scholarly journals Conformational Transitions of Double-Stranded DNA in Thin Films

2021 ◽  
Vol 11 (5) ◽  
pp. 2360
Author(s):  
Kristina Serec ◽  
Nikola Šegedin ◽  
Maria Krajačić ◽  
Sanja Dolanski Babić
Keyword(s):  
Thin Films ◽  
Conformational Changes ◽  
Conformational Transitions ◽  
Ambient Conditions ◽  
Band Shape ◽  
Double Stranded Dna ◽  
Analysis Of Dynamics ◽  
Fitting In

Conformational transitions of double-stranded DNA in different environments have long been studied as vital parts of both in vitro and in vivo processes. In this study, utilizing Fourier transform infrared spectroscopy (FTIR), we provide detailed analysis of dynamics of A- to B-form transitions in DNA thin films of different hydrated states based on a statistical analysis of a substantial number of spectra and band shape analysis (peak fitting) in both the phosphate (1150–1000 cm−1) and sugar–phosphate (900–750 cm−1) region. Hydration of DNA thin films is systematically controlled by the time spent in the desiccator chamber (from 3 min to 40 min) allowing conformation and hydration signatures, in addition to variations due to ambient conditions, to be resolved in the spectra. Conformation transition from A-form to more ordered B-form is observed if sufficient time in the desiccator chamber is allowed and is confirmed by changes on the bands at ≈890, 860, 837, and 805 cm−1. Phosphate vibrations at ≈1230 cm−1 and 1089 cm−1, and backbone vibrations at ≈1030 cm−1 and 765 cm−1 were found to be sensitive to changes in hydration rather than conformation. Additionally, we found that spectral variations caused by ambient conditions can be significantly reduced without inducing conformational changes, which serves as a good basis for quality assurance.

scholarly journals GroEL2 of Mycobacterium tuberculosis Reveals the Importance of Structural Pliability in Chaperonin Function

2015 ◽  
Vol 198 (3) ◽  
pp. 486-497 ◽  
Author(s):  
Neeraja Chilukoti ◽  
C. M. Santosh Kumar ◽  
Shekhar C. Mande
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Conformational Changes ◽  
Normal Mode Analysis ◽  
Conformational Transitions ◽  
Mode Analysis ◽  
Content Type ◽  
E Coli ◽  
Allosteric Transitions

ABSTRACTIntracellular protein folding is mediated by molecular chaperones, the best studied among which are the chaperonins GroEL and GroES. Conformational changes and allosteric transitions between different metastable states are hallmarks of the chaperonin mechanism. These conformational transitions between three structural domains of GroEL are anchored at two hinges. Although hinges are known to be critical for mediating the communication between different domains of GroEL, the relative importance of hinges on GroEL oligomeric assembly, ATPase activity, conformational changes, and functional activity is not fully characterized. We have exploited the inability ofMycobacterium tuberculosisGroEL2 to functionally complement anEscherichia coligroELmutant to address the importance of hinge residues in the GroEL mechanism. Various chimeras ofM. tuberculosisGroEL2 andE. coliGroEL allowed us to understand the role of hinges and dissect the consequences of oligomerization and substrate binding capability on conformational transitions. The present study explains the concomitant conformational changes observed with GroEL hinge variants and is best supported by the normal mode analysis.IMPORTANCEConformational changes and allosteric transitions are hallmarks of the chaperonin mechanism. We have exploited the inability ofM. tuberculosisGroEL2 to functionally complement a strain ofE. coliin whichgroELexpression is repressed to address the importance of hinges. The significance of conservation at the hinge regions stands out as a prominent feature of the GroEL mechanism in binding to GroES and substrate polypeptides. The hinge residues play a significant role in the chaperonin activityin vivoandin vitro.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

scholarly journals E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

scholarly journals The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sabrina Dietz ◽  
Miguel Vasconcelos Almeida ◽  
Emily Nischwitz ◽  
Jan Schreier ◽  
Nikenza Viceconte ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Wild Type ◽  
C Elegans ◽  
Double Stranded Dna ◽  
Telomere Sequence ◽  
Proteomics Approach ◽  
Telomeric Protein ◽  
Regulate Telomere Length

AbstractTelomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.

scholarly journals Furin Processing and Proteolytic Activation of Semliki Forest Virus

2003 ◽  
Vol 77 (5) ◽  
pp. 2981-2989 ◽  
Author(s):  
Xinyong Zhang ◽  
Martin Fugère ◽  
Robert Day ◽  
Margaret Kielian
Keyword(s):  
Conformational Changes ◽  
Secretory Pathway ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Protein Fusion ◽  
Proteolytic Activation ◽  
Control Virus

ABSTRACT The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed “p62,” which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.

BAF is required for emerin assembly into the reforming nuclear envelope

2001 ◽  
Vol 114 (24) ◽  
pp. 4575-4585 ◽  
Author(s):  
Tokuko Haraguchi ◽  
Takako Koujin ◽  
Miriam Segura-Totten ◽  
Kenneth K. Lee ◽  
Yosuke Matsuoka ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Hela Cells ◽  
Core Region ◽  
Lamin B ◽  
The Core ◽  
Double Stranded Dna ◽  
Recessive Form ◽  
Nuclear Envelope Assembly

Mutations in emerin cause the X-linked recessive form of Emery-Dreifuss muscular dystrophy (EDMD). Emerin localizes at the inner membrane of the nuclear envelope (NE) during interphase, and diffuses into the ER when the NE disassembles during mitosis. We analyzed the recruitment of wildtype and mutant GFP-tagged emerin proteins during nuclear envelope assembly in living HeLa cells. During telophase, emerin accumulates briefly at the ‘core’ region of telophase chromosomes, and later distributes over the entire nuclear rim. Barrier-to-autointegration factor (BAF), a protein that binds nonspecifically to double-stranded DNA in vitro, co-localized with emerin at the ‘core’ region of chromosomes during telophase. An emerin mutant defective for binding to BAF in vitro failed to localize at the ‘core’ in vivo, and subsequently failed to localize at the reformed NE. In HeLa cells that expressed BAF mutant G25E, which did not show ‘core’ localization, the endogenous emerin proteins failed to localize at the ‘core’ region during telophase, and did not assemble into the NE during the subsequent interphase. BAF mutant G25E also dominantly dislocalized LAP2β and lamin A from the NE, but had no effect on the localization of lamin B. We conclude that BAF is required for the assembly of emerin and A-type lamins at the reforming NE during telophase, and may mediate their stability in the subsequent interphase.

scholarly journals The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

2020 ◽  
Author(s):  
Sabrina Dietz ◽  
Miguel Vasconcelos Almeida ◽  
Emily Nischwitz ◽  
Jan Schreier ◽  
Nikenza Viceconte ◽  
...  
Keyword(s):  
Quantitative Proteomics ◽  
Binding Proteins ◽  
Protein Complexes ◽  
Wild Type ◽  
C Elegans ◽  
Double Stranded Dna ◽  
Telomere Sequence ◽  
Proteomics Approach

AbstractTelomeres are bound by dedicated protein complexes, like shelterin in mammals, which protect telomeres from DNA damage. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to screen for proteins binding to C. elegans telomeres, and identified TEBP-1 and TEBP-2, two paralogs that associate to telomeres in vitro and in vivo. TEBP-1 and TEBP-2 are expressed in the germline and during embryogenesis. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a mortal germline, a phenotype characterized by transgenerational germline deterioration. Notably, tebp-1; tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. TEBP-1 and TEBP-2 form a telomeric complex with the known single-stranded telomere-binding proteins POT-1, POT-2, and MRT-1. Furthermore, we find that POT-1 bridges the double- stranded binders TEBP-1 and TEBP-2, with the single-stranded binders POT-2 and MRT-1. These results describe the first telomere-binding complex in C. elegans, with TEBP-1 and TEBP-2, two double-stranded telomere binders required for fertility and that mediate opposite telomere dynamics.

