scholarly journals Osmosensing by WNK Kinases

2021 ◽  
pp. mbc.E20-01-0089
Author(s):  
Radha Akella ◽  
John M. Humphreys ◽  
Kamil Sekulski ◽  
Haixia He ◽  
Mateusz Durbacz ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Ethylene Glycol ◽  
Conformational Equilibrium ◽  
Bound Water ◽  
Kinase Domain ◽  
Polyethylene Glycol 400 ◽  
X Ray ◽  
X Ray Scattering ◽  
Wnk Kinases

WNK kinases regulate electro-neutral cotransporters that are controlled by osmotic stress and chloride. We showed previously that autophosphorylation of WNK1 is inhibited by chloride, raising the possibility that WNKs are activated by osmotic stress. Here we demonstrate that unphosphorylated WNK isoforms 3 and 1 autophosphorylate in response to osmotic pressure in vitro, applied with the crowding agent polyethylene glycol 400 or osmolyte ethylene glycol, and that this activation is opposed by chloride. Small Angle X-ray Scattering of WNK3 in the presence and absence of PEG400, static light scattering in ethylene glycol, and crystallography of WNK1 were used to understand mechanism. Osmosensing in WNK3 and WNK1 appear to occur through a conformational equilibrium between an inactive, unphosphorylated, chloride-binding dimer and an autophosphorylation-competent monomer. An improved structure of the inactive kinase domain of WNK1, and a comparison with the structure of a monophosphorylated form of WNK1, suggests that large cavities, greater hydration, and specific bound water may participate in the osmosensing mechanism. Our prior work showed that osmolytes have effects on the structure of phosphorylated WNK1, suggestive of multiple stages of osmotic regulation in WNKs.

Time-Resolved Cryo-Electron Microscopy of Oscillating Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 502-503
Author(s):  
Eva-Maria Mandelkow ◽  
Ron Milligan
Keyword(s):  
Electron Microscopy ◽  
Dynamic Instability ◽  
Entire Solution ◽  
Reaction Scheme ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
A Chain ◽  
Ray Scattering

Microtubules form part of the cytoskeleton of eukaryotic cells. They are hollow libers of about 25 nm diameter made up of 13 protofilaments, each of which consists of a chain of heterodimers of α-and β-tubulin. Microtubules can be assembled in vitro at 37°C in the presence of GTP which is hydrolyzed during the reaction, and they are disassembled at 4°C. In contrast to most other polymers microtubules show the behavior of “dynamic instability”, i.e. they can switch between phases of growth and phases of shrinkage, even at an overall steady state [1]. In certain conditions an entire solution can be synchronized, leading to autonomous oscillations in the degree of assembly which can be observed by X-ray scattering (Fig. 1), light scattering, or electron microscopy [2-5]. In addition such solutions are capable of generating spontaneous spatial patterns [6].In an earlier study we have analyzed the structure of microtubules and their cold-induced disassembly by cryo-EM [7]. One result was that disassembly takes place by loss of protofilament fragments (tubulin oligomers) which fray apart at the microtubule ends. We also looked at microtubule oscillations by time-resolved X-ray scattering and proposed a reaction scheme [4] which involves a cyclic interconversion of tubulin, microtubules, and oligomers (Fig. 2). The present study was undertaken to answer two questions: (a) What is the nature of the oscillations as seen by time-resolved cryo-EM? (b) Do microtubules disassemble by fraying protofilament fragments during oscillations at 37°C?

scholarly journals Polymorphic Protective Dps–DNA Co-Crystals by Cryo Electron Tomography and Small Angle X-Ray Scattering

Biomolecules ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 39 ◽  
Author(s):  
Roman Kamyshinsky ◽  
Yury Chesnokov ◽  
Liubov Dadinova ◽  
Andrey Mozhaev ◽  
Ivan Orlov ◽  
...  
Keyword(s):  
Small Angle ◽  
Electron Tomography ◽  
Actual Structure ◽  
X Ray ◽  
Cell Parameters ◽  
X Ray Scattering ◽  
Cryo Electron Tomography ◽  
Dps Protein ◽  
Ray Scattering

