scholarly journals The polyglutamine amyloid nucleus in living cells is monomeric and has competing dimensions of order

2021 ◽  
Author(s):  
Tej Kandola ◽  
Jiahui Zhang ◽  
Shriram Venkatesan ◽  
Brooklyn Lerbakken ◽  
Jillian F Blanck ◽  
...  
Keyword(s):  
Physical Nature ◽  
Living Cells ◽  
Sequence Variants ◽  
Amyloid Formation ◽  
Structural Mechanism ◽  
Low Concentrations ◽  
Molecular Etiology ◽  
Dynamics Simulations ◽  
Heterogeneous Ensemble ◽  
Polyq Diseases

A long-standing goal of the study of amyloids has been to characterize the physical nature of the rate-determining nucleating event. However, the transience and rarity of that event within the heterogeneous ensemble of states populated by amyloid-forming proteins make it inaccessible to classical biochemistry, structural biology, and computational approaches. Here, we address these limitations by measuring the dependence of amyloid formation on concentration and conformational templates in living cells, whose volumes are sufficiently small to resolve independent nucleation events. We characterized over one hundred rationally designed sequence variants of polyglutamine (polyQ), a polypeptide that precipitates Huntingtons and other amyloid-associated neurodegenerative diseases when its length exceeds a characteristic threshold. We deduce that amyloid formation by polyQ begins with a steric zipper embryo of approximately twelve interdigitated glutamine side chains within an individual polypeptide molecule. Formation of the embryo was limited to polypeptides longer than the pathogenic threshold, and involved neither phase separation nor oligomerization. We found that different amyloid propensities of polyQ sequence variants can be rationalized by steric zipper ordering orthogonally to the axis of polymerization, and validated this intuition using all-atom molecular dynamics simulations. The unique ability of the polyQ sequence to fold in this fashion not only allowed for polyQ amyloid to nucleate from low concentrations; it also stalled amyloid growth with a concomitant accumulation of partially-ordered oligomers. By illuminating the structural mechanism of polyQ amyloid formation in cells, our findings reveal a potential molecular etiology for polyQ diseases, and may provide a roadmap for the design of new therapies.

scholarly journals Mechanism of calcium potentiation of the α7 nicotinic acetylcholine receptor

2020 ◽  
Vol 152 (9) ◽  
Author(s):  
Kathiresan Natarajan ◽  
Nuriya Mukhtasimova ◽  
Jeremías Corradi ◽  
Matías Lasala ◽  
Cecilia Bouzat ◽  
...  
Keyword(s):  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Single Channel ◽  
Structural Motif ◽  
Site Mutation ◽  
Α7 Nicotinic Acetylcholine Receptor ◽  
Nicotinic Acetylcholine ◽  
Low Concentrations ◽  
Channel Opening ◽  
Dynamics Simulations

The α7 nicotinic acetylcholine receptor (nAChR) is among the most abundant types of nAChR in the brain, yet the ability of nerve-released ACh to activate α7 remains enigmatic. In particular, a major population of α7 resides in extra-synaptic regions where the ACh concentration is reduced, owing to dilution and enzymatic hydrolysis, yet ACh shows low potency in activating α7. Using high-resolution single-channel recording techniques, we show that extracellular calcium is a powerful potentiator of α7 activated by low concentrations of ACh. Potentiation manifests as robust increases in the frequency of channel opening and the average duration of the openings. Molecular dynamics simulations reveal that calcium binds to the periphery of the five ligand binding sites and is framed by a pair of anionic residues from the principal and complementary faces of each site. Mutation of residues identified by simulation prevents calcium from potentiating ACh-elicited channel opening. An anionic residue is conserved at each of the identified positions in all vertebrate species of α7. Thus, calcium associates with a novel structural motif on α7 and is an obligate cofactor in regions of limited ACh concentration.

scholarly journals Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sean P. Carney ◽  
Wen Ma ◽  
Kevin D. Whitley ◽  
Haifeng Jia ◽  
Timothy M. Lohman ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Variable Number ◽  
Structural Basis ◽  
Variable Step Size ◽  
Dna Unwinding ◽  
Step Size ◽  
Structural Mechanism ◽  
Dynamics Simulations

AbstractUvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging ~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

3. Making sense of genes and genomes

2018 ◽  
pp. 28-43
Author(s):  
John Archibald
Keyword(s):  
Dna Sequencing ◽  
Computational Methods ◽  
Genome Assembly ◽  
Physical Nature ◽  
Living Cells ◽  
Shotgun Sequencing ◽  
Significant Challenge ◽  
Making Sense ◽  
Problems And Solutions ◽  
Balancing Efficiency

While DNA sequencing is faster and cheaper than ever before, genome assembly remains a significant challenge. ‘Making sense of genes and genomes’ explores how laboratory and computational methods are used in combination to elucidate the true physical nature of DNA molecules inside living cells, and how genes are identified among the vast quantities of chemical letters making up an organism’s genome. It begins with shotgun sequencing—a method that has stood the test of time in balancing efficiency and accuracy. It then considers the problems and solutions of genome assembly; gene finding with transcriptomics; the BLAST algorithm; how to find where proteins carry out their functions; and genome re-sequencing.

Crystal structures and snapshots along the reaction pathway of human phosphoserine phosphatase

2019 ◽  
Vol 75 (6) ◽  
pp. 592-604 ◽  
Author(s):  
Marie Haufroid ◽  
Manon Mirgaux ◽  
Laurence Leherte ◽  
Johan Wouters
Keyword(s):  
Molecular Dynamics ◽  
Reaction Mechanism ◽  
Crystal Structures ◽  
Reaction Pathway ◽  
Living Cells ◽  
Homocysteic Acid ◽  
Phosphoserine Phosphatase ◽  
Key Enzyme ◽  
The Subject ◽  
Dynamics Simulations

The equilibrium between phosphorylation and dephosphorylation is one of the most important processes that takes place in living cells. Human phosphoserine phosphatase (hPSP) is a key enzyme in the production of serine by the dephosphorylation of phospho-L-serine. It is directly involved in the biosynthesis of other important metabolites such as glycine and D-serine (a neuromodulator). hPSP is involved in the survival mechanism of cancer cells and has recently been found to be an essential biomarker. Here, three new high-resolution crystal structures of hPSP (1.5–2.0 Å) in complexes with phosphoserine and with serine, which are the substrate and the product of the reaction, respectively, and in complex with a noncleavable substrate analogue (homocysteic acid) are presented. New types of interactions take place between the enzyme and its ligands. Moreover, the loop involved in the open/closed state of the enzyme is fully refined in a totally unfolded conformation. This loop is further studied through molecular-dynamics simulations. Finally, all of these analyses allow a more complete reaction mechanism for this enzyme to be proposed which is consistent with previous publications on the subject.

scholarly journals Single molecule microscopy reveals key physical features of repair foci in living cells

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Judith Miné-Hattab ◽  
Mathias Heltberg ◽  
Marie Villemeur ◽  
Chloé Guedj ◽  
Thierry Mora ◽  
...  
Keyword(s):  
Single Molecule ◽  
Liquid Droplet ◽  
Physical Nature ◽  
Living Cells ◽  
Double Strand Breaks ◽  
Single Molecule Microscopy ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Functional Homolog ◽  
Physical Features

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus reflecting the existence of a liquid droplet around damaged DNA.

Transmembrane penetration mechanism of Cyclic Pollutants Inspected by Molecular Dynamics and Metadynamics. Case of Morpholine, Phenol, 1,4-Dioxane and Oxane.

2021 ◽  
Author(s):  
Zsófia Borbála Rózsa ◽  
Emma Szőri-Dorogházi ◽  
Béla Viskolcz ◽  
Milán Szőri
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Living Cells ◽  
Passive Transport ◽  
Penetration Mechanism ◽  
Dynamics Simulations

The presence of industrially produced chemicals in water is often not monitored while their passive transport and accumulation can cause serious damage in living cells. Molecular dynamics simulations make an...

scholarly journals Molecular structure and interactions within amyloid-like fibrils formed by a low-complexity protein sequence from FUS

