scholarly journals Time-course, negative-stain electron microscopy–based analysis for investigating protein–protein interactions at the single-molecule level

2017 ◽  
Vol 292 (47) ◽  
pp. 19400-19410 ◽  
Author(s):  
Bartek Nogal ◽  
Charles A. Bowman ◽  
Andrew B. Ward
Keyword(s):  
Electron Microscopy ◽  
Single Molecule ◽  
Protein Interactions ◽  
Time Course ◽  
Protein Protein Interactions ◽  
Single Molecule Level ◽  
Negative Stain ◽  
Negative Stain Electron Microscopy

scholarly journals Quantifying protein-protein interactions by molecular counting with mass photometry

2020 ◽  
Author(s):  
Fabian Soltermann ◽  
Eric D.B. Foley ◽  
Veronica Pagnoni ◽  
Martin R. Galpin ◽  
Justin L.P. Benesch ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Single Molecule ◽  
Protein Interactions ◽  
Analytical Techniques ◽  
Binding Affinities ◽  
Label Free ◽  
Protein Protein Interactions ◽  
Single Molecule Level ◽  
Minimal Invasiveness ◽  
Universal Approach

AbstractInteractions between biomolecules control the processes of life in health, and their malfunction in disease, making their characterization and quantification essential. Immobilization- and label-free analytical techniques are particular desirable because of their simplicity and minimal invasiveness, but struggle to quantify tight interactions. Here, we show that we can accurately count, distinguish by molecular mass, and thereby reveal the relative abundances of different un-labelled biomolecules and their complexes in mixtures at the single-molecule level by mass photometry. These measurements enable us to quantify binding affinities over four orders of magnitude at equilibrium for both simple and complex stoichiometries within minutes, as well as to determine the associated kinetics. Our results introduce mass photometry as a rapid, simple and label-free method for studying sub-μM binding affinities, with potential to be extended towards a universal approach for characterising complex biomolecular interactions.

scholarly journals Structural titration of receptor ion channel GLIC gating by HS-AFM

2018 ◽  
Vol 115 (41) ◽  
pp. 10333-10338 ◽  
Author(s):  
Yi Ruan ◽  
Kevin Kao ◽  
Solène Lefebvre ◽  
Arin Marchesi ◽  
Pierre-Jean Corringer ◽  
...  
Keyword(s):  
Ion Channel ◽  
Single Molecule ◽  
Conformational Changes ◽  
Protein Interactions ◽  
High Speed ◽  
Conformational Dynamics ◽  
Protein Protein Interactions ◽  
Ion Conductance ◽  
Single Molecule Level ◽  
Selective Channel

Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated, cation-selective channel, is a prokaryotic homolog of the pentameric Cys-loop receptor ligand-gated ion channel family. Despite large changes in ion conductance, small conformational changes were detected in X-ray structures of detergent-solubilized GLIC at pH 4 (active/desensitized state) and pH 7 (closed state). Here, we used high-speed atomic force microscopy (HS-AFM) combined with a buffer exchange system to perform structural titration experiments to visualize GLIC gating at the single-molecule level under native conditions. Reference-free 2D classification revealed channels in multiple conformational states during pH gating. We find changes of protein–protein interactions so far elusive and conformational dynamics much larger than previously assumed. Asymmetric pentamers populate early stages of activation, which provides evidence for an intermediate preactivated state.

scholarly journals TagBiFC technique allows long-term single-molecule tracking of protein-protein interactions in living cells

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shipeng Shao ◽  
Hongchen Zhang ◽  
Yong Zeng ◽  
Yongliang Li ◽  
Chaoying Sun ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Time Course ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Protein Protein Interactions ◽  
Spatiotemporal Resolution ◽  
Dynamic Interactions ◽  
Single Molecule Tracking

AbstractProtein-protein interactions (PPIs) are critical for cellular activity regulation. Visualization of PPIs using bimolecular fluorescence complementation (BiFC) techniques helps to understand how PPIs implement their functions. However, current BiFC is based on fluorescent proteins and the brightness and photostability are suboptimal for single molecule tracking experiments, resulting in either low spatiotemporal resolution or incapability of tracking for extended time course. Here, we developed the TagBiFC technique based on split HaloTag, a self-labeling tag that could conjugate an organic dye molecule and thus offered better brightness and photostability than fluorescent proteins for PPI visualization inside living cells. Through screening and optimization, we demonstrated that the reconstituted HaloTag exhibited higher localization precision and longer tracking length than previous methods. Using TagBiFC, we reveal that the dynamic interactions of transcription factor dimers with chromatin DNA are distinct and closely related to their dimeric states, indicating a general regulatory mechanism for these kinds of transcription factors. In addition, we also demonstrated the advantageous applications of TagBiFC in single nucleosome imaging, light-burden imaging of single mRNA, low background imaging of cellular structures. We believe these superior properties of our TagBiFC system will have broad applications in the studies of single molecule imaging inside living cells.

Nanopore-based measurement of the interaction of P450cam monooxygenase and putidaredoxin at single-molecule level

2021 ◽  
Author(s):  
Hui Chen ◽  
Yao Lin ◽  
Yi-Tao Long ◽  
Shelley Minteer ◽  
Yi-Lun Ying
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Single Molecule Level ◽  
P Protein ◽  
Wide Range ◽  
Significant Barrier ◽  
Function Characterization ◽  
Life Function

Protein-protein interactions occur in a wide range of biological processes and are of great significance to life function. Characterization of transient protein-protein interactions remains a significant barrier to our understanding...

scholarly journals Temperature-dependent reversible assembly of taxol-treated microtubules.

1987 ◽  
Vol 105 (6) ◽  
pp. 2847-2854 ◽  
Author(s):  
C A Collins ◽  
R B Vallee
Keyword(s):  
Electron Microscopy ◽  
Self Assembly ◽  
Temperature Dependent ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Microtubule Associated Proteins ◽  
Negative Stain ◽  
Microtubule Protein ◽  
Associated Proteins ◽  
Negative Stain Electron Microscopy

Taxol is a plant alkaloid that binds to and strongly stabilizes microtubules. Taxol-treated microtubules resist depolymerization under a variety of conditions that readily disassemble untreated microtubules. We report here that taxol-treated microtubules can be induced to disassemble by a combination of depolymerizating conditions. Reversible cycles of disassembly and reassembly were carried out using taxol-containing microtubules from calf brain and sea urchin eggs by shifting temperature in the presence of millimolar levels of Ca2+. Microtubules depolymerized completely, yielding dimers and ring-shaped oligomers as revealed by negative stain electron microscopy and Bio-Gel A-15m chromatography, and reassembled into well-formed microtubule polymer structures. Microtubule-associated proteins (MAPs), including species previously identified only by taxol-based purification such as MAP 1B and kinesin, were found to copurify with tubulin through reversible assembly cycles. To determine whether taxol remained bound to tubulin subunits, we subjected depolymerized taxol-treated microtubule protein to Sephadex G-25 chromatography, and the fractions were assayed for taxol content by reverse-phase HPLC. Taxol was found to be dissociated from the depolymerized microtubules. Protein treated in this way was found to be competent to reassemble, but now required conditions comparable with those for protein that had never been exposed to taxol. Thus, the binding of taxol to tubulin can be reversed. This has implications for the mechanism of taxol action and for the purification of microtubules from a wide variety of sources for use in self-assembly experiments.

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