scholarly journals Accurate Transfer Efficiencies, Distance Distributions, and Ensembles of Unfolded and Intrinsically Disordered Proteins From Single-Molecule FRET

2018 ◽  
pp. 287-325 ◽  
Author(s):  
Erik D. Holmstrom ◽  
Andrea Holla ◽  
Wenwei Zheng ◽  
Daniel Nettels ◽  
Robert B. Best ◽  
...  
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Single Molecule Fret ◽  
Distance Distributions

scholarly journals Single Molecule FRET: A Powerful Tool to Study Intrinsically Disordered Proteins

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 140 ◽  
Author(s):  
Sharonda LeBlanc ◽  
Prakash Kulkarni ◽  
Keith Weninger
Keyword(s):  
Single Molecule ◽  
Biological Networks ◽  
Intrinsically Disordered Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Polymer Physics ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Single Molecule Fret ◽  
Random Fluctuations

Intrinsically disordered proteins (IDPs) are often modeled using ideas from polymer physics that suggest they smoothly explore all corners of configuration space. Experimental verification of this random, dynamic behavior is difficult as random fluctuations of IDPs cannot be synchronized across an ensemble. Single molecule fluorescence (or Förster) resonance energy transfer (smFRET) is one of the few approaches that are sensitive to transient populations of sub-states within molecular ensembles. In some implementations, smFRET has sufficient time resolution to resolve transitions in IDP behaviors. Here we present experimental issues to consider when applying smFRET to study IDP configuration. We illustrate the power of applying smFRET to IDPs by discussing two cases in the literature of protein systems for which smFRET has successfully reported phosphorylation-induced modification (but not elimination) of the disordered properties that have been connected to impacts on the related biological function. The examples we discuss, PAGE4 and a disordered segment of the GluN2B subunit of the NMDA receptor, illustrate the great potential of smFRET to inform how IDP function can be regulated by controlling the detailed ensemble of disordered states within biological networks.

Single-Molecule FRET of Intrinsically Disordered Proteins

2020 ◽  
Vol 71 (1) ◽  
pp. 391-414 ◽  
Author(s):  
Lauren Ann Metskas ◽  
Elizabeth Rhoades
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Intrinsically Disordered Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Protein Protein Interactions ◽  
Cellular Functions ◽  
Intrinsically Disordered ◽  
Single Molecule Fret

Intrinsically disordered proteins (IDPs) are now widely recognized as playing critical roles in a broad range of cellular functions as well as being implicated in diverse diseases. Their lack of stable secondary structure and tertiary interactions, coupled with their sensitivity to measurement conditions, stymies many traditional structural biology approaches. Single-molecule Förster resonance energy transfer (smFRET) is now widely used to characterize the physicochemical properties of these proteins in isolation and is being increasingly applied to more complex assemblies and experimental environments. This review provides an overview of confocal diffusion-based smFRET as an experimental tool, including descriptions of instrumentation, data analysis, and protein labeling. Recent papers are discussed that illustrate the unique capability of smFRET to provide insight into aggregation-prone IDPs, protein–protein interactions involving IDPs, and IDPs in complex experimental milieus.

scholarly journals Simulating freely-diffusing single-molecule FRET data with consideration of protein conformational dynamics

2021 ◽  
Author(s):  
James Losey ◽  
Michael Jauch ◽  
David S. Matteson ◽  
Mahmoud Moradi
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Analysis Techniques ◽  
Intrinsically Disordered ◽  
Single Molecule Fret ◽  
Great Progress

AbstractSingle molecule Förster resonance energy transfer experiments have added a great deal to the under-standing of conformational states of biologically important molecules. While great progress has been made, much is still unknown in systems that are highly flexible such as intrinsically disordered proteins because of the high degeneracy of distance states, particularly when freely diffusing smFRET experiments are used. Simulated smFRET data allows for the control of underlying process that generates the data to examine if analytic techniques can detect these underlying differences. We have extended the PyBroMo software that simulates the freely diffusing smFRET data to include a distribution of inter-dye distances generated using Langevin dynamics in order to model proteins with greater flexibility or disorder in structure. Standard analysis techniques for smFRET data compared highlighted the differences observed between data generated with the base software and data that included the distribution of inter-dye distance.

scholarly journals Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET

2017 ◽  
Vol 114 (33) ◽  
pp. E6812-E6821 ◽  
Author(s):  
Hoi Sung Chung ◽  
Fanjie Meng ◽  
Jae-Yeol Kim ◽  
Kevin McHale ◽  
Irina V. Gopich ◽  
...  
Keyword(s):  
Single Molecule ◽  
Intrinsically Disordered Proteins ◽  
Dissociation Constants ◽  
Structural Information ◽  
Ensemble Methods ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Single Molecule Fret ◽  
C Terminus ◽  
Tetramerization Domain

We describe a method that combines two- and three-color single-molecule FRET spectroscopy with 2D FRET efficiency–lifetime analysis to probe the oligomerization process of intrinsically disordered proteins. This method is applied to the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. TD exists as a monomer at subnanomolar concentrations and forms a dimer and a tetramer at higher concentrations. Because the dissociation constants of the dimer and tetramer are very close, as we determine in this paper, it is not possible to characterize different oligomeric species by ensemble methods, especially the dimer that cannot be readily separated. However, by using single-molecule FRET spectroscopy that includes measurements of fluorescence lifetime and two- and three-color FRET efficiencies with corrections for submillisecond acceptor blinking, we show that it is possible to obtain structural information for individual oligomers at equilibrium and to determine the dimerization kinetics. From these analyses, we show that the monomer is intrinsically disordered and that the dimer conformation is very similar to that of the tetramer but the C terminus of the dimer is more flexible.

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