Combining Rapid Microfluidic Mixing and Three-Color Single-Molecule FRET for Probing the Kinetics of Protein Conformational Changes

2021 ◽  
Author(s):  
Stephan Benke ◽  
Andrea Holla ◽  
Bengt Wunderlich ◽  
Andrea Soranno ◽  
Daniel Nettels ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Microfluidic Mixing ◽  
Single Molecule Fret ◽  
Protein Conformational Changes ◽  
Kinetics Of

scholarly journals Single-Molecule Fret Analysis of Key Protein Conformational Changes During Promoter Escape by RNA Polymerase

2021 ◽  
Vol 120 (3) ◽  
pp. 109a
Author(s):  
Anna Wang ◽  
Abhishek Mazumder ◽  
Achillefs N. Kapanidis
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Conformational Changes ◽  
Promoter Escape ◽  
Single Molecule Fret ◽  
Protein Conformational Changes

scholarly journals Real-time analysis of RAG complex activity in V(D)J recombination

2016 ◽  
Vol 113 (42) ◽  
pp. 11853-11858 ◽  
Author(s):  
Jennifer Zagelbaum ◽  
Noriko Shimazaki ◽  
Zitadel Anne Esguerra ◽  
Go Watanabe ◽  
Michael R. Lieber ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Conformational Changes ◽  
Signal Sequence ◽  
High Mobility ◽  
Time Analysis ◽  
Complex Activity ◽  
Synaptic Complex ◽  
Single Molecule Fret ◽  
Real Time Analysis

Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS–RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.

scholarly journals Probing Conformational Changes and Dynamics in eIF4A Helicase during RNA Unwinding by Single-Molecule FRET

2013 ◽  
Vol 104 (2) ◽  
pp. 421a
Author(s):  
Yingjie Sun ◽  
Amit Meller
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Single Molecule Fret ◽  
Rna Unwinding

scholarly journals Using single-molecule FRET to probe the nucleotide-dependent conformational landscape of polymerase β-DNA complexes

2020 ◽  
Vol 295 (27) ◽  
pp. 9012-9020
Author(s):  
Carel Fijen ◽  
Mariam M. Mahmoud ◽  
Meike Kronenberg ◽  
Rebecca Kaup ◽  
Mattia Fontana ◽  
...  
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Conformational Changes ◽  
Dna Complexes ◽  
Single Molecule Fret ◽  
Nucleotide Incorporation ◽  
Eukaryotic Dna ◽  
Cellular Dna ◽  
Dna Complex ◽  
Gapped Dna

Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as “fingers closing.” Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol β. First, using a doubly labeled DNA construct, we show that Pol β bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol β and donor-labeled DNA, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol β, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol β reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

scholarly journals Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions

2016 ◽  
Vol 44 (22) ◽  
pp. 10691-10710 ◽  
Author(s):  
Wonbae Lee ◽  
John P. Gillies ◽  
Davis Jose ◽  
Brett A. Israels ◽  
Peter H. von Hippel ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Replication Fork ◽  
Binding Protein ◽  
Bacteriophage T4 ◽  
Dna Binding Protein ◽  
Binding Kinetics ◽  
Cooperative Binding ◽  
Single Molecule Fret ◽  
Kinetics Of

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 Detailed Kinetics of RNA Folding Pathways and Thermodynamic Origins of Crowding by Single-Molecule FRET

2019 ◽  
Vol 116 (3) ◽  
pp. 278a
Author(s):  
Hsuan-Lei Sung ◽  
Abhigyan Sengupta ◽  
David J. Nesbitt
Keyword(s):  
Single Molecule ◽  
Rna Folding ◽  
Single Molecule Fret ◽  
Folding Pathways ◽  
Detailed Kinetics ◽  
Kinetics Of

scholarly journals Single Molecule FRET Reveals Lipid Induced Conformational Changes in Cytoplasmic Domain of Kir2.1

2019 ◽  
Vol 116 (3) ◽  
pp. 109a
Author(s):  
Joshua B. Brettmann ◽  
Sun Joo Lee ◽  
Shizhen Wang ◽  
Colin G. Nichols
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Cytoplasmic Domain ◽  
Single Molecule Fret
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