Single-Molecule FRET Kinetics of the Mn2+ Riboswitch: Evidence for Allosteric Mg2+ Control of “Induced-Fit” vs “Conformational Selection” Folding Pathways

2019 ◽  
Vol 123 (9) ◽  
pp. 2005-2015 ◽  
Author(s):  
Hsuan-Lei Sung ◽  
David J. Nesbitt
Keyword(s):  
Single Molecule ◽  
Induced Fit ◽  
Conformational Selection ◽  
Single Molecule Fret ◽  
Folding Pathways ◽  
Kinetics Of

scholarly journals Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Fahad Rashid ◽  
Paul D Harris ◽  
Manal S Zaher ◽  
Mohamed A Sobhy ◽  
Luay I Joudeh ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genome Stability ◽  
Substrate Recognition ◽  
Induced Fit ◽  
Flap Endonuclease 1 ◽  
Catalytic Selectivity ◽  
Single Molecule Fret ◽  
Diffusion Limited ◽  
Separate Control ◽  
Dna Substrate

Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never misses cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.

Domain-Specific Folding Kinetics of Staphylococcal Nuclease Observed through Single-Molecule FRET in a Microfluidic Mixer

ChemPhysChem ◽  
2011 ◽  
Vol 12 (18) ◽  
pp. 3515-3518 ◽  
Author(s):  
Zeyong Zhi ◽  
Pengcheng Liu ◽  
Peng Wang ◽  
Yanyi Huang ◽  
Xin Sheng Zhao
Keyword(s):  
Single Molecule ◽  
Staphylococcal Nuclease ◽  
Folding Kinetics ◽  
Microfluidic Mixer ◽  
Domain Specific ◽  
Single Molecule Fret ◽  
Kinetics Of

scholarly journals Multispot single-molecule FRET: high-throughput analysis of freely diffusing molecules

2016 ◽  
Author(s):  
A. Ingargiola ◽  
E. Lerner ◽  
S. Chung ◽  
F. Panzeri ◽  
A. Gulinatti ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
High Throughput Analysis ◽  
Parallel Acquisition ◽  
Promoter Escape ◽  
Single Molecule Fret ◽  
Bacterial Rna ◽  
Measured Sample ◽  
Outstanding Question ◽  
Kinetics Of

AbstractWe describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics identical to those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription.Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.

scholarly journals Author response: Single-molecule FRET unveils induced-fit mechanism for substrate selectivity in flap endonuclease 1

2017 ◽  
Author(s):  
Fahad Rashid ◽  
Paul D Harris ◽  
Manal S Zaher ◽  
Mohamed A Sobhy ◽  
Luay I Joudeh ◽  
...  
Keyword(s):  
Single Molecule ◽  
Author Response ◽  
Substrate Selectivity ◽  
Induced Fit ◽  
Flap Endonuclease 1 ◽  
Single Molecule Fret
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