Rapid increase of intracellular synthesis of specific histone-like Dps protein that binds DNA to protect the genome against deleterious factors leads to in cellulo crystallization—one of the most curious processes in the area of life science at the moment. However, the actual structure of the Dps–DNA co-crystals remained uncertain in the details for more than two decades. Cryo-electron tomography and small-angle X-ray scattering revealed polymorphous modifications of the co-crystals depending on the buffer parameters. Two different types of the Dps–DNA co-crystals are formed in vitro: triclinic and cubic. Three-dimensional reconstruction revealed DNA and Dps molecules in cubic co-crystals, and the unit cell parameters of cubic lattice were determined consistently by both methods.

scholarly journals Protective Dps–DNA co‐crystallization in stressed cells: an in vitro structural study by small‐angle X‐ray scattering and cryo‐electron tomography

FEBS Letters ◽  
2019 ◽  
Vol 593 (12) ◽  
pp. 1360-1371 ◽  
Author(s):  
Liubov A. Dadinova ◽  
Yurii M. Chesnokov ◽  
Roman A. Kamyshinsky ◽  
Ivan A. Orlov ◽  
Maxim V. Petoukhov ◽  
...  
Keyword(s):  
Structural Study ◽  
Small Angle ◽  
Electron Tomography ◽  
X Ray ◽  
X Ray Scattering ◽  
Cryo Electron Tomography ◽  
Stressed Cells ◽  
Ray Scattering

scholarly journals Decellularized pericardium tissues at increasing glucose, galactose and ribose concentrations and at different time points studied using scanning X-ray microscopy

IUCrJ ◽  
2021 ◽  
Vol 8 (4) ◽  
Author(s):  
Cinzia Giannini ◽  
Liberato De Caro ◽  
Alberta Terzi ◽  
Luca Fusaro ◽  
Davide Altamura ◽  
...  
Keyword(s):  
Molecular Level ◽  
Wide Angle ◽  
Collagen Structure ◽  
Tissue Samples ◽  
X Ray ◽  
X Ray Scattering ◽  
Aging Populations ◽  
Secondary Diseases

Diseases like widespread diabetes or rare galactosemia may lead to high sugar concentrations in the human body, thereby promoting the formation of glycoconjugates. Glycation of collagen, i.e. the formation of glucose bridges, is nonenzymatic and therefore cannot be prevented in any other way than keeping the sugar level low. It relates to secondary diseases, abundantly occurring in aging populations and diabetics. However, little is known about the effects of glycation of collagen on the molecular level. We studied in vitro the effect of glycation, with D-glucose and D-galactose as well as D-ribose, on the structure of type 1 collagen by preparing decellularized matrices of bovine pericardia soaked in different sugar solutions, at increasing concentrations (0, 2.5, 5, 10, 20 and 40 mg ml−1), and incubated at 37°C for 3, 14, 30 and 90 days. The tissue samples were analyzed with small- and wide-angle X-ray scattering in scanning mode. We found that glucose and galactose produce similar changes in collagen, i.e. they mainly affect the lateral packing between macromolecules. However, ribose is much faster in glycation, provoking a larger effect on the lateral packing, but also seems to cause qualitatively different effects on the collagen structure.

scholarly journals Direct Correlation Between Ligand-Induced α-Synuclein Oligomers and Amyloid-like Fibril Growth

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Martin Nors Pedersen ◽  
Vito Foderà ◽  
Istvan Horvath ◽  
Andreas van Maarschalkerweerd ◽  
Katrine Nørgaard Toft ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Small Angle ◽  
Alpha Synuclein ◽  
X Ray ◽  
Transient Nature ◽  
X Ray Scattering ◽  
Amyloid Deposits ◽  
Potential Link ◽  
Presence And Absence

