scholarly journals Computational Tool for Ensemble Averaging of Single-Molecule Data

2020 ◽  
Author(s):  
Thomas Blackwell ◽  
W. Tom Stump ◽  
Sarah R. Clippinger ◽  
Michael J. Greenberg
Keyword(s):  
Brownian Motion ◽  
User Interface ◽  
Single Molecule ◽  
Conformational Changes ◽  
Graphical User Interface ◽  
Molecular Motions ◽  
Computational Tool ◽  
Ensemble Averaging ◽  
Mechanochemical Coupling ◽  
Kinetics Of

AbstractMolecular motors couple chemical transitions to conformational changes that perform mechanical work in a wide variety of biological processes. Disruption of this coupling can lead to diseases, and therefore there is a need to accurately measure mechanochemical coupling in motors in both health and disease. Optical tweezers, with nanometer spatial and millisecond temporal resolution, have provided valuable insights into these processes. However, fluctuations due to Brownian motion can make it difficult to precisely resolve these conformational changes. One powerful analysis technique that has improved our ability to accurately measure mechanochemical coupling in motor proteins is ensemble averaging of individual trajectories. Here, we present a user-friendly computational tool, Software for Precise Analysis of Single Molecules (SPASM), for generating ensemble averages of single-molecule data. This tool utilizes several conceptual advances, including optimized procedures for identifying single-molecule interactions and the implementation of a change point algorithm, to more precisely resolve molecular transitions. Using both simulated and experimental data, we demonstrate that these advances allow for accurate determination of the mechanics and kinetics of the myosin working stroke with a smaller set of data. Importantly, we provide our open source MATLAB-based program with a graphical user interface that enables others to readily apply these advances to the analysis of their own data.Statement of SignificanceSingle molecule optical trapping experiments have given unprecedented insights into the mechanisms of molecular machines. Analysis of these experiments is often challenging because Brownian motion-induced fluctuations introduce noise that can obscure molecular motions. A powerful technique for analyzing these noisy traces is ensemble averaging of individual binding interactions, which can uncover information about the mechanics and kinetics of molecular motions that are typically obscured by Brownian motion. Here, we provide an open source, easy-to-use computational tool, SPASM, with a graphical user interface for ensemble averaging of single molecule data. This computational tool utilizes several conceptual advances that significantly improve the accuracy and resolution of ensemble averages, enabling the generation of high-resolution averages from a smaller number of binding interactions.

scholarly journals Computational Tool for Ensemble Averaging of Single-Molecule Data

2021 ◽  
Vol 120 (1) ◽  
pp. 10-20
Author(s):  
Thomas Blackwell ◽  
W. Tom Stump ◽  
Sarah R. Clippinger ◽  
Michael J. Greenberg
Keyword(s):  
Single Molecule ◽  
Computational Tool ◽  
Ensemble Averaging

scholarly journals Single molecule kinetics of bacteriorhodopsin by HS-AFM

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alma P. Perrino ◽  
Atsushi Miyagi ◽  
Simon Scheuring
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
High Speed ◽  
Continuous Light ◽  
Photon Absorption ◽  
Short Pulses ◽  
Reaction Cycle ◽  
Force Microscopy ◽  
Molecule Reaction ◽  
Kinetics Of

AbstractBacteriorhodopsin is a seven-helix light-driven proton-pump that was structurally and functionally extensively studied. Despite a wealth of data, the single molecule kinetics of the reaction cycle remain unknown. Here, we use high-speed atomic force microscopy methods to characterize the single molecule kinetics of wild-type bR exposed to continuous light and short pulses. Monitoring bR conformational changes with millisecond temporal resolution, we determine that the cytoplasmic gate opens 2.9 ms after photon absorption, and stays open for proton capture for 13.2 ms. Surprisingly, a previously active protomer cannot be reactivated for another 37.6 ms, even under excess continuous light, giving a single molecule reaction cycle of ~20 s−1. The reaction cycle slows at low light where the closed state is prolonged, and at basic or acidic pH where the open state is extended.

A Software Tool for Simulating Motions and Loading in Planar Tensegrity Structures

2014 ◽  
Author(s):  
Carl A. Nelson
Keyword(s):  
User Interface ◽  
Graphical User Interface ◽  
Software Tool ◽  
Computational Tool ◽  
Tensegrity Structures ◽  
Indeterminate Structures

Tensegrity structures are unique in the sense that they tend to be overconstrained as mechanisms, yet they can be mobile due to the elastic nature of their tensile elements. The ability to easily visualize these motion properties would help designers to create new and useful tensegrity mechanisms. In this paper, a simple and interactive computational tool with a graphical user interface is presented for visualizing loading and displacements in tensegrity structures. This tool can also be extended for use with traditional static structures, including both determinate and indeterminate structures, as well as mechanisms.

scholarly journals F1-ATPase conformational cycle from simultaneous single-molecule FRET and rotation measurements

2016 ◽  
Vol 113 (21) ◽  
pp. E2916-E2924 ◽  
Author(s):  
Mitsuhiro Sugawa ◽  
Kei-ichi Okazaki ◽  
Masaru Kobayashi ◽  
Takashi Matsui ◽  
Gerhard Hummer ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Single Molecule ◽  
Conformational Changes ◽  
Molecular Motor ◽  
Principal Component ◽  
Structural Basis ◽  
F1 Atpase ◽  
Single Molecule Fret ◽  
Mechanochemical Coupling ◽  
Central Shaft

