scholarly journals POT1 stability and binding measured by fluorescence thermal shift assays

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0245675
Author(s):  
Lynn W. DeLeeuw ◽  
Robert C. Monsen ◽  
Vytautas Petrauskas ◽  
Robert D. Gray ◽  
Lina Baranauskiene ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Dna Binding ◽  
Competitive Inhibition ◽  
Single Stranded Dna ◽  
Biophysical Characterization ◽  
Drug Candidates ◽  
G Quadruplex ◽  
And Function ◽  
Thermal Shift

The protein POT1 (Protection of Telomeres 1) is an integral part of the shelterin complex that protects the ends of human chromosomes from degradation or end fusions. It is the only component of shelterin that binds single-stranded DNA. We describe here the application of two separate fluorescent thermal shift assays (FTSA) that provide quantitative biophysical characterization of POT1 stability and its interactions. The first assay uses Sypro Orange™ and monitors the thermal stability of POT1 and its binding under a variety of conditions. This assay is useful for the quality control of POT1 preparations, for biophysical characterization of its DNA binding and, potentially, as an efficient screening tool for binding of small molecule drug candidates. The second assay uses a FRET-labeled human telomeric G-quadruplex structure that reveals the effects of POT1 binding on thermal stability from the DNA frame of reference. These complementary assays provide efficient biophysical approaches for the quantitative characterization of multiple aspects of POT1 structure and function. The results from these assays provide thermodynamics details of POT1 folding, the sequence selectivity of its DNA binding and the thermodynamic profile for its binding to its preferred DNA binding sequence. Most significantly, results from these assays elucidate two mechanisms for the inhibition of POT1 –DNA interactions. The first is by competitive inhibition at the POT1 DNA binding site. The second is indirect and is by stabilization of G-quadruplex formation within the normal POT1 single-stranded DNA sequence to prevent POT1 binding.

scholarly journals POT1 Stability and Binding Measured by Fluorescence Thermal Shift Assays

2021 ◽  
Author(s):  
Lynn W. DeLeeuw ◽  
Robert C. Monsen ◽  
Vytautas Petrauskas ◽  
Robert D. Gray ◽  
Lina Baranauskiene ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Dna Binding ◽  
Competitive Inhibition ◽  
Single Stranded Dna ◽  
Biophysical Characterization ◽  
Drug Candidates ◽  
G Quadruplex ◽  
And Function ◽  
Thermal Shift

AbstractThe protein POT1 (Protection of Telomeres 1) is an integral part of the shelterin complex that protects the ends of human chromosomes from degradation or end fusions. It is the only component of shelterin that binds single-stranded DNA. We describe here the application of two separate fluorescent thermal shift assays (FTSA) that provide quantitative biophysical characterization of POT1 stability and its interactions. The first assay uses Sypro Orange™ and monitors the thermal stability of POT1 and its binding under a variety of conditions. This assay is useful for the quality control of POT1 preparations, for biophysical characterization of its DNA binding and, potentially, as an efficient screening tool for binding of small molecule drug candidates. The second assay uses a FRET-labeled human telomeric G-quadruplex structure that reveals the effects of POT1 binding on thermal stability from the DNA frame of reference. These complementary assays provide efficient biophysical approaches for the quantitative characterization of multiple aspects of POT1 structure and function. The results from these assays provide thermodynamics details of POT1 folding, the sequence selectivity of its DNA binding and the thermodynamic profile for its binding to its preferred DNA binding sequence. Most significantly, results from these assays elucidate two mechanisms for the inhibition of POT1 – DNA interactions. The first is by competitive inhibition at the POT1 DNA binding site. The second is indirect and is by stabilization of G-quadruplex formation within the normal POT1 single-stranded DNA sequence to prevent POT1 binding.

scholarly journals Nanomechanics and co-transcriptional folding of Spinach and Mango

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jaba Mitra ◽  
Taekjip Ha
Keyword(s):  
Single Molecule ◽  
Rna Aptamers ◽  
Biophysical Characterization ◽  
Mechanical Responses ◽  
Discrete Step ◽  
Force Relaxation ◽  
G Quadruplex ◽  
And Function

Abstract Recent advances in fluorogen-binding “light-up” RNA aptamers have enabled protein-free detection of RNA in cells. Detailed biophysical characterization of folding of G-Quadruplex (GQ)-based light-up aptamers such as Spinach, Mango and Corn is still lacking despite the potential implications on their folding and function. In this work we employ single-molecule fluorescence-force spectroscopy to examine mechanical responses of Spinach2, iMangoIII and MangoIV. Spinach2 unfolds in four discrete steps as force is increased to 7 pN and refolds in reciprocal steps upon force relaxation. In contrast, GQ-core unfolding in iMangoIII and MangoIV occurs in one discrete step at forces >10 pN and refolding occurred at lower forces showing hysteresis. Co-transcriptional folding using superhelicases shows reduced misfolding propensity and allowed a folding pathway different from refolding. Under physiologically relevant pico-Newton levels of force, these aptamers may unfold in vivo and subsequently misfold. Understanding of the dynamics of RNA aptamers will aid engineering of improved fluorogenic modules for cellular applications.

scholarly journals Purification and Characterization of the Coliphage N4-coded Single-stranded DNA Binding Protein

1989 ◽  
Vol 264 (21) ◽  
pp. 12700-12708
Author(s):  
G Lindberg ◽  
S C Kowalczykowski ◽  
J K Rist ◽  
A Sugino ◽  
L B Rothman-Denes
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Purification And Characterization ◽  
Single Stranded Dna

scholarly journals Biophysical characterization of DNA binding from single molecule force measurements

2010 ◽  
Vol 7 (3) ◽  
pp. 299-341 ◽  
Author(s):  
Kathy R. Chaurasiya ◽  
Thayaparan Paramanathan ◽  
Micah J. McCauley ◽  
Mark C. Williams
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Force Measurements ◽  
Biophysical Characterization

Biophysical methods toolbox to study ABC exporter structure and function

2017 ◽  
Vol 398 (2) ◽  
pp. 229-235
Author(s):  
Thomas Marcellino ◽  
Vasundara Srinivasan
Keyword(s):  
Membrane Proteins ◽  
Abc Transporter ◽  
Integrated Approach ◽  
Structure And Function ◽  
Dynamic Membrane ◽  
Biophysical Characterization ◽  
Intermediate States ◽  
And Function

Abstract ABC exporters are highly dynamic membrane proteins that span a huge spectrum of different conformations. A detailed integrated approach of cellular, biochemical and biophysical characterization of these ‘open’, ‘closed’ and other intermediate states is central to understanding their function. Almost 40 years after the discovery of the first ABC transporter, thanks to the enormous development in methodologies, a picture is slowly emerging to visualize how these fascinating molecules transport their substrates. This mini review summarizes some of the biophysical tools that have made a major impact in understanding the function of the ABC exporters.

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