scholarly journals Experiment-friendly kinetic analysis of single molecule data in and out of equilibrium

2016 ◽  
Author(s):  
Sonja Schmid ◽  
Markus Götz ◽  
Thorsten Hugel
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Thermodynamic Quantities ◽  
Kinetic Rate ◽  
Complete Dataset ◽  
Likelihood Approach ◽  
Single Molecule Analysis ◽  
Kinetics Of

We present a simple and robust technique to extract kinetic rate models and thermodynamic quantities from single molecule time traces. SMACKS (Single Molecule Analysis of Complex Kinetic Sequences) is a maximum likelihood approach that works equally well for long trajectories as for a set of short ones. It resolves all statistically relevant rates and also their uncertainties. This is achieved by optimizing one global kinetic model based on the complete dataset, while allowing for experimental variations between individual trajectories. In particular, neither a priori models nor equilibrium have to be assumed. The power of SMACKS is demonstrated on the kinetics of the multi-domain protein Hsp90 measured by smFRET (single molecule Förster resonance energy transfer). Experiments in and out of equilibrium are analyzed and compared to simulations, shedding new light on the role of Hsp90’s ATPase function. SMACKS pushes the boundaries of single molecule kinetics far beyond current methods.

scholarly journals Affinity of Skp to OmpC revealed by single-molecule detection

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sichen Pan ◽  
Chen Yang ◽  
Xin Sheng Zhao
Keyword(s):  
Single Molecule ◽  
Molecular Chaperones ◽  
Outer Membrane Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Hill Coefficient ◽  
Stoichiometric Ratio ◽  
Single Molecule Level

Abstract Outer membrane proteins (OMPs) are essential to gram-negative bacteria, and molecular chaperones prevent the OMPs from aggregation in the periplasm during the OMPs biogenesis. Skp is one of the molecular chaperones for this purpose. Here, we combined single-molecule fluorescence resonance energy transfer and fluorescence correlation spectroscopy to study the affinity and stoichiometric ratio of Skp in its binding with OmpC at the single-molecule level. The half concentration of the Skp self-trimerization (C1/2) was measured to be (2.5 ± 0.7) × 102 nM. Under an Skp concentration far below the C1/2, OmpC could recruit Skp monomers to form OmpC·Skp3. The affinity to form the OmpC·Skp3 complex was determined to be (5.5 ± 0.4) × 102 pM with a Hill coefficient of 1.6 ± 0.2. Under the micromolar concentrations of Skp, the formation of OmpC·(Skp3)2 was confirmed, and the dissociation constant of OmpC·(Skp3)2 was determined to be 1.2 ± 0.4 μM. The precise information will help us to quantitatively depict the role of Skp in the biogenesis of OMPs.

scholarly journals A Single Molecule Investigation of I-Motif: Stability, Folding Kinetics, and Potential as an In-situ pH Sensor

2021 ◽  
Author(s):  
Golam Mustafa ◽  
Prabesh Gyawali ◽  
Jacob A. Taylor ◽  
Parastoo Maleki ◽  
Marlon V. Nunez ◽  
...  
Keyword(s):  
Single Molecule ◽  
Ph Sensor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Telomeric Sequence ◽  
Folding Kinetics ◽  
Single Molecule Level ◽  
Oxygen Scavenging ◽  
Kinetics Of

We present a collection of single molecule work on the i-motif structure formed by the human telomeric sequence. Even though it was largely ignored in earlier years of its discovery due to its modest stability and requirement for physiologically low pH levels (pH<6.5), the i-motif has been attracting more attention recently as both a physiologically relevant structure and as a potent pH sensor. In this manuscript, we establish single molecule F&oumlrster resonance energy transfer (smFRET) as a tool to study the i-motif over a broad pH and ionic conditions. We demonstrate pH and salt dependence of i-motif formation under steady state conditions and illustrate the kinetics of i-motif folding in real time at the single molecule level. We also show the prominence of intermediate folding states and reversible folding/unfolding transitions. We present an example of using the i-motif as an in-situ pH sensor and use this sensor establish the time scale for the pH drop in a commonly used oxygen scavenging system.

scholarly journals Determinants of cyclization–decyclization kinetics of short DNA with sticky ends

2020 ◽  
Vol 48 (9) ◽  
pp. 5147-5156 ◽  
Author(s):  
Jiyoun Jeong ◽  
Harold D Kim
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Helical Axis ◽  
Finite Width ◽  
Base Pairs ◽  
Technical Issues ◽  
Sticky Ends ◽  
Helical Turn ◽  
Kinetics Of