In Vivo Retrieval of a Trapped Merci Embolectomy Device: Technical Case Report

2010 ◽  
Vol 67 (3) ◽  
pp. onsE304-onsE304 ◽  
Author(s):  
Ajeet Gordhan ◽  
John Soliman
Keyword(s):  
Vertebral Artery ◽  
Conformational Changes ◽  
Basilar Artery ◽  
Technical Note ◽  
Vessel Injury ◽  
Artery Segment ◽  
In Vitro Assessment ◽  
Artery Flow

Abstract BACKGROUND AND IMPORTANCE: This technical note describes a complication related to the use of the Merci embolectomy device not previously reported. The device can induce critical flow limitation within an accessed vessel because of a combination of vasospasm and anatomic conformational changes. Furthermore, this can limit the safe removal of the device from intracranial vasculature. We present a novel rescue technique that can be used to safely retrieve the entrapped Merci device without inciting localized vessel injury. CLINICAL PRESENTATION: A 51-year-old male with embolic occlusion of the distal basilar artery and dissection-related occlusion of the left cervical vertebral underwent mechanical thrombolysis. Flow-limiting vasospasm and/or anatomic conformational changes/ telescoping of the intracranial right vertebral artery segment was induced during deployment with subsequent entrapment of the device. Reclamation of the entrapped device was performed by initially removing the Merci microcatheter. The entrapped and fixated device was then resheathed into a 4F slip catheter within the intracranial vertebral artery. The Merci device and the slip catheter were then removed. Right vertebral and proximal basilar artery flow was reestablished after removal of the Merci device. Successful clot extraction was thereafter performed using a microsnare. CONCLUSION: In vitro assessment of the device has demonstrated its propensity to induce vasospasm. In vivo entrapment of the device has not been previously reported. Successful retrieval can be achieved if the Merci device becomes entrapped and fixated. This may be an important consideration as increased utilization of the device occurs.

scholarly journals Functional Analysis of Coordinated Cleavage in V(D)J Recombination

1998 ◽  
Vol 18 (8) ◽  
pp. 4679-4688 ◽  
Author(s):  
Deok Ryong Kim ◽  
Marjorie A. Oettinger
Keyword(s):  
Functional Analysis ◽  
Divalent Cation ◽  
Chromosomal Translocations ◽  
Cleavage Reaction ◽  
Chemically Modified ◽  
Double Stranded Dna ◽  
Cleavage Complex ◽  
Helical Turn

ABSTRACT V(D)J recombination in vivo requires a pair of signals with distinct spacer elements of 12 and 23 bp that separate conserved heptamer and nonamer motifs. Cleavage in vitro by the RAG1 and RAG2 proteins can occur at individual signals when the reaction buffer contains Mn2+, but cleavage is restricted to substrates containing two signals when Mg2+ is the divalent cation. By using a novel V(D)J cleavage substrate, we show that while the RAG proteins alone establish a moderate preference for a 12/23 pair versus a 12/12 pair, a much stricter dependence of cleavage on the 12/23 signal pair is produced by the inclusion of HMG1 and competitor double-stranded DNA. The competitor DNA serves to inhibit the cleavage of substrates carrying a 12/12 or 23/23 pair, as well as the cutting at individual signals in 12/23 substrates. We show that a 23/33 pair is more efficiently recombined than a 12/33 pair, suggesting that the 12/23 rule can be generalized to a requirement for spacers that differ from each other by a single helical turn. Furthermore, we suggest that a fixed spatial orientation of signals is required for cleavage. In general, the same signal variants that can be cleaved singly can function under conditions in which a signal pair is required. However, a chemically modified substrate with one noncleavable signal enables us to show that formation of a functional cleavage complex is mechanistically separable from the cleavage reaction itself and that although cleavage requires a pair of signals, cutting does not have to occur simultaneously at both. The implications of these results are discussed with respect to the mechanism of V(D)J recombination and the generation of chromosomal translocations.

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