as the salt-bridge formation between Asp408 and Lys940 and other sidechain rearrangements among essential residues.Consistent with the crystallographic differences between the O and L protomers, simulations offer dynamic details of how proton release drives the O-to-L transition in AcrB and address the controversy regarding the proton/drug stoichiometry. This work offers a significant step towards characterizing the complete cycle of proton-coupled drug transport in AcrB and further validates the membrane hybrid-solvent CpHMD technique for studies of proton-coupled transmembrane proteins which are currently poorly understood. <p><br></p>

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

2018 ◽  
Author(s):  
Alexander Carl DeHaven
Keyword(s):  
Energy Transfer ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
U2 Snrna ◽  
Folding Dynamics ◽  
The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

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