Abstract Cyclization of DNA with sticky ends is commonly used to measure DNA bendability as a function of length and sequence, but how its kinetics depend on the rotational positioning of the sticky ends around the helical axis is less clear. Here, we measured cyclization (looping) and decyclization (unlooping) rates (kloop and kunloop) of DNA with sticky ends over three helical periods (100-130 bp) using single-molecule fluorescence resonance energy transfer (FRET). kloop showed a nontrivial undulation as a function of DNA length whereas kunloop showed a clear oscillation with a period close to the helical turn of DNA (∼10.5 bp). The oscillation of kunloop was almost completely suppressed in the presence of gaps around the sticky ends. We explain these findings by modeling double-helical DNA as a twisted wormlike chain with a finite width, intrinsic curvature, and stacking interaction between the end base pairs. We also discuss technical issues for converting the FRET-based cyclization/decyclization rates to an equilibrium quantity known as the J factor that is widely used to characterize DNA bending mechanics.

scholarly journals Kinetics of the conformational cycle of Hsp70 reveals the importance of the dynamic and heterogeneous nature of Hsp70 for its function

2020 ◽  
Vol 117 (14) ◽  
pp. 7814-7823 ◽  
Author(s):  
Si Wu ◽  
Liu Hong ◽  
Yuqing Wang ◽  
Jieqiong Yu ◽  
Jie Yang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Substrate Binding ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bound State ◽  
Kinetic Measurements ◽  
Kinetics Of ◽  
Conformational Distribution

Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an “ADP-like” undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70–Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.

scholarly journals Monitoring RNA dynamics in native transcriptional complexes

2021 ◽  
Vol 118 (45) ◽  
pp. e2106564118
Author(s):  
Adrien Chauvier ◽  
Patrick St-Pierre ◽  
Jean-François Nadon ◽  
Elsa D. M. Hien ◽  
Cibrán Pérez-González ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Start Codon ◽  
Biological Processes ◽  
Rna Structures ◽  
Transient Nature ◽  
Translation Start ◽  
Metabolite Binding

Cotranscriptional RNA folding is crucial for the timely control of biological processes, but because of its transient nature, its study has remained challenging. While single-molecule Förster resonance energy transfer (smFRET) is unique to investigate transient RNA structures, its application to cotranscriptional studies has been limited to nonnative systems lacking RNA polymerase (RNAP)–dependent features, which are crucial for gene regulation. Here, we present an approach that enables site-specific labeling and smFRET studies of kilobase-length transcripts within native bacterial complexes. By monitoring Escherichia coli nascent riboswitches, we reveal an inverse relationship between elongation speed and metabolite-sensing efficiency and show that pause sites upstream of the translation start codon delimit a sequence hotspot for metabolite sensing during transcription. Furthermore, we demonstrate a crucial role of the bacterial RNAP actively delaying the formation, within the hotspot sequence, of competing structures precluding metabolite binding. Our approach allows the investigation of cotranscriptional regulatory mechanisms in bacterial and eukaryotic elongation complexes.

scholarly journals Determinants of cyclization-decyclization kinetics of short DNA with sticky ends

2018 ◽  
Author(s):  
Jiyoun Jeong ◽  
Harold D Kim
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Helical Axis ◽  
Chain Model ◽  
Sticky Ends ◽  
Cyclization Rate ◽  
Kinetics Of ◽  
Shed Light ◽  
Good Agreement

Cyclization of DNA with sticky ends is commonly used to construct DNA minicircles and to measure DNA bendability. The cyclization probability of short DNA (<150 bp) has a strong length dependence, but how it depends on the rotational positioning of the sticky ends around the helical axis is less clear. To shed light upon the determinants of the cyclization probability of short DNA, we measured cyclization and decyclization rates of ~100-bp DNA with sticky ends over two helical periods using single-molecule Fluorescence Resonance Energy Transfer (FRET). The cyclization rate increases monotonically with length, indicating no excess twisting, while the decyclization rate oscillates with length, higher at half-integer helical turns and lower at integer helical turns. The oscillation profile is kinetically and thermodynamically consistent with a three-state cyclization model in which sticky-ended short DNA first bends into a torsionally-relaxed teardrop, and subsequently transitions to a more stable loop upon terminal base stacking. We also show that the looping probability density (the J factor) extracted from this study is in good agreement with the worm-like chain model near 100 bp. For shorter DNA, we discuss various experimental factors that prevent an accurate measurement of the J factor.

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.

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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