scholarly journals Structural Characterization of Arabidopsis thaliana NAP1-Related Protein 2 (AtNRP2) and Comparison with its Homolog AtNRP1

Molecules ◽  
2019 ◽  
Vol 24 (12) ◽  
pp. 2258 ◽  
Author(s):  
Kumar ◽  
Kumar Singh ◽  
Chandrakant Bobde ◽  
Vasudevan
Keyword(s):  
Crystal Structure ◽  
Arabidopsis Thaliana ◽  
Titration Calorimetry ◽  
Histone Chaperones ◽  
Nucleosome Assembly ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
The Stability ◽  
Structural Architecture ◽  
And Function

Nucleosome Assembly Protein (NAP) is a highly conserved family of histone chaperones present in yeast, animals, and plants. Unlike other organisms, plants possess an additional class of proteins in its NAP family, known as the NAP1-related proteins or NRP. Arabidopsis thaliana possesses two NRP isoforms, namely AtNRP1 and AtNRP2, that share 87% sequence identity. Both AtNRP1 and AtNRP2 get expressed in all the plant tissues. Most works in the past, including structural studies, have focused on AtNRP1. We wanted to do a comparative study of the two proteins to find why the plant would have two very similar proteins and whether there is any difference between the two for their structure and function as histone chaperones. Here we report the crystal structure of AtNRP2 and a comparative analysis of its structural architecture with other NAP family proteins. The crystal structure of AtNRP2 shows it to be a homodimer, with its fold similar to that of other structurally characterized NAP family proteins. Although AtNRP1 and AtNRP2 have a similar fold, upon structural superposition, we find an offset in the dimerization helix of the two proteins. We evaluated the stability, oligomerization status, and histone chaperoning properties of the two proteins, for a comparison. The thermal melting experiments suggest that AtNRP2 is more stable than AtNRP1 at higher temperatures. In addition, electrophoretic mobility shift assay and isothermal titration calorimetry experiments suggest histone binding ability of AtNRP2 is higher than that of AtNRP1. Overall, these results provide insights about the specific function and relevance of AtNRP2 in plants through structural and biophysical studies.

scholarly journals RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains

2014 ◽  
Vol 42 (15) ◽  
pp. 10185-10195 ◽  
Author(s):  
Constanze Schelhorn ◽  
James M.B. Gordon ◽  
Lidia Ruiz ◽  
Javier Alguacil ◽  
Enrique Pedroso ◽  
...  
Keyword(s):  
Rna Binding ◽  
Titration Calorimetry ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Mobility Shift ◽  
Cytoplasmic Polyadenylation ◽  
C Terminus ◽  
Self Association ◽  
Mobility Shift Assay ◽  
A Minor

Abstract Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1–4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1–RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins’ ability to interact with RNA as demonstrated by ion mobility–mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1–RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the β-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1–RRM2–CPE complex that illustrates the experimental data.

scholarly journals Single-Myb-histone proteins from Arabidopsis thaliana: a quantitative study of telomere-binding specificity and kinetics

2009 ◽  
Vol 419 (1) ◽  
pp. 221-230 ◽  
Author(s):  
Ctirad Hofr ◽  
Pavla Šultesová ◽  
Michal Zimmermann ◽  
Iva Mozgová ◽  
Petra Procházková Schrumpfová ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Telomeric Dna ◽  
Mobility Shift ◽  
Telomere Repeat ◽  
Binding Stoichiometry ◽  
Binding Factor ◽  
Telomere Function ◽  
Telomere Capping ◽  
Mobility Shift Assay ◽  
And Control

Proteins that bind telomeric DNA modulate the structure of chromosome ends and control telomere function and maintenance. It has been shown that AtTRB (Arabidopsis thaliana telomere-repeat-binding factor) proteins from the SMH (single-Myb-histone) family selectively bind double-stranded telomeric DNA and interact with the telomeric protein AtPOT1b (A. thaliana protection of telomeres 1b), which is involved in telomere capping. In the present study, we performed the first quantitative DNA-binding study of this plant-specific family of proteins. Interactions of full-length proteins AtTRB1 and AtTRB3 with telomeric DNA were analysed by electrophoretic mobility-shift assay, fluorescence anisotropy and surface plasmon resonance to reveal their binding stoichiometry and kinetics. Kinetic analyses at different salt conditions enabled us to estimate the electrostatic component of binding and explain different affinities of the two proteins to telomeric DNA. On the basis of available data, a putative model explaining the binding stoichiometry and the protein arrangement on telomeric DNA is presented.

