scholarly journals Prokaryotic Argonaute from Archaeoglobus fulgidus interacts with DNA as a homodimer

2020 ◽  
Author(s):  
Edvardas Golovinas ◽  
Danielis Rutkauskas ◽  
Elena Manakova ◽  
Marija Jankunec ◽  
Arunas Silanskas ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Full Length ◽  
Archaeoglobus Fulgidus ◽  
Piwi Domain ◽  
Single Molecule Fret ◽  
Dna Loop ◽  
Domains Of Life ◽  
Monomeric Proteins ◽  
And Function

ABSTRACTBackgroundArgonaute (Ago) proteins are found in all three domains of life. The best characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are monomeric proteins, all composed of four major structural/functional domains (N, PAZ, MID and PIWI) and thereby closely resemble eAgos. It is believed that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown.ResultsIn this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal β-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including single-molecule FRET, SAXS and AFM, to test the possible dimerization of AfAgo. SAXS results confirm that WT AfAgo, but not the dimerization surface mutant AfAgoΔ, forms a homodimer both in the apo-form and when bound to a nucleic acid. Single molecule FRET and AFM studies demonstrate that the dimeric WT AfAgo binds two ends of a linear DNA fragment, forming a relatively stable DNA loop.ConclusionOur results show that contrary to other characterized Ago proteins, AfAgo is a stable homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding broadens the range of currently known Argonaute-nucleic acid interaction mechanisms.

scholarly journals Prokaryotic Argonaute From Archaeoglobus Fulgidus Interacts With DNA as a Homodimer

2020 ◽  
Author(s):  
Edvardas Golovinas ◽  
Danielis Rutkauskas ◽  
Elena Manakova ◽  
Marija Jankunec ◽  
Arunas Silanskas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Full Length ◽  
Archaeoglobus Fulgidus ◽  
Acid Interaction ◽  
Action Mechanism ◽  
Piwi Domain ◽  
The Core ◽  
Single Molecule Fret ◽  
Domains Of Life ◽  
And Function

Abstract Argonaute (Ago) proteins are found in all three domains of life. The best characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are composed of four major structural/functional domains (N, PAZ, MID and PIWI) and thereby closely resemble eAgos. It was demonstrated that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown. In this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal β-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including SEC-MALS, SAXS, single-molecule FRET and AFM, to show that AfAgo is indeed a homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding underscores the diversity of prokaryotic Agos and broadens the range of currently known Argonaute-nucleic acid interaction mechanisms.

scholarly journals Prokaryotic Argonaute from Archaeoglobus fulgidus interacts with DNA as a homodimer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Edvardas Golovinas ◽  
Danielis Rutkauskas ◽  
Elena Manakova ◽  
Marija Jankunec ◽  
Arunas Silanskas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Full Length ◽  
Archaeoglobus Fulgidus ◽  
Acid Interaction ◽  
Action Mechanism ◽  
Piwi Domain ◽  
The Core ◽  
Single Molecule Fret ◽  
Domains Of Life ◽  
And Function

AbstractArgonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are composed of four major structural/functional domains (N, PAZ, MID, and PIWI) and thereby closely resemble eAgos. It was demonstrated that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown. In this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal β-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including SEC-MALS, SAXS, single-molecule FRET, and AFM, to show that AfAgo is indeed a homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding underscores the diversity of prokaryotic Agos and broadens the range of currently known Argonaute-nucleic acid interaction mechanisms.

scholarly journals Single Molecule FRET Studies of Protein Conformational Landscapes: 3 Prototypic Examples for the Relation Between Conformational Dynamics and Function

2011 ◽  
Vol 100 (3) ◽  
pp. 474a-475a
Author(s):  
Markus Richert ◽  
Dymitro Rodnin ◽  
Carola S. Hengstenberg ◽  
Thomas Peulen ◽  
Alessandro Valeri ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Single Molecule Fret ◽  
Dynamics And Function ◽  
And Function

scholarly journals Biased cytochrome P450-mediated metabolism via small-molecule ligands binding P450 oxidoreductase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Simon Bo Jensen ◽  
Sara Thodberg ◽  
Shaheena Parween ◽  
Matias E. Moses ◽  
Cecilie C. Hansen ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Single Molecule ◽  
Cytochromes P450 ◽  
Conformational Sampling ◽  
P450 Oxidoreductase ◽  
Single Molecule Fret ◽  
Biased Signaling ◽  
Small Molecule Ligands ◽  
Cell Assays ◽  
And Function

