Complexity of the Folding Transition of the B Domain of Protein A Revealed by the High-Speed Tracking of Single-Molecule Fluorescence Time Series

2015 ◽  
Vol 119 (20) ◽  
pp. 6081-6091 ◽  
Author(s):  
Hiroyuki Oikawa ◽  
Kiyoto Kamagata ◽  
Munehito Arai ◽  
Satoshi Takahashi
Keyword(s):  
Time Series ◽  
Single Molecule ◽  
High Speed ◽  
Protein A ◽  
Single Molecule Fluorescence ◽  
Speed Tracking

scholarly journals Stretching DNA to twice the normal length with single-molecule hydrodynamic trapping

2019 ◽  
Author(s):  
Yan Jiang ◽  
Theodore Feldman ◽  
Julia A.M. Bakx ◽  
Darren Yang ◽  
Wesley P. Wong
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Force Spectroscopy ◽  
Soft Materials ◽  
Covalent Attachment ◽  
Extended State ◽  
Single Molecule Fluorescence ◽  
Uv Curable ◽  
Force Application ◽  
Hydrodynamic Trapping

AbstractSingle-molecule force spectroscopy has brought many new insights into nanoscale biology, from the functioning of molecular motors, to the mechanical response of soft materials within the cell. To expand the single-molecule toolbox, we have developed a surface-free force spectroscopy assay based on a high-speed hydrodynamic trap capable of applying extremely high tensions for long periods of time. High-speed single-molecule trapping is enabled by a rigid and gas-impermeable microfluidic chip, rapidly and inexpensively fabricated out of glass, double-sided tape and UV-curable adhesive. Our approach does not require difficult covalent attachment chemistries, and enables simultaneous force application and single-molecule fluorescence. Using this approach, we have induced a highly extended state with twice the contour length of B-DNA in regions of partially intercalated double-stranded (dsDNA) by applying forces up to 250 pN. This highly extended state resembles the hyperstretched state of dsDNA, which was initially discovered as a structure fully intercalated by dyes under high tension. It has been hypothesized that hyperstretched DNA could also be induced without the aid of intercalators if high-enough forces were applied, which matches our observation. Combining force application with single-molecule fluorescence imaging is critical for distinguishing hyperstretched DNA from single-stranded DNA that can result from peeling. High-speed hydrodynamic trapping is a powerful yet accessible force spectroscopy method that enables the mechanics of biomolecules to be probed in previously difficult to access regimes.

High-speed atomic force microscope combined with single-molecule fluorescence microscope

2013 ◽  
Vol 84 (7) ◽  
pp. 073706 ◽  
Author(s):  
Shingo Fukuda ◽  
Takayuki Uchihashi ◽  
Ryota Iino ◽  
Yasutaka Okazaki ◽  
Masato Yoshida ◽  
...  
Keyword(s):  
Atomic Force Microscope ◽  
Single Molecule ◽  
High Speed ◽  
Fluorescence Microscope ◽  
Single Molecule Fluorescence ◽  
Atomic Force

Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

1992 ◽  
Vol 67 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Anne M Aakhus ◽  
J Michael Wilkinson ◽  
Nils Olav Solum
Keyword(s):  
Actin Filaments ◽  
High Speed ◽  
Binding Protein ◽  
Protein A ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

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