scholarly journals Structure of the human histone chaperone FACT Spt16 N-terminal domain

2016 ◽  
Vol 72 (2) ◽  
pp. 121-128 ◽  
Author(s):  
G. Marcianò ◽  
D. T. Huang
Keyword(s):  
Crystal Structure ◽  
Saccharomyces Cerevisiae ◽  
Complex Surface ◽  
Histone Chaperone ◽  
Titration Calorimetry ◽  
Nucleosome Assembly ◽  
Surface Residue ◽  
Histone Binding ◽  
Terminal Domain ◽  
Heterodimeric Complex

The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of theSaccharomyces cerevisiaeandSchizosaccharomyces pombeSpt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.

scholarly journals Viroplasm Protein P9-1 ofRice Black-Streaked Dwarf VirusPreferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site

2013 ◽  
Vol 87 (23) ◽  
pp. 12885-12899 ◽  
Author(s):  
Jianyan Wu ◽  
Jia Li ◽  
Xiang Mao ◽  
Weiwu Wang ◽  
Zhaobang Cheng ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Rna Binding ◽  
Structure And Function ◽  
Interior Structure ◽  
Mobility Shift ◽  
Chromatography Analysis ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
And Function

The P9-1 protein ofRice black-streaked dwarf virus(RBSDV) is an essential part of the viroplasm. However, little is known about its nature or biological function in the viroplasm. In this study, the structure and function of P9-1 were analyzed forin vitrobinding to nucleic acids. We found that the P9-1 protein preferentially bound to single-stranded versus double-stranded nucleic acids; however, the protein displayed no preference for RBSDV versus non-RBSDV single-stranded ssRNA (ssRNA). A gel mobility shift assay revealed that the RNA gradually shifted as increasing amounts of P9-1 were added, suggesting that multiple subunits of P9-1 bind to ssRNA. By using discontinuous blue native gel and chromatography analysis, we found that the P9-1 protein was capable of forming dimers, tetramers, and octamers. Strikingly, we demonstrated that P9-1 preferentially bound to ssRNA in the octamer, rather than the dimer, form. Deletion of the C-terminal arm resulted in P9-1 no longer forming octamers; consequently, the deletion mutant protein bound to ssRNA with significantly lower affinity and with fewer copies bound per ssRNA. Alanine substitution analysis revealed that electropositive amino acids among residues 25 to 44 are important for RNA binding and map to the central interior structure that was formed only by P9-1 octamers. Collectively, our findings provide novel insights into the structure and function of RBSDV viroplasm protein P9-1 binding to RNA.

Computational and structural studies of MeCP2 and associated mutants

2020 ◽  
Vol 19 (06) ◽  
pp. 2041001
Author(s):  
Tugba G. Kucukkal ◽  
Rijul U. Amin
Keyword(s):  
Protein Structure ◽  
Rett Syndrome ◽  
Current Knowledge ◽  
Genetic Disorder ◽  
Mobility Shift ◽  
Different Types ◽  
Mecp2 Protein ◽  
The Stability ◽  
And Function ◽  
Electrophoretic Mobility Shift Assays

Rett Syndrome is a rare genetic disorder exclusively seen in girls. Approximately 95% of RTT cases is caused by mutations in the MeCP2 gene which codes for Methyl-CpG-binding protein 2 (MeCP2). In this review, first, a brief introductory review of Rett Syndrome, MeCP2 protein structure and function, mutation types and frequencies, and phenotype–genotype relationships were provided. After that, the current knowledge on the wild-type and mutant MeCP2 protein structure and dynamics as well as its binding to DNA is reviewed. The review particularly focuses on computational (such as molecular dynamics) and experimental (such as electrophoretic mobility shift assays) studies on the MeCP2 binding to different types of DNA as well as the computational and experimental (such as circular dichroism) studies on the stability changes upon mutations. In the end, a brief opinion on future outlook for further computational studies is provided.

