Multi-parameter measurements of conformational dynamics in nucleic acids and nucleoprotein complexes

Methods ◽  
2019 ◽  
Vol 169 ◽  
pp. 69-77 ◽  
Author(s):  
Ivan E. Ivanov ◽  
Zev Bryant
Keyword(s):  
Nucleic Acids ◽  
Conformational Dynamics ◽  
Nucleoprotein Complexes

Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

1992 ◽  
Vol 50 (1) ◽  
pp. 762-763
Author(s):  
Stephen D. Jett
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Nucleic Acid Binding ◽  
Mobility Shift ◽  
Carbon Coated ◽  
Nucleoprotein Complexes ◽  
Mobility Shift Assay ◽  
Protein Nucleic Acid ◽  
Gel Mobility Shift ◽  
Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

scholarly journals High-resolution EPR distance measurements on RNA and DNA with the non-covalent Ǵ spin label

2019 ◽  
Vol 48 (2) ◽  
pp. 924-933 ◽  
Author(s):  
Marcel Heinz ◽  
Nicole Erlenbach ◽  
Lukas S Stelzl ◽  
Grace Thierolf ◽  
Nilesh R Kamble ◽  
...  
Keyword(s):  
High Resolution ◽  
Nucleic Acids ◽  
Spin Label ◽  
Double Resonance ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Spin Labels ◽  
Abasic Site ◽  
Abasic Sites ◽  
Rna And Dna

Abstract Pulsed electron paramagnetic resonance (EPR) experiments, among them most prominently pulsed electron-electron double resonance experiments (PELDOR/DEER), resolve the conformational dynamics of nucleic acids with high resolution. The wide application of these powerful experiments is limited by the synthetic complexity of some of the best-performing spin labels. The recently developed $\bf\acute{G}$ (G-spin) label, an isoindoline-nitroxide derivative of guanine, can be incorporated non-covalently into DNA and RNA duplexes via Watson-Crick base pairing in an abasic site. We used PELDOR and molecular dynamics (MD) simulations to characterize $\bf\acute{G}$, obtaining excellent agreement between experiments and time traces calculated from MD simulations of RNA and DNA double helices with explicitly modeled $\bf\acute{G}$ bound in two abasic sites. The MD simulations reveal stable hydrogen bonds between the spin labels and the paired cytosines. The abasic sites do not significantly perturb the helical structure. $\bf\acute{G}$ remains rigidly bound to helical RNA and DNA. The distance distributions between the two bound $\bf\acute{G}$ labels are not substantially broadened by spin-label motions in the abasic site and agree well between experiment and MD. $\bf\acute{G}$ and similar non-covalently attached spin labels promise high-quality distance and orientation information, also of complexes of nucleic acids and proteins.

scholarly journals Helicase Mechanisms During Homologous Recombination inSaccharomyces cerevisiae

2019 ◽  
Vol 48 (1) ◽  
pp. 255-273 ◽  
Author(s):  
J. Brooks Crickard ◽  
Eric C. Greene
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleic Acid ◽  
Homologous Recombination ◽  
Nucleic Acids ◽  
Acid Metabolism ◽  
Model Organism ◽  
Nucleic Acid Metabolism ◽  
Repair Pathway ◽  
Unidirectional Movement ◽  
Nucleoprotein Complexes

Helicases are enzymes that move, manage, and manipulate nucleic acids. They can be subdivided into six super families and are required for all aspects of nucleic acid metabolism. In general, all helicases function by converting the chemical energy stored in the bond between the gamma and beta phosphates of adenosine triphosphate into mechanical work, which results in the unidirectional movement of the helicase protein along one strand of a nucleic acid. The results of this translocation activity can range from separation of strands within duplex nucleic acids to the physical remodeling or removal of nucleoprotein complexes. In this review, we focus on describing key helicases from the model organism Saccharomyces cerevisiae that contribute to the regulation of homologous recombination, which is an essential DNA repair pathway for fixing damaged chromosomes.

scholarly journals Stabilization and ATP-binding for tandem RRM domains of ALS-causing TDP-43 and hnRNPA1

