scholarly journals Towards monitoring conformational changes of the GPCR neurotensin receptor 1 by single-molecule FRET

2018 ◽  
Author(s):  
Michael Börsch ◽  
Thomas Heitkamp ◽  
Reinhard Grisshammer
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Neurotensin Receptor ◽  
Single Molecule Fret ◽  
Neurotensin Receptor 1

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 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

Single-molecule fluorescence resonance energy transfer techniques on rotary ATP synthases

2011 ◽  
Vol 392 (1-2) ◽  
Author(s):  
Michael Börsch
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Molecule Fret ◽  
Intensity Changes ◽  
Fluorescence Resonance

Abstract Conformational changes of proteins can be monitored in real time by fluorescence resonance energy transfer (FRET). Two different fluorophores have to be attached to those protein domains which move during function. Distance fluctuations between the fluorophores are measured by relative fluorescence intensity changes or fluorescence lifetime changes. The rotary mechanics of the two motors of FoF1-ATP synthase have been studied in vitro by single-molecule FRET. The results are summarized and perspectives for other transport ATPases are discussed.

scholarly journals Conformational Changes in MutS during Mismtach Repair Signaling Determined with Single Molecule FRET

2012 ◽  
Vol 102 (3) ◽  
pp. 283a
Author(s):  
Ruoyi Qiu ◽  
Keith Weninger
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Single Molecule Fret

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 Conformational Changes in Tyrosine 11 of Neurotensin Are Required to Activate the Neurotensin Receptor 1

2020 ◽  
Vol 3 (4) ◽  
pp. 690-705 ◽  
Author(s):  
Fabian Bumbak ◽  
Trayder Thomas ◽  
Billy J. Noonan-Williams ◽  
Tasneem M. Vaid ◽  
Fei Yan ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Neurotensin Receptor ◽  
Neurotensin Receptor 1

scholarly journals Detection of actin conformational changes by single molecule FRET measurement

2001 ◽  
Vol 41 (supplement) ◽  
pp. S55
Author(s):  
H. Yokota ◽  
J. Kozuka ◽  
Y. Ishii ◽  
T. Yanagida
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Single Molecule Fret
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