Structure and function of nucleosome assembly proteinsThis paper is one of a selection of papers published in this Special Issue, entitled 27th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

2006 ◽  
Vol 84 (4) ◽  
pp. 549-549 ◽  
Author(s):  
Young-Jun Park ◽  
Karolin Luger
Keyword(s):  
Current Knowledge ◽  
Peer Review Process ◽  
Histone Chaperones ◽  
Nucleosome Assembly ◽  
Nucleosome Assembly Protein ◽  
Nucleosome Assembly Protein 1 ◽  
Assembly Factor ◽  
And Function ◽  
Cycle Regulation ◽  
Selection Of

Homologues of nucleosome assembly protein 1 (NAP1) have been identified in all eukaryotes. Although initially identified as histone chaperones and chromatin-assembly factors, additional functions include roles in tissue-specific transcription regulation, apoptosis, histone shuttling, and cell-cycle regulation, and extend beyond those of a simple chaperone and assembly factor. NAP1 family members share a structurally conserved fold, the NAP domain. Here we review current knowledge of the NAP family of proteins within the context of the recently determined crystal structure of the NAP1 family's first representative, NAP1 from yeast.

scholarly journals Functional Analysis of the Stability Determinant AlfB of pBET131, a Miniplasmid Derivative of Bacillus subtilis (natto) Plasmid pLS32

2009 ◽  
Vol 192 (5) ◽  
pp. 1221-1230 ◽  
Author(s):  
Teruo Tanaka
Keyword(s):  
Bacillus Subtilis ◽  
Gene Product ◽  
Tandem Repeats ◽  
Plasmid Stability ◽  
Mobility Shift ◽  
Segregational Stability ◽  
Yeast Two Hybrid System ◽  
Target Dna ◽  
Mobility Shift Assay ◽  
The Stability

ABSTRACT Bacillus subtilis plasmid pBET131 is a derivative of pLS32, which was isolated from a natto strain of Bacillus subtilis. The DNA region in pBET131 that confers segregational stability contains an operon consisting of three genes, of which alfA, encoding an actin-like ATPase, and alfB are essential for plasmid stability. In this work, the alfB gene product and its target DNA region were studied in detail. Transcription of the alf operon initiated from a σA-type promoter was repressed by the alfB gene product. Overproduction of AlfA was inhibitory to cell growth, suggesting that the repression of the alf operon by AlfB is important for maintaining appropriate levels of AlfA. An electrophoretic mobility shift assay and footprinting analysis with purified His-tagged AlfB showed that it bound to a DNA region containing three tandem repeats of 8-bp AT-rich sequence (here designated parN), which partially overlaps the −35 sequence of the promoter. A sequence alteration in the first or third repeat did not affect the AlfB binding and plasmid stability, whereas that in the second repeat resulted in inhibition of these phenomena. The repression of alfA-lacZ expression was observed in the constructs carrying a mutation in either the first or third repeat, but not in the second repeat, indicating a correlation between plasmid stability, AlfB binding, and repression. It was also demonstrated by the yeast two-hybrid system that AlfA and AlfB interact with each other and among themselves. From these results, it was concluded that AlfB participates in partitioning pBET131 by forming a complex with AlfA and parN, the mode of which is typified by the type II partition mechanism.

scholarly journals P-element Somatic Inhibitor Protein Binding a Target Sequence in dsx Pre-mRNA Conserved in Bombyx mori and Spodoptera litura

2019 ◽  
Vol 20 (9) ◽  
pp. 2361 ◽  
Author(s):  
Yao Wang ◽  
Qin Zhao ◽  
Qiu-Xing Wan ◽  
Kai-Xuan Wang ◽  
Xing-Fu Zha
Keyword(s):  
Sex Determination ◽  
Bombyx Mori ◽  
Active Sites ◽  
Cd Spectroscopy ◽  
P Element ◽  
Titration Calorimetry ◽  
Target Sequence ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Double Switch

Bombyx mori doublesex (Bmdsx) functions as a double-switch gene in the final step of the sex-determination cascade in the silkworm Bombyx mori. The P-element somatic inhibitor (PSI) protein in B. mori interacts with Bmdsx pre-mRNA in CE1 as an exonic splicing silencer to promote male-specific splicing of Bmdsx. However, the character of the interaction between BmPSI and Bmdsx pre-mRNA remains unclear. Electrophoretic mobility shift assay (EMSA) results showed that the four KH_1 motifs in BmPSI are all essential for the binding, especially the former two KH_1 motifs. Three active sites (I116, L127, and IGGI) in the KH_1 motif were found to be necessary for the binding through EMSA, circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). The PSI homologous protein in S. litura (SlPSI) was purified and the binding of SlPSI and CE1 was verified. Compared with BmPSI, the mutant SlPSI proteins of I116 and IGGI lost their ability to bind to CE1. In conclusion, the binding of PSI and dsx pre-mRNA are generally conserved in both B. mori and S. litura. These findings provide clues for sex determination in Lepidoptera.