Abstract Aggregation of proteins into amyloid deposits is the hallmark of several neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. The suggestion that intermediate oligomeric species may be cytotoxic has led to intensified investigations of pre-fibrillar oligomers, which are complicated by their transient nature and low population. Here we investigate alpha-synuclein oligomers, enriched by a 2-pyridone molecule (FN075) and the conversion of oligomers into fibrils. As probed by leakage assays, the FN075 induced oligomers potently disrupt vesicles in vitro, suggesting a potential link to disease related degenerative activity. Fibrils formed in the presence and absence of FN075 are indistinguishable on microscopic and macroscopic levels. Using small angle X-ray scattering, we reveal that FN075 induced oligomers are similar, but not identical, to oligomers previously observed during alpha-synuclein fibrillation. Since the levels of FN075 induced oligomers correlate with the amounts of fibrils among different FN075:protein ratios, the oligomers appear to be on-pathway and modeling supports an ‘oligomer stacking model’ for alpha-synuclein fibril elongation.

scholarly journals Structural and Dynamic Markers of Membrane Osmotic Stress From X-Ray Scattering and Solid-State 2H NMR

2009 ◽  
Vol 96 (3) ◽  
pp. 356a
Author(s):  
Avigdor Leftin ◽  
Matthew J. Justice ◽  
Jacob G. Kinnun ◽  
Horia I. Petrache ◽  
Michael F. Brown
Keyword(s):  
Solid State ◽  
Osmotic Stress ◽  
2H Nmr ◽  
X Ray ◽  
X Ray Scattering ◽  
Ray Scattering

scholarly journals The domain architecture of JBP1 suggests synergy between J-base DNA binding and thymidine hydroxylase activity

2018 ◽  
Author(s):  
Athanassios Adamopoulos ◽  
Tatjana Heidebrecht ◽  
Jeroen Roosendaal ◽  
Wouter G Touw ◽  
Isabelle Phan ◽  
...  
Keyword(s):  
Dna Binding ◽  
Domain Architecture ◽  
Three Dimensional ◽  
Specific Sequence ◽  
X Ray ◽  
Tet Proteins ◽  
X Ray Scattering ◽  
Sequence Patterns ◽  
Three Dimensional Models

JBP1 (J-DNA Binding Protein 1) contributes to biosynthesis and maintenance of base J (β-D-glucosyl-hydroxymethyluracil), a modification of thymidine (T) confined to pathogenic protozoa. JBP1 has two known functional domains: an N-terminal thymidine hydroxylase (TH) homologous to the 5-methylcytosine hydroxylase domain in TET proteins; and a J-DNA binding domain (JDBD) that resides in the middle of JBP1. Here we show that removing JDBD from JBP1 results in a soluble protein (Δ-JDBD) with the N- and C-terminal regions tightly associated together in a well-ordered domain. This Δ-JDBD domain retains thymidine hydroxylation activity in vitro, but displays a fifteen-fold lower apparent rate of hydroxylation compared to JBP1. Small Angle X-ray Scattering (SAXS) experiments on JBP1 and JDBD in the presence and absence of J-DNA, and on Δ-JDBD, allowed us to generate low-resolution three-dimensional models. We conclude that Δ-JDBD, and not the N-terminal region of JBP1 alone, is a distinct folding unit. Our SAXS-based model supports the notion that binding of JDBD specifically to J-DNA can facilitate hydroxylation a T 12-14 bp downstream on the complementary strand of the J-recognition site. We postulate that insertion of the JDBD module in the Δ-JDBD scaffold during evolution provided a mechanism to synergize between J recognition and T hydroxylation, ensuring inheritance of J in specific sequence patterns following DNA replication.

scholarly journals Biophysical analysis of Pseudomonas-phage PaP3 small terminase suggests a mechanism for sequence-specific DNA-binding by lateral interdigitation

