Single-Molecule Multicolor FRET Assay for Studying Structural Dynamics of Biomolecules

2016 ◽  
pp. 461-486 ◽  
Author(s):  
S. Lee ◽  
Y. Jang ◽  
S.-J. Lee ◽  
S. Hohng
Keyword(s):  
Structural Dynamics ◽  
Single Molecule

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

2018 ◽  
Author(s):  
Alexander Carl DeHaven
Keyword(s):  
Energy Transfer ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
U2 Snrna ◽  
Folding Dynamics ◽  
The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

scholarly journals mRNA structural dynamics shape Argonaute-target interactions

2019 ◽  
Author(s):  
Suzan Ruijtenberg ◽  
Stijn Sonneveld ◽  
Tao Ju Cui ◽  
Ive Logister ◽  
Dion de Steenwinkel ◽  
...  
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
Protein Interactions ◽  
Small Rnas ◽  
Mrna Degradation ◽  
Translational Repression ◽  
Modeling And Experiments ◽  
Regulate Protein ◽  
Sirna Target

AbstractSmall RNAs (such as miRNAs, siRNAs and piRNAs) regulate protein expression in a wide variety of biological processes and play an important role in cellular function, development and disease. Association of small RNAs with Argonaute (AGO) family proteins guide AGO to target RNAs, generally resulting in target silencing through transcriptional silencing, translational repression or mRNA degradation. Here we develop a live-cell single-molecule imaging assay to simultaneously visualize translation of individual mRNA molecules and their silencing by human AGO2-siRNA complexes. We find that siRNA target sites are commonly masked in vivo by RNA secondary structures, which inhibit AGO2-target interactions. Translating ribosomes unmask AGO2 binding sites, stimulating AGO2-target interactions and promoting mRNA degradation. Using a combination of mathematical modeling and experiments, we find that mRNA structures are highly heterogeneous and continuously refolding. We show that structural dynamics of mRNAs shape AGO2-target recognition, which may be a common feature controlling mRNA-protein interactions.

scholarly journals Investigating The Structural Dynamics Of The Lict Transcriptional Antiterminator And Its RNA Target By Single Molecule FRET

2009 ◽  
Vol 96 (3) ◽  
pp. 56a
Author(s):  
Matthew L. Ferguson ◽  
Caroline Clerté ◽  
Emmanuel Margeat ◽  
Nathalie Declerck ◽  
Catherine A. Royer
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
Single Molecule Fret

Single molecule FRET for the study on structural dynamics of biomolecules

Biosystems ◽  
2007 ◽  
Vol 88 (3) ◽  
pp. 243-250 ◽  
Author(s):  
Mitsuhiro Sugawa ◽  
Yoshiyuki Arai ◽  
Atsuko Hikikoshi Iwane ◽  
Yoshiharu Ishii ◽  
Toshio Yanagida
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
Single Molecule Fret

scholarly journals Single-molecule stepping and structural dynamics of myosin X

2010 ◽  
Vol 17 (4) ◽  
pp. 485-491 ◽  
Author(s):  
Yujie Sun ◽  
Osamu Sato ◽  
Felix Ruhnow ◽  
Mark E Arsenault ◽  
Mitsuo Ikebe ◽  
...  
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
Myosin X

scholarly journals Multiplexed, bioorthogonal labeling of multicomponent, biomolecular complexes using genomically encoded, non-canonical amino acids

2019 ◽  
Author(s):  
Bijoy J. Desai ◽  
Ruben L. Gonzalez
Keyword(s):  
Amino Acids ◽  
Structural Biology ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Functional Studies ◽  
Bioorthogonal Labeling ◽  
Biomolecular Complexes ◽  
And Function

Stunning advances in the structural biology of multicomponent biomolecular complexes (MBCs) have ushered in an era of intense, structure-guided mechanistic and functional studies of these complexes. Nonetheless, existing methods to site-specifically conjugate MBCs with biochemical and biophysical labels are notoriously impracticable and/or significantly perturb MBC assembly and function. To overcome these limitations, we have developed a general, multiplexed method in which we genomically encode non-canonical amino acids (ncAAs) into multiple, structure-informed, individual sites within a target MBC; select for ncAA-containing MBC variants that assemble and function like the wildtype MBC; and site-specifically conjugate biochemical or biophysical labels to these ncAAs. As a proof-of-principle, we have used this method to generate unique single-molecule fluorescence resonance energy transfer (smFRET) signals reporting on ribosome structural dynamics that have thus far remained inaccessible to smFRET studies of translation.

scholarly journals Caught in the act: structural dynamics of replication origin activation and fork progression

2020 ◽  
Vol 48 (3) ◽  
pp. 1057-1066 ◽  
Author(s):  
Jacob S. Lewis ◽  
Alessandro Costa
Keyword(s):  
Dna Replication ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Single Particle ◽  
Replication Origin ◽  
Origin Activation ◽  
Recent Advances ◽  
Eukaryotic Dna ◽  
New Challenges

This review discusses recent advances in single-particle cryo-EM and single-molecule approaches used to visualise eukaryotic DNA replication reactions reconstituted in vitro. We comment on the new challenges facing structural biologists, as they turn to describing the dynamic cascade of events that lead to replication origin activation and fork progression.

scholarly journals Evolution of structural dynamics in bilobed proteins

2020 ◽  
Author(s):  
Giorgos Gouridis ◽  
Yusran A. Muthahari ◽  
Marijn de Boer ◽  
Konstantinos Tassis ◽  
Alexandra Tsirigotaki ◽  
...  
Keyword(s):  
Structural Dynamics ◽  
Protein Evolution ◽  
Single Molecule ◽  
Protein Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Domain ◽  
Divergent Evolution ◽  
Exchange Mass Spectrometry ◽  
Cytoplasmic Transport

AbstractNovel biophysical tools allow the structural dynamics of proteins, and the regulation of such dynamics by binding partners, to be explored in unprecedented detail. Although this has provided critical insights into protein function, the means by which structural dynamics direct protein evolution remains poorly understood. Here, we investigated how proteins with a bilobed structure, composed of two related domains from the type-II periplasmic binding protein domain family, have undergone divergent evolution leading to modification of their structural dynamics and function. We performed a structural analysis of ~600 bilobed proteins with a common primordial structural core, which we complemented with biophysical studies to explore the structural dynamics of selected examples by single-molecule Förster resonance energy transfer and Hydrogen-Deuterium exchange mass spectrometry. We show that evolutionary modifications of the structural core, largely at its termini, enables distinct structural dynamics, allowing the diversification of these proteins into transcription factors, enzymes, and extra-cytoplasmic transport-related proteins. Structural embellishments of the core created new interdomain interactions that stabilized structural states, reshaping the active site geometry, and ultimately, altered substrate specificity. Our findings reveal an as yet unrecognized mechanism for the emergence of functional promiscuity during long periods of protein evolution and are applicable to a large number of domain architectures.

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