scholarly journals Crystal structure of the nucleoid-associated protein Fis (PA4853) from Pseudomonas aeruginosa

2020 ◽  
Vol 76 (5) ◽  
pp. 209-215
Author(s):  
Juan Zhou ◽  
Zengqiang Gao ◽  
Heng Zhang ◽  
Yuhui Dong
Keyword(s):  
Crystal Structure ◽  
Pseudomonas Aeruginosa ◽  
Promoter Region ◽  
Mobility Shift ◽  
Dna Inversion ◽  
Terminal Loop ◽  
Factor For Inversion Stimulation ◽  
Critical Residues ◽  
Mobility Shift Assay

Factor for inversion stimulation (Fis) is a versatile bacterial nucleoid-associated protein that can directly bind and bend DNA to influence DNA topology. It also plays crucial roles in regulating bacterial virulence factors and in optimizing bacterial adaptation to various environments. Fis from Pseudomonas aeruginosa (PA4853, referred to as PaFis) has recently been found to be required for virulence by regulating the expression of type III secretion system (T3SS) genes. PaFis can specifically bind to the promoter region of exsA, which functions as a T3SS master regulator, to regulate its expression and plays an essential role in transcription elongation from exsB to exsA. Here, the crystal structure of PaFis, which is composed of a four-helix bundle and forms a homodimer, is reported. PaFis shows remarkable structural similarities to the well studied Escherichia coli Fis (EcFis), including an N-terminal flexible loop and a C-terminal helix–turn–helix (HTH) motif. However, the critical residues for Hin-catalyzed DNA inversion in the N-terminal loop of EcFis are not conserved in PaFis and further studies are required to investigate its exact role. A gel-electrophoresis mobility-shift assay showed that PaFis can efficiently bind to the promoter region of exsA. Structure-based mutagenesis revealed that several conserved basic residues in the HTH motif play essential roles in DNA binding. These structural and biochemical studies may help in understanding the role of PaFis in the regulation of T3SS expression and in virulence.

scholarly journals Faster diffusive dynamics of histone-like nucleoid structuring proteins in live bacteria caused by silver ions

2019 ◽  
Author(s):  
Asmaa A. Sadoon ◽  
Prabhat Khadka ◽  
Jack Freeland ◽  
Ravi Kumar Gundampati ◽  
Ryan Manso ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Silver Ions ◽  
Single Particle Tracking ◽  
Live Cells ◽  
Titration Calorimetry ◽  
Mobility Shift ◽  
Bent Dna ◽  
Diffusive Dynamics ◽  
Live Bacteria ◽  
Mobility Shift Assay

AbstractThe antimicrobial activity and mechanism of silver ions (Ag+) have gained broad attention in recent years. However, dynamic studies are rare in this field. Here, we report our measurement of the effects of Ag+ ions on the dynamics of histone-like nucleoid structuring (H-NS) proteins in live bacteria using single-particle tracking photoactivated localization microscopy (sptPALM). It was found that treating the bacteria with Ag+ ions led to faster diffusive dynamics of H-NS proteins. Several techniques were used to understand the mechanism of the observed faster dynamics. Electrophoretic mobility shift assay on purified H-NS proteins indicated that Ag+ ions weaken the binding between H-NS proteins and DNA. Isothermal titration calorimetry confirmed that DNA and Ag+ ions interact directly. Our recently developed sensing method based on bent DNA suggested that Ag+ ions caused dehybridization of double-stranded DNA (i.e., dissociation into single strands). These evidences led us to a plausible mechanism for the observed faster dynamics of H-NS proteins in live bacteria when subjected to Ag+ ions: Ag+-induced DNA dehybridization weakens the binding between H-NS proteins and DNA. This work highlighted the importance of dynamic study of single proteins in the live cells for understanding the functions of antimicrobial agents to the bacteria.

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