2020 ◽  
Author(s):  
Mei Dang ◽  
Yifan Li ◽  
Jianxing Song
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acids ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Human Diseases ◽  
Atp Binding ◽  
Nucleic Acid Binding ◽  
General Role ◽  
Nmr Characterization

AbstractTDP-43 and hnRNPA1 contain tandemly-tethered RRM domains, which not only functionally bind an array of nucleic acids, but also participate in aggregation/fibrillation, a pathological hallmark of various human diseases including ALS, FTD, AD and MSP. Here, by DSF, NMR and MD simulations we systematically characterized stability, ATP-binding and conformational dynamics of TDP-43 and hnRNPA1 RRM domains in both tethered and isolated forms. The results reveal three key findings: 1) very unexpectedly, upon tethering TDP-43 RRM domains become dramatically coupled and destabilized with Tm reduced to only 49 °C. 2) ATP specifically binds TDP-43 and hnRNPA1 RRM domains, in which ATP occupies the similar pockets within the conserved nucleic-acid-binding surfaces, with the affinity higher to the tethered than isolated forms. 3) MD simulations indicate that the tethered RRM domains of TDP-43 and hnRNPA1 have higher conformational dynamics than the isolated forms. Two RRM domains become coupled as shown by NMR characterization and analysis of inter-domain correlation motions. The study explains the long-standing puzzle that the tethered TDP-43 RRM1-RRM2 is particularly prone to aggregation/fibrillation, and underscores the general role of ATP in inhibiting aggregation/fibrillation of RRM-containing proteins. The results also rationalize the observation that the risk of aggregation-causing diseases increases with aging.

Antagonistic effects of natural osmolyte mixtures and hydrostatic pressure on the conformational dynamics of a DNA hairpin probed at the single-molecule level

2018 ◽  
Vol 20 (19) ◽  
pp. 13159-13170 ◽  
Author(s):  
Satyajit Patra ◽  
Christian Anders ◽  
Paul Hendrik Schummel ◽  
Roland Winter
Keyword(s):  
Hydrostatic Pressure ◽  
Nucleic Acids ◽  
Single Molecule ◽  
Deep Sea ◽  
Conformational Dynamics ◽  
Dna Hairpin ◽  
Antagonistic Effects ◽  
Single Molecule Level

Osmolyte mixtures from deep sea organisms are able to rescue nucleic acids from pressure-induced unfolding.

Site-directed spin labeling EPR spectroscopy in protein research

2013 ◽  
Vol 394 (10) ◽  
pp. 1281-1300 ◽  
Author(s):  
Johann P. Klare
Keyword(s):  
Nucleic Acids ◽  
Epr Spectroscopy ◽  
Protein Function ◽  
Conformational Dynamics ◽  
Spin Labeling ◽  
Paramagnetic Resonance ◽  
Protein Research ◽  
Site Directed Spin Labeling ◽  
Electron Paramagnetic ◽  
Natronomonas Pharaonis

Abstract Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has emerged as an efficient tool to elucidate the structure and the conformational dynamics of proteins under conditions close to the native state. This review article summarizes the basics as well as the recent progress in SDSL and EPR methods, especially for investigations on protein structure, protein function, and interaction of proteins with other proteins or nucleic acids. Labeling techniques as well as EPR methods are introduced and exemplified with applications to systems that have been studied in the author’s laboratory in the past 15 years, headmost the sensory rhodopsin-transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis. Further examples underline the application of SDSL EPR spectroscopy to answer specific questions about the system under investigation, such as the nature and influence of interactions of proteins with other proteins or nucleic acids. Finally, it is discussed how SDSL EPR can be combined with other biophysical techniques to combine the strengths of the different methodologies.

Conformational Dynamics of Cyanocobalamin and Its Conjugates with Peptide Nucleic Acids

2017 ◽  
Vol 121 (14) ◽  
pp. 2968-2979 ◽  
Author(s):  
Tomasz Pieńko ◽  
Aleksandra J. Wierzba ◽  
Monika Wojciechowska ◽  
Dorota Gryko ◽  
Joanna Trylska
Keyword(s):  
Nucleic Acids ◽  
Peptide Nucleic Acids ◽  
Conformational Dynamics
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