AbstractMetabolic control is mediated by the dynamic assemblies and function of multiple redox enzymes. A key element in these assemblies, the P450 oxidoreductase (POR), donates electrons and selectively activates numerous (>50 in humans and >300 in plants) cytochromes P450 (CYPs) controlling metabolism of drugs, steroids and xenobiotics in humans and natural product biosynthesis in plants. The mechanisms underlying POR-mediated CYP metabolism remain poorly understood and to date no ligand binding has been described to regulate the specificity of POR. Here, using a combination of computational modeling and functional assays, we identify ligands that dock on POR and bias its specificity towards CYP redox partners, across mammal and plant kingdom. Single molecule FRET studies reveal ligand binding to alter POR conformational sampling, which results in biased activation of metabolic cascades in whole cell assays. We propose the model of biased metabolism, a mechanism akin to biased signaling of GPCRs, where ligand binding on POR stabilizes different conformational states that are linked to distinct metabolic outcomes. Biased metabolism may allow designing pathway-specific therapeutics or personalized food suppressing undesired, disease-related, metabolic pathways.

scholarly journals Ligand-modulated folding of the full-length adenine riboswitch probed by NMR and single-molecule FRET spectroscopy

2017 ◽  
Vol 45 (9) ◽  
pp. 5512-5522 ◽  
Author(s):  
Sven Warhaut ◽  
Klara Rebecca Mertinkus ◽  
Philipp Höllthaler ◽  
Boris Fürtig ◽  
Mike Heilemann ◽  
...  
Keyword(s):  
Single Molecule ◽  
Full Length ◽  
Single Molecule Fret

scholarly journals Argonaute bypasses cellular obstacles without hindrance during target search

2019 ◽  
Author(s):  
Tao Ju Cui ◽  
Misha Klein ◽  
Jorrit W. Hegge ◽  
Stanley D. Chandradoss ◽  
John van der Oost ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
High Efficiency ◽  
Lateral Diffusion ◽  
Secondary Structures ◽  
Single Molecule Fret ◽  
Tight Contact ◽  
Dna Backbone ◽  
Protein Nucleic Acid ◽  
Nucleic Acid Interactions

Argonaute (Ago) proteins are key players in gene regulation in eukaryotes and host defense in prokaryotes. For specific interference, Ago relies on base pairing between small nucleic acid guides and complementary target sequences. To efficiently scan nucleic acid chains for potential targets, Ago must bypass both secondary structures in mRNA and single stranded DNA as well as protein barriers. Through single-molecule FRET, we reveal that lateral diffusion is mediated mainly through protein-nucleic acid interactions, rather than interactions between the guide and targeted strand. This allows Ago to scan for targets with high efficiency but without maintaining tight contact with the DNA backbone. Real-time observations show that Ago “glides” short distances over secondary structures while using intersegmental jumps to reduce scanning redundancy and bypass protein barriers. Our single-molecule method in combination with kinetic analysis may serve as a novel platform to study the effect of sequence on search kinetics for other nucleic acid-guided proteins.

scholarly journals Kinetic and thermodynamic framework for P4-P6 RNA reveals tertiary motif modularity and modulation of the folding preferred pathway

2016 ◽  
Vol 113 (34) ◽  
pp. E4956-E4965 ◽  
Author(s):  
Namita Bisaria ◽  
Max Greenfeld ◽  
Charles Limouse ◽  
Dmitri S. Pavlichin ◽  
Hideo Mabuchi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Structural Changes ◽  
Structural Information ◽  
Equilibrium Constants ◽  
Single Point ◽  
Single Point Mutation ◽  
Single Molecule Fret ◽  
Predictive Understanding ◽  
And Function ◽  
And Behavior

The past decade has seen a wealth of 3D structural information about complex structured RNAs and identification of functional intermediates. Nevertheless, developing a complete and predictive understanding of the folding and function of these RNAs in biology will require connection of individual rate and equilibrium constants to structural changes that occur in individual folding steps and further relating these steps to the properties and behavior of isolated, simplified systems. To accomplish these goals we used the considerable structural knowledge of the folded, unfolded, and intermediate states of P4-P6 RNA. We enumerated structural states and possible folding transitions and determined rate and equilibrium constants for the transitions between these states using single-molecule FRET with a series of mutant P4-P6 variants. Comparisons with simplified constructs containing an isolated tertiary contact suggest that a given tertiary interaction has a stereotyped rate for breaking that may help identify structural transitions within complex RNAs and simplify the prediction of folding kinetics and thermodynamics for structured RNAs from their parts. The preferred folding pathway involves initial formation of the proximal tertiary contact. However, this preference was only ∼10 fold and could be reversed by a single point mutation, indicating that a model akin to a protein-folding contact order model will not suffice to describe RNA folding. Instead, our results suggest a strong analogy with a modified RNA diffusion-collision model in which tertiary elements within preformed secondary structures collide, with the success of these collisions dependent on whether the tertiary elements are in their rare binding-competent conformations.

Single-molecule FRET reveals the energy landscape of the full-length SAM-I riboswitch

2017 ◽  
Vol 13 (11) ◽  
pp. 1172-1178 ◽  
Author(s):  
Christoph Manz ◽  
Andrei Yu Kobitski ◽  
Ayan Samanta ◽  
Bettina G Keller ◽  
Andres Jäschke ◽  
...  
Keyword(s):  
Single Molecule ◽  
Energy Landscape ◽  
Full Length ◽  
Single Molecule Fret
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