scholarly journals A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity

2021 ◽  
Vol 22 (6) ◽  
pp. 2848
Author(s):  
Liu He ◽  
Zoe Jelić Matošević ◽  
Damjan Mitić ◽  
Dora Markulin ◽  
Tom Killelea ◽  
...  
Keyword(s):  
Active Site ◽  
Nuclease Activity ◽  
Human Cells ◽  
Effect Of Temperature ◽  
Tryptophan Residue ◽  
Allosteric Control ◽  
Class 1 ◽  
Higher Temperature ◽  
Dynamics Simulations ◽  
Induced Changes

Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp-406 forms a ‘gate’ for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells.

scholarly journals CRISPR-Cas3 induces broad and unidirectional genome editing in human cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Hiroyuki Morisaka ◽  
Kazuto Yoshimi ◽  
Yuya Okuzaki ◽  
Peter Gee ◽  
Yayoi Kunihiro ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Cleavage ◽  
Exon Skipping ◽  
Nuclease Activity ◽  
Human Cells ◽  
Eukaryotic Cells ◽  
Type I ◽  
Crispr System ◽  
Class 1 ◽  
Component Class

AbstractAlthough single-component Class 2 CRISPR systems, such as type II Cas9 or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multi-component Class 1 CRISPR has been less developed. Here we demonstrate that type I-E CRISPR mediates distinct DNA cleavage activity in human cells. Notably, Cas3, which possesses helicase and nuclease activity, predominantly triggered several thousand base pair deletions upstream of the 5′-ARG protospacer adjacent motif (PAM), without prominent off-target activity. This Cas3-mediated directional and broad DNA degradation can be used to introduce functional gene knockouts and knock-ins. As an example of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne muscular dystrophy (DMD) gene in patient-induced pluripotent stem cells (iPSCs). These findings broaden our understanding of the Class 1 CRISPR system, which may serve as a unique genome editing tool in eukaryotic cells distinct from the Class 2 CRISPR system.

scholarly journals Thermodynamic Studies on the Sorption of Lead (II), Chromium (III) and Manganese (II) ions onto Acid-Activated Shale

2021 ◽  
Vol 2 ◽  
pp. 10-18
Author(s):  
I. R. Ilaboya ◽  
J. S. Okpoko
Keyword(