Kinetics of Photoinduced Reactions at the Single‐Molecule Level: The KACB Method

2020 ◽  
Vol 26 (35) ◽  
pp. 7740-7746
Author(s):  
Kiyohiko Kawai ◽  
Atsushi Maruyama
Keyword(s):  
Single Molecule ◽  
Single Molecule Level ◽  
Photoinduced Reactions ◽  
Kinetics Of

scholarly journals Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution

2020 ◽  
Author(s):  
Ilina Bareja ◽  
Hugo Wioland ◽  
Miro Janco ◽  
Philip R. Nicovich ◽  
Antoine Jégou ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
High Probability ◽  
Actin Binding ◽  
Single Molecule Level ◽  
Filament Dynamics ◽  
Kinetics Of ◽  
Insight Into

ABSTRACTTropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

scholarly journals Bottom-up single-molecule strategy for understanding subunit function of tetrameric β-galactosidase

2018 ◽  
Vol 115 (33) ◽  
pp. 8346-8351 ◽  
Author(s):  
Xiang Li ◽  
Yu Jiang ◽  
Shaorong Chong ◽  
David R. Walt
Keyword(s):  
Single Molecule ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Single Molecule Level ◽  
Subunit Function ◽  
Enzyme Molecules ◽  
Individual Enzyme ◽  
Kinetics Of

In this paper, we report an example of the engineered expression of tetrameric β-galactosidase (β-gal) containing varying numbers of active monomers. Specifically, by combining wild-type and single-nucleotide polymorphism plasmids at varying ratios, tetrameric β-gal was expressed in vitro with one to four active monomers. The kinetics of individual enzyme molecules revealed four distinct populations, corresponding to the number of active monomers in the enzyme. Using single-molecule-level enzyme kinetics, we were able to measure an accurate in vitro mistranslation frequency (5.8 × 10−4 per base). In addition, we studied the kinetics of the mistranslated β-gal at the single-molecule level.

Revealing Kinetics of Two-Electron Oxygen Reduction Reaction at Single-Molecule Level

2020 ◽  
Vol 142 (30) ◽  
pp. 13201-13209
Author(s):  
Yi Xiao ◽  
Jaeyoung Hong ◽  
Xiao Wang ◽  
Tao Chen ◽  
Taeghwan Hyeon ◽  
...  
Keyword(s):  
Oxygen Reduction Reaction ◽  
Oxygen Reduction ◽  
Single Molecule ◽  
Reduction Reaction ◽  
Single Molecule Level ◽  
Kinetics Of

scholarly journals Controlling load-dependent kinetics of β-cardiac myosin at the single-molecule level

2018 ◽  
Vol 25 (6) ◽  
pp. 505-514 ◽  
Author(s):  
Chao Liu ◽  
Masataka Kawana ◽  
Dan Song ◽  
Kathleen M. Ruppel ◽  
James A. Spudich
Keyword(s):  
Single Molecule ◽  
Cardiac Myosin ◽  
Single Molecule Level ◽  
Kinetics Of

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 Archaeal Chromatin Proteins Cren7 and Sul7d Compact DNA by Bending and Bridging

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Zhenfeng Zhang ◽  
Zhengyan Zhan ◽  
Bing Wang ◽  
Yuanyuan Chen ◽  
Xiuqiang Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Bending ◽  
Chromosomal Dna ◽  
Content Type ◽  
Single Molecule Level ◽  
Dna Compaction ◽  
Dna Organization ◽  
Chromatin Proteins ◽  
Mechanism And Kinetics ◽  
Kinetics Of

ABSTRACT Archaeal chromatin proteins Cren7 and Sul7d from Sulfolobus are DNA benders. To better understand their architectural roles in chromosomal DNA organization, we analyzed DNA compaction by Cren7 and Sis7d, a Sul7d family member, from Sulfolobus islandicus at the single-molecule (SM) level by total single-molecule internal reflection fluorescence microscopy (SM-TIRFM) and atomic force microscopy (AFM). We show that both Cren7 and Sis7d were able to compact singly tethered λ DNA into a highly condensed structure in a three-step process and that Cren7 was over an order of magnitude more efficient than Sis7d in DNA compaction. The two proteins were similar in DNA bending kinetics but different in DNA condensation patterns. At saturating concentrations, Sis7d formed randomly distributed clusters whereas Cren7 generated a single and highly condensed core on plasmid DNA. This observation is consistent with the greater ability of Cren7 than of Sis7d to bridge DNA. Our results offer significant insights into the mechanism and kinetics of chromosomal DNA organization in Crenarchaea. IMPORTANCE A long-standing question is how chromosomal DNA is packaged in Crenarchaeota, a major group of archaea, which synthesize large amounts of unique small DNA-binding proteins but in general contain no archaeal histones. In the present work, we tested our hypothesis that the two well-studied crenarchaeal chromatin proteins Cren7 and Sul7d compact DNA by both DNA bending and bridging. We show that the two proteins are capable of compacting DNA, albeit with different efficiencies and in different manners, at the single molecule level. We demonstrate for the first time that the two proteins, which have long been regarded as DNA binders and benders, are able to mediate DNA bridging, and this previously unknown property of the proteins allows DNA to be packaged into highly condensed structures. Therefore, our results provide significant insights into the mechanism and kinetics of chromosomal DNA organization in Crenarchaeota.

Kinetics of a Three-Step Reaction Observed at the Single-Molecule Level

2003 ◽  
Vol 42 (17) ◽  
pp. 1926-1929 ◽  
Author(s):  
Tudor Luchian ◽  
Seong-Ho Shin ◽  
Hagan Bayley
Keyword(s):  
Single Molecule ◽  
Single Molecule Level ◽  
Kinetics Of
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