Shape-Dependent Macromolecular Crowding on the Thermodynamics and Microsecond Conformational Dynamics of Protein Unfolding Revealed at the Single-Molecule Level

2020 ◽  
Vol 124 (28) ◽  
pp. 5858-5871
Author(s):  
Nilimesh Das ◽  
Pratik Sen
Keyword(s):  
Single Molecule ◽  
Protein Unfolding ◽  
Conformational Dynamics ◽  
Macromolecular Crowding ◽  
Single Molecule Level

Antagonistic effects of natural osmolyte mixtures and hydrostatic pressure on the conformational dynamics of a DNA hairpin probed at the single-molecule level

2018 ◽  
Vol 20 (19) ◽  
pp. 13159-13170 ◽  
Author(s):  
Satyajit Patra ◽  
Christian Anders ◽  
Paul Hendrik Schummel ◽  
Roland Winter
Keyword(s):  
Hydrostatic Pressure ◽  
Nucleic Acids ◽  
Single Molecule ◽  
Deep Sea ◽  
Conformational Dynamics ◽  
Dna Hairpin ◽  
Antagonistic Effects ◽  
Single Molecule Level

Osmolyte mixtures from deep sea organisms are able to rescue nucleic acids from pressure-induced unfolding.

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 Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy: Concepts and Applications

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2972 ◽  
Author(s):  
Takuhiro Otosu ◽  
Shoichi Yamaguchi
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Fluorescence Correlation Spectroscopy ◽  
Conformational Dynamics ◽  
Correlation Spectroscopy ◽  
Two Dimensional ◽  
Single Molecule Level ◽  
Fluorescence Correlation ◽  
Promising Tool ◽  
Molecular Ruler

We review the basic concepts and recent applications of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), which is the extension of fluorescence correlation spectroscopy (FCS) to analyze the correlation of fluorescence lifetime in addition to fluorescence intensity. Fluorescence lifetime is sensitive to the microenvironment and can be a “molecular ruler” when combined with FRET. Utilization of fluorescence lifetime in 2D FLCS thus enables us to quantify the inhomogeneity of the system and the interconversion dynamics among different species with a higher time resolution than other single-molecule techniques. Recent applications of 2D FLCS to various biological systems demonstrate that 2D FLCS is a unique and promising tool to quantitatively analyze the microsecond conformational dynamics of macromolecules at the single-molecule level.

scholarly journals KERA: Analysis Tool for Multi-Process, Multi-State Single-Molecule Data

2021 ◽  
Author(s):  
Joseph Tibbs ◽  
Mohamed Ghoneim ◽  
Colleen C. Caldwell ◽  
Troy Buzynski ◽  
Wayne Bowie ◽  
...  
Keyword(s):  
Single Molecule ◽  
Molecular Interactions ◽  
Conformational Dynamics ◽  
Software Tool ◽  
Underlying Mechanism ◽  
Temporal Organization ◽  
Analysis Tool ◽  
Time Data ◽  
Single Molecule Level ◽  
Complex Architectures

ABSTRACTMolecular machines within cells dynamically assemble, disassemble, and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy (CoSMoS), in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy (TIRFM) approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell-time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERA’s utility by analyzing conformational dynamics of two DNA binding proteins: RPA and XPD helicase.

scholarly journals Conformational Dynamics of a G Protein-Coupled Receptor at the Single-Molecule Level

2015 ◽  
Vol 108 (2) ◽  
pp. 350a
Author(s):  
Rajan Lamichhane ◽  
Jeffrey J. Liu ◽  
Raymond C. Stevens ◽  
David P. Millar
Keyword(s):  
G Protein ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
G Protein Coupled Receptor ◽  
Single Molecule Level ◽  
G Protein Coupled

scholarly journals Direct Experimental Evidence of Surface-induced Protein Unfolding at the Single-molecule Level

2020 ◽  
Vol 26 (S2) ◽  
pp. 312-313
Author(s):  
Evgeny Dubrovin ◽  
Nikolay Barinov ◽  
Tilman Schäffer ◽  
Dmitry Klinov
Keyword(s):  
Experimental Evidence ◽  
Single Molecule ◽  
Protein Unfolding ◽  
Single Molecule Level ◽  
Direct Experimental Evidence

scholarly journals Free-energy landscapes of membrane co-translocational protein unfolding

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Christian Bech Rosen ◽  
Hagan Bayley ◽  
David Rodriguez-Larrea
Keyword(s):  
Free Energy ◽  
Single Molecule ◽  
Protein Unfolding ◽  
Protein Import ◽  
Cation Binding ◽  
Energy Landscapes ◽  
Native State ◽  
Single Molecule Level ◽  
Model Protein ◽  
Free Energy Landscapes

AbstractProtein post-translational translocation is found at the plasma membrane of prokaryotes and protein import into organellae. Translocon structures are becoming available, however the dynamics of proteins during membrane translocation remain largely obscure. Here we study, at the single-molecule level, the folding landscape of a model protein while forced to translocate a transmembrane pore. We use a DNA tag to drive the protein into the α-hemolysin pore under a quantifiable force produced by an applied electric potential. Using a voltage-quench approach we find that the protein fluctuates between the native state and an intermediate in the translocation process at estimated forces as low as 1.9 pN. The fluctuation kinetics provide the free energy landscape as a function of force. We show that our stable, ≈15 kBT, substrate can be unfolded and translocated with physiological membrane potentials and that selective divalent cation binding may have a profound effect on the translocation kinetics.

scholarly journals Developing an Assay to Probe Activtion and Conformational Dynamics of β2-Adrenergic Receptor on Single Molecule Level

2013 ◽  
Vol 104 (2) ◽  
pp. 61a ◽  
Author(s):  
Salome Veshaguri ◽  
Marijonas Tutkus ◽  
Christian V. Lundgaard ◽  
Asger Tønnesen ◽  
Signe Mathiasen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Adrenergic Receptor ◽  
Conformational Dynamics ◽  
Single Molecule Level ◽  
Β2 Adrenergic Receptor
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