Despite extensive studies, the structural basis for the mechanochemical coupling in the rotary molecular motor F1-ATPase (F1) is still incomplete. We performed single-molecule FRET measurements to monitor conformational changes in the stator ring-α3β3, while simultaneously monitoring rotations of the central shaft-γ. In the ATP waiting dwell, two of three β-subunits simultaneously adopt low FRET nonclosed forms. By contrast, in the catalytic intermediate dwell, two β-subunits are simultaneously in a high FRET closed form. These differences allow us to assign crystal structures directly to both major dwell states, thus resolving a long-standing issue and establishing a firm connection between F1 structure and the rotation angle of the motor. Remarkably, a structure of F1 in an ε-inhibited state is consistent with the unique FRET signature of the ATP waiting dwell, while most crystal structures capture the structure in the catalytic dwell. Principal component analysis of the available crystal structures further clarifies the five-step conformational transitions of the αβ-dimer in the ATPase cycle, highlighting the two dominant modes: the opening/closing motions of β and the loosening/tightening motions at the αβ-interface. These results provide a new view of tripartite coupling among chemical reactions, stator conformations, and rotary angles in F1-ATPase.

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 Nanopores: a versatile tool to study protein dynamics

2020 ◽  
Author(s):  
Sonja Schmid ◽  
Cees Dekker
Keyword(s):  
Protein Dynamics ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Function ◽  
Label Free ◽  
Cellular Functions ◽  
Ensemble Averaging ◽  
Time Resolved ◽  
Wide Range ◽  
Versatile Tool

Abstract Proteins are the active workhorses in our body. These biomolecules perform all vital cellular functions from DNA replication and general biosynthesis to metabolic signaling and environmental sensing. While static 3D structures are now readily available, observing the functional cycle of proteins – involving conformational changes and interactions – remains very challenging, e.g., due to ensemble averaging. However, time-resolved information is crucial to gain a mechanistic understanding of protein function. Single-molecule techniques such as FRET and force spectroscopies provide answers but can be limited by the required labelling, a narrow time bandwidth, and more. Here, we describe electrical nanopore detection as a tool for probing protein dynamics. With a time bandwidth ranging from microseconds to hours, nanopore experiments cover an exceptionally wide range of timescales that is very relevant for protein function. First, we discuss the working principle of label-free nanopore experiments, various pore designs, instrumentation, and the characteristics of nanopore signals. In the second part, we review a few nanopore experiments that solved research questions in protein science, and we compare nanopores to other single-molecule techniques. We hope to make electrical nanopore sensing more accessible to the biochemical community, and to inspire new creative solutions to resolve a variety of protein dynamics – one molecule at a time.

scholarly journals High-throughput smFRET analysis of freely diffusing nucleic acid molecules and associated proteins

2019 ◽  
Author(s):  
Maya Segal ◽  
Antonino Ingargiola ◽  
Eitan Lerner ◽  
Sang Yoon Chung ◽  
Jonathan A. White ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Conformational Changes ◽  
Single Photon ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Kinetic Studies ◽  
Ensemble Averaging ◽  
Single Spot ◽  
Biologically Relevant

AbstractSingle-molecule Förster resonance energy transfer (smFRET) is a powerful technique for nanometer-scale studies of single molecules. Solution-based smFRET, in particular, can be used to study equilibrium intra- and intermolecular conformations, binding/unbinding events and conformational changes under biologically relevant conditions without ensemble averaging. However, single-spot smFRET measurements in solution are slow. Here, we detail a high-throughput smFRET approach that extends the traditional single-spot confocal geometry to a multispot one. The excitation spots are optically conjugated to two custom silicon single photon avalanche diode (SPAD) arrays. Two-color excitation is implemented using a periodic acceptor excitation (PAX), allowing distinguishing between singly- and doubly-labeled molecules. We demonstrate the ability of this setup to rapidly and accurately determine FRET efficiencies and population stoichiometries by pooling the data collected independently from the multiple spots. We also show how the high throughput of this approach can be used to increase the temporal resolution of single-molecule FRET population characterization from minutes to seconds. Combined with microfluidics, this high-throughput approach will enable simple real-time kinetic studies as well as powerful molecular screening applications.

scholarly journals Harvesting Mechanical Work From Folding-Based Protein Engines: From Single-Molecule Mechanochemical Cycles to Macroscopic Devices

CCS Chemistry ◽  
2019 ◽  
pp. 138-147 ◽  
Author(s):  
Linglan Fu ◽  
Han Wang ◽  
Hongbin Li
Keyword(s):  
Protein Folding ◽  
Single Molecule ◽  
Conformational Changes ◽  
Molecular Motors ◽  
Building Blocks ◽  
Mechanical Work ◽  
Energy Output ◽  
Mechanochemical Coupling ◽  
Soft Actuators ◽  
Generation Mechanisms

Mechanochemical coupling cycles underlie the work-generation mechanisms of biological systems and are realized by highly regulated conformational changes of the protein machineries. However, it has been challenging to utilize protein conformational changes to do mechanical work at the macroscopic level in biomaterials, and it remains elusive to construct macroscopic mechanochemical devices based on molecular-level mechanochemical coupling systems. Here, the authors demonstrate that protein folding can be utilized to realize protein’s mechanochemical cycles at both single-molecule and macroscopic levels. Our results demonstrate, for the first time, the successful harnessing of mechanical work generated by protein folding in a macroscopic protein hydrogel device, and the work generated by protein folding compares favorably with the energy output of molecular motors. Our work bridges a gap between single-molecule and macroscopic levels, and paves the way to utilizing proteins as building blocks to design protein-based artificial muscles and soft actuators.

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