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Myungwoon Lee ◽  
Ujjayini Ghosh ◽  
Kent R. Thurber ◽  
Masato Kato ◽  
Robert Tycko
Keyword(s):  
Self Assembly ◽  
Structural Model ◽  
Rna Binding ◽  
Low Complexity ◽  
Growth Direction ◽  
Structural Basis ◽  
Amyloid Formation ◽  
Hydrophobic Residues ◽  
Dynamics Simulations ◽  
Lateral Sclerosis

AbstractProtein domains without the usual distribution of amino acids, called low complexity (LC) domains, can be prone to self-assembly into amyloid-like fibrils. Self-assembly of LC domains that are nearly devoid of hydrophobic residues, such as the 214-residue LC domain of the RNA-binding protein FUS, is particularly intriguing from the biophysical perspective and is biomedically relevant due to its occurrence within neurons in amyotrophic lateral sclerosis, frontotemporal dementia, and other neurodegenerative diseases. We report a high-resolution molecular structural model for fibrils formed by the C-terminal half of the FUS LC domain (FUS-LC-C, residues 111-214), based on a density map with 2.62 Å resolution from cryo-electron microscopy (cryo-EM). In the FUS-LC-C fibril core, residues 112-150 adopt U-shaped conformations and form two subunits with in-register, parallel cross-β structures, arranged with quasi-21 symmetry. All-atom molecular dynamics simulations indicate that the FUS-LC-C fibril core is stabilized by a plethora of hydrogen bonds involving sidechains of Gln, Asn, Ser, and Tyr residues, both along and transverse to the fibril growth direction, including diverse sidechain-to-backbone, sidechain-to-sidechain, and sidechain-to-water interactions. Nuclear magnetic resonance measurements additionally show that portions of disordered residues 151-214 remain highly dynamic in FUS-LC-C fibrils and that fibrils formed by the N-terminal half of the FUS LC domain (FUS-LC-N, residues 2-108) have the same core structure as fibrils formed by the full-length LC domain. These results contribute to our understanding of the molecular structural basis for amyloid formation by FUS and by LC domains in general.

Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged α-synuclein

2007 ◽  
Vol 4 (4) ◽  
pp. 345-351 ◽  
Author(s):  
María J Roberti ◽  
Carlos W Bertoncini ◽  
Reinhard Klement ◽  
Elizabeth A Jares-Erijman ◽  
Thomas M Jovin
Keyword(s):  
Fluorescence Imaging ◽  
Living Cells ◽  
Amyloid Formation

scholarly journals Symbiotic Bradyrhizobium japonicum Reduces N2O Surrounding the Soybean Root System via Nitrous Oxide Reductase

2006 ◽  
Vol 72 (4) ◽  
pp. 2526-2532 ◽  
Author(s):  
Reiko Sameshima-Saito ◽  
Kaori Chiba ◽  
Junta Hirayama ◽  
Manabu Itakura ◽  
Hisayuki Mitsui ◽  
...  
Keyword(s):  
Nitrous Oxide ◽  
Root System ◽  
Bradyrhizobium Japonicum ◽  
Reductase Activity ◽  
Ambient Air ◽  
Living Cells ◽  
Nitrous Oxide Reductase ◽  
Free Living ◽  
Low Concentrations ◽  
Soybean Roots

ABSTRACT N2O reductase activity in soybean nodules formed with Bradyrhizobium japonicum was evaluated from N2O uptake and conversion of 15N-N2O into 15N-N2. Free-living cells of USDA110 showed N2O reductase activity, whereas a nosZ mutant did not. Complementation of the nosZ mutant with two cosmids containing the nosRZDFYLX genes of B. japonicum USDA110 restored the N2O reductase activity. When detached soybean nodules formed with USDA110 were fed with 15N-N2O, they rapidly emitted 15N-N2 outside the nodules at a ratio of 98.5% of 15N-N2O uptake, but nodules inoculated with the nosZ mutant did not. Surprisingly, N2O uptake by soybean roots nodulated with USDA110 was observed even in ambient air containing a low concentration of N2O (0.34 ppm). These results indicate that the conversion of N2O to N2 depends exclusively on the respiratory N2O reductase and that soybean roots nodulated with B. japonicum carrying the nos genes are able to remove very low concentrations of N2O.

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