2020 ◽  
Vol 48 (20) ◽  
pp. 11721-11736
Author(s):  
Marzia Niazi ◽  
Tyler J Florio ◽  
Ruoyu Yang ◽  
Ravi K Lokareddy ◽  
Nicholas A Swanson ◽  
...  
Keyword(s):  
Heparin Binding ◽  
Genome Packaging ◽  
X Ray ◽  
X Ray Scattering ◽  
Open Conformation ◽  
Terminase Subunit ◽  
Biophysical Analysis ◽  
Specific Manner ◽  
Genome Encapsidation

Abstract The genome packaging motor of tailed bacteriophages and herpesviruses is a powerful nanomachine built by several copies of a large (TerL) and a small (TerS) terminase subunit. The motor assembles transiently at the portal vertex of an empty precursor capsid (or procapsid) to power genome encapsidation. Terminase subunits have been studied in-depth, especially in classical bacteriophages that infect Escherichia coli or Salmonella, yet, less is known about the packaging motor of Pseudomonas-phages that have increasing biomedical relevance. Here, we investigated the small terminase subunit from three Podoviridae phages that infect Pseudomonas aeruginosa. We found TerS is polymorphic in solution but assembles into a nonamer in its high-affinity heparin-binding conformation. The atomic structure of Pseudomonas phage PaP3 TerS, the first complete structure for a TerS from a cos phage, reveals nine helix-turn-helix (HTH) motifs asymmetrically arranged around a β-stranded channel, too narrow to accommodate DNA. PaP3 TerS binds DNA in a sequence-specific manner in vitro. X-ray scattering and molecular modeling suggest TerS adopts an open conformation in solution, characterized by dynamic HTHs that move around an oligomerization core, generating discrete binding crevices for DNA. We propose a model for sequence-specific recognition of packaging initiation sites by lateral interdigitation of DNA.

scholarly journals Site-specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription

2020 ◽  
Vol 117 (37) ◽  
pp. 22823-22832
Author(s):  
Yan Wang ◽  
Yaoyi Chen ◽  
Yanping Hu ◽  
Xianyang Fang
Keyword(s):  
Structural Biology ◽  
Solid Phase ◽  
Site Specific ◽  
X Ray ◽  
X Ray Scattering ◽  
Rna Labeling ◽  
Virus Serotype ◽  
Genetic Alphabet ◽  
Ray Scattering

Conjugation of RNAs with nanoparticles (NPs) is of significant importance because of numerous applications in biology and medicine, which, however, remains challenging especially for large ones. So far, the majority of RNA labeling relies on solid-phase chemical synthesis, which is generally limited to RNAs smaller than 100 nucleotides (nts). We, here, present an efficient and generally applicable labeling strategy for site-specific covalent conjugation of large RNAs with a gold nanoparticle (Nanogold) empowered by transcription of an expanded genetic alphabet containing the A-T/U and G-C natural base pairs (bps) and the TPT3-NaM unnatural base pair (UBP). We synthesize an amine-derivatized TPT3 (TPT3A), which is site specifically incorporated into a 97-nt 3′SL RNA and a 719-nt minigenomic RNA (DENV-mini) from Dengue virus serotype 2 (DENV2) by in vitro T7 transcription. The TPT3A-modified RNAs are covalently conjugated with mono-Sulfo-N-hydroxysuccinimidyl (NHS)-Nanogold NPs via an amine and NHS ester reaction and further purified under nondenaturing conditions. TPT3 modification and Nanogold labeling cause minimal structural perturbations to the RNAs by circular dichroism, small angle X-ray scattering (SAXS), and binding activity assay. We demonstrate the application of the Nanogold-RNA conjugates in large RNA structural biology by an emerging molecular ruler, X-ray scattering interferometry (XSI). The internanoparticle distance distributions in the 3′SL and DENV-mini RNAs derived from XSI measurements support the hypothetical model of flavivirus genome circularization, thus, validate the applicability of this labeling strategy. The presented strategy overcomes the size constraints in conventional RNA labeling strategies and is expected to have wide applications in large RNA structural biology and RNA nanotechnology.

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