scholarly journals Insight into dynamics of APOBEC3G protein in complexes with DNA assessed by high speed AFM

2019 ◽  
Author(s):  
Yangang Pan ◽  
Luda S. Shlyakhtenko ◽  
Yuri L. Lyubchenko
Keyword(s):  
High Speed ◽  
Molecular Mechanisms ◽  
Catalytic Domain ◽  
Structural Information ◽  
Dynamic Structure ◽  
Time Lapse ◽  
Ssdna Binding ◽  
Binding Domains ◽  
Ssdna Binding Protein ◽  
Dumbbell Structure

AbstractAPOBEC3G (A3G) is a single-stranded DNA (ssDNA) binding protein that restricts the HIV virus by deamination of dC to dU during reverse transcription of the viral genome. A3G has two zing-binding domains: the N-terminal domain (NTD), which efficiently binds ssDNA, and the C-terminal catalytic domain (CTD), which supports deaminase activity of A3G. Until now, structural information on A3G has lacked, preventing elucidation of the molecular mechanisms underlying its interaction with ssDNA and deaminase activity. We have recently built computational model for the full-length A3G monomer and validated its structure by data obtained from time-lapse High-Speed Atomic Force Microscopy (HS AFM). Here time-lapse HS AFM was applied to directly visualize the structure and dynamics of A3G in complexes with ssDNA. Our results demonstrate a highly dynamic structure of A3G, where two domains of the protein fluctuate between compact globular and extended dumbbell structures. Quantitative analysis of our data revealed a substantial increase in the number of A3G dumbbell structures in the presence of the DNA substrate, suggesting the interaction of A3G with the ssDNA substrate stabilizes this dumbbell structure. Based on these data, we developed a model explaining the interaction of globular and dumbbell structures of A3G with ssDNA and suggested a possible role of the dumbbell structure in A3G function.

scholarly journals Site-Search Process for Synaptic Protein-DNA Complexes

2021 ◽  
Vol 23 (1) ◽  
pp. 212
Author(s):  
Sridhar Vemulapalli ◽  
Mohtadin Hashemi ◽  
Yuri L. Lyubchenko
Keyword(s):  
Dna Cleavage ◽  
High Speed ◽  
Molecular Mechanisms ◽  
Experimental System ◽  
Time Lapse ◽  
Single Site ◽  
Search Process ◽  
Synaptic Protein ◽  
Dna Complexes ◽  
Site Search

The assembly of synaptic protein-DNA complexes by specialized proteins is critical for bringing together two distant sites within a DNA molecule or bridging two DNA molecules. The assembly of such synaptosomes is needed in numerous genetic processes requiring the interactions of two or more sites. The molecular mechanisms by which the protein brings the sites together, enabling the assembly of synaptosomes, remain unknown. Such proteins can utilize sliding, jumping, and segmental transfer pathways proposed for the single-site search process, but none of these pathways explains how the synaptosome assembles. Here we used restriction enzyme SfiI, that requires the assembly of synaptosome for DNA cleavage, as our experimental system and applied time-lapse, high-speed AFM (HS-AFM) to directly visualize the site search process accomplished by the SfiI enzyme. For the single-site SfiI-DNA complexes, we were able to directly visualize such pathways as sliding, jumping, and segmental site transfer. However, within the synaptic looped complexes, we visualized the threading and site-bound segment transfer as the synaptosome-specific search pathways for SfiI. In addition, we visualised sliding and jumping pathways for the loop dissociated complexes. Based on our data, we propose the site-search model for synaptic protein-DNA systems.

scholarly journals Evidence That the Adenovirus Single-Stranded DNA Binding Protein Mediates the Assembly of Biomolecular Condensates to Form Viral Replication Compartments

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1778
Author(s):  
Paloma Hidalgo ◽  
Arturo Pimentel ◽  
Diana Mojica-Santamaría ◽  
Konstantin von Stromberg ◽  
Helga Hofmann-Sieber ◽  
...  
Keyword(s):  
Viral Replication ◽  
Defense Mechanisms ◽  
Binding Protein ◽  
Human Adenovirus ◽  
Time Lapse ◽  
Low Complexity ◽  
Ssdna Binding ◽  
Intrinsically Disordered ◽  
Ssdna Binding Protein ◽  
Infected Cells

A common viral replication strategy is characterized by the assembly of intracellular compartments that concentrate factors needed for viral replication and simultaneously conceal the viral genome from host-defense mechanisms. Recently, various membrane-less virus-induced compartments and cellular organelles have been shown to represent biomolecular condensates (BMCs) that assemble through liquid-liquid phase separation (LLPS). In the present work, we analyze biophysical properties of intranuclear replication compartments (RCs) induced during human adenovirus (HAdV) infection. The viral ssDNA-binding protein (DBP) is a major component of RCs that contains intrinsically disordered and low complexity proline-rich regions, features shared with proteins that drive phase transitions. Using fluorescence recovery after photobleaching (FRAP) and time-lapse studies in living HAdV-infected cells, we show that DBP-positive RCs display properties of liquid BMCs, which can fuse and divide, and eventually form an intranuclear mesh with less fluid-like features. Moreover, the transient expression of DBP recapitulates the assembly and liquid-like properties of RCs in HAdV-infected cells. These results are of relevance as they indicate that DBP may be a scaffold protein for the assembly of HAdV-RCs and should contribute to future studies on the role of BMCs in virus-host cell interactions.

scholarly journals Nuclear Hormone Receptor Architecture - Form and Dynamics: The 2009 FASEB Summer Conference on Dynamic Structure of the Nuclear Hormone Receptors

2009 ◽  
Vol 7 (1) ◽  
pp. nrs.07011 ◽  
Author(s):  
Iain J. McEwan ◽  
Ann M. Nardulli
Keyword(s):  
Hormone Receptors ◽  
Structural Information ◽  
Dynamic Structure ◽  
Structural Features ◽  
Nuclear Hormone Receptor ◽  
Nuclear Hormone Receptors ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Fatty Acid Derivatives ◽  
Agonist And Antagonist

Nuclear hormone receptors (NHRs) represent a large and diverse family of ligand-activated transcription factors involved in regulating development, metabolic homeostasis, salt balance and reproductive health. The ligands for these receptors are typically small hydrophobic molecules such as steroid hormones, thyroid hormone, vitamin D3 and fatty acid derivatives. The first NHR structural information appeared ∼20 years ago with the solution and crystal structures of the DNA binding domains and was followed by the structure of the agonist and antagonist bound ligand binding domains of different NHR members. Interestingly, in addition to these defined structural features, it has become clear that NHRs also possess significant structural plasticity. Thus, the dynamic structure of the NHRs was the topic of a recent stimulating and informative FASEB Summer Research Conference held in Vermont.

scholarly journals A key interaction with RPA orients XPA in NER complexes

2020 ◽  
Vol 48 (4) ◽  
pp. 2173-2188 ◽  
Author(s):  
Agnieszka M Topolska-Woś ◽  
Norie Sugitani ◽  
John J Cordoba ◽  
Kateryna V Le Meur ◽  
Rémy A Le Meur ◽  
...  
Keyword(s):  
Structural Model ◽  
Excision Repair ◽  
Mechanical Action ◽  
Scattering Data ◽  
Missense Mutations ◽  
Ssdna Binding ◽  
Binding Domains ◽  
X Ray Scattering ◽  
Protein Functions ◽  
Ssdna Binding Protein

Abstract The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD–RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.

scholarly journals Ligand discrimination and gating in cyclic nucleotide-gated ion channels from apo and partial agonist-bound cryo-EM structures

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Jan Rheinberger ◽  
Xiaolong Gao ◽  
Philipp AM Schmidpeter ◽  
Crina M Nimigean
Keyword(s):  
Cyclic Nucleotide ◽  
Molecular Mechanisms ◽  
Single Channel ◽  
Structural Information ◽  
Partial Agonist ◽  
Visual Signal ◽  
Functional Responses ◽  
Binding Domains ◽  
Single Channel Activity ◽  
Functional States

Cyclic nucleotide-modulated channels have important roles in visual signal transduction and pacemaking. Binding of cyclic nucleotides (cAMP/cGMP) elicits diverse functional responses in different channels within the family despite their high sequence and structure homology. The molecular mechanisms responsible for ligand discrimination and gating are unknown due to lack of correspondence between structural information and functional states. Using single particle cryo-electron microscopy and single-channel recording, we assigned functional states to high-resolution structures of SthK, a prokaryotic cyclic nucleotide-gated channel. The structures for apo, cAMP-bound, and cGMP-bound SthK in lipid nanodiscs, correspond to no, moderate, and low single-channel activity, respectively, consistent with the observation that all structures are in resting, closed states. The similarity between apo and ligand-bound structures indicates that ligand-binding domains are strongly coupled to pore and SthK gates in an allosteric, concerted fashion. The different orientations of cAMP and cGMP in the ‘resting’ and ‘activated’ structures suggest a mechanism for ligand discrimination.

scholarly journals Insight into the dynamics of APOBEC3G protein in complexes with DNA assessed by high speed AFM

2019 ◽  
Vol 1 (10) ◽  
pp. 4016-4024 ◽  
Author(s):  
Yangang Pan ◽  
Luda S. Shlyakhtenko ◽  
Yuri L. Lyubchenko
Keyword(s):  
Reverse Transcription ◽  
Viral Genome ◽  
High Speed ◽  
Binding Protein ◽  
Single Stranded Dna ◽  
Ssdna Binding ◽  
Hiv Virus ◽  
Ssdna Binding Protein ◽  
Insight Into

APOBEC3G (A3G) is a single-stranded DNA (ssDNA) binding protein that restricts the HIV virus by deamination of dC to dU during reverse transcription of the viral genome.

scholarly journals Phosphoproteomics Reveals a Distinct Mode of Mec1/ATR Signaling in Response to DNA End Hyper-Resection

2020 ◽  
Author(s):  
Ethan J. Sanford ◽  
Vitor M. Faça ◽  
Stephanie C. Vega ◽  
William J. Comstock ◽  
Marcus B. Smolka
Keyword(s):  
Dna Repair ◽  
Scaffolding Protein ◽  
Genome Maintenance ◽  
Distinct Mode ◽  
Checkpoint Signaling ◽  
Ssdna Binding ◽  
Binding Domains ◽  
Ssdna Binding Protein ◽  
Atr Kinase ◽  
Dna Ends

ABSTRACTThe Mec1/ATR kinase is crucial for genome maintenance in response to a range of genotoxic insults, although how it promotes context-dependent signaling and DNA repair remains elusive. Here we uncovered a specialized mode of Mec1/ATR signaling triggered by the extensive nucleolytic processing (resection) of DNA ends. Cells lacking RAD9, a checkpoint activator and an inhibitor of resection, exhibit a selective increase in Mec1-dependent phosphorylation of proteins associated with single strand DNA transactions, including the ssDNA binding protein Rfa2, the translocase/ubiquitin ligase Uls1 and the HR-regulatory Sgs1-Top3-Rmi1 (STR) complex. Extensive Mec1-dependent phosphorylation of the STR complex, mostly on the Sgs1 helicase subunit, promotes an interaction between STR and the DNA repair scaffolding protein Dpb11. Fusion of Sgs1 to phosphopeptide-binding domains of Dpb11 strongly impairs HR-mediated repair, supporting a model whereby Mec1 signaling regulates STR upon hyper-resection to influence recombination outcomes. Overall, the identification of a distinct mode of Mec1 signaling triggered by hyper-resection highlights the multi-faceted action of this kinase in the coordination of checkpoint signaling and HR-mediated DNA repair.

Five-Dimensional Microscopy using Widefield-Deconvolution: Practical Considerations and Biological Applications

1996 ◽  
Vol 54 ◽  
pp. 268-269
Author(s):  
W.F. Marshall ◽  
K. Oegema ◽  
J. Nunnari ◽  
A.F. Straight ◽  
D.A. Agard ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
High Speed ◽  
Laser Scanning ◽  
Ccd Camera ◽  
Image Plane ◽  
Time Lapse ◽  
Laser Scanning Confocal Microscopy ◽  
Three Dimensions ◽  
Excitation Light ◽  
Cooled Ccd

The ability to image cells in three dimensions has brought about a revolution in biological microscopy, enabling many questions to be asked which would be inaccessible without this capability. There are currently two major methods of three dimensional microscopy: laser-scanning confocal microscopy and widefield-deconvolution microscopy. The method of widefield-deconvolution uses a cooled CCD to acquire images from a standard widefield microscope, and then computationally removes out of focus blur. Using such a scheme, it is easy to acquire time-lapse 3D images of living cells without killing them, and to do so for multiple wavelengths (using computer-controlled filter wheels). Thus, it is now not only feasible, but routine, to perform five dimensional microscopy (three spatial dimensions, plus time, plus wavelength).Widefield-deconvolution has several advantages over confocal microscopy. The two main advantages are high speed of acquisition (because there is no scanning, a single optical section is acquired at a time by using a cooled CCD camera) and the use of low excitation light levels Excitation intensity can be much lower than in a confocal microscope for three reasons: 1) longer exposures can be taken since the entire 512x512 image plane is acquired in parallel, so that dwell time is not an issue, 2) the higher quantum efficiently of a CCD detect over those typically used in confocal microscopy (although this is expected to change due to advances in confocal detector technology), and 3) because no pinhole is used to reject light, a much larger fraction of the emitted light is collected. Thus we can typically acquire images with thousands of photons per pixel using a mercury lamp, instead of a laser, for illumination. The use of low excitation light is critical for living samples, and also reduces bleaching. The high speed of widefield microscopy is also essential for time-lapse 3D microscopy, since one must acquire images quickly enough to resolve interesting events.

Rapid Online Buffer Exchange: A Method for Screening of Proteins, Protein Complexes, and Cell Lysates by Native Mass Spectrometry

2019 ◽  
Author(s):  
Zachary VanAernum ◽  
Florian Busch ◽  
Benjamin J. Jones ◽  
Mengxuan Jia ◽  
Zibo Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Speed ◽  
Structural Information ◽  
Protein Complexes ◽  
High Sensitivity ◽  
Native Mass Spectrometry ◽  
Structural Features ◽  
Consumer Products ◽  
Cell Lysates ◽  
Protein Expression And Purification

It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and structural characterization, as well as for use in consumer products, chemical processes, and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is timeconsuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes, or clarified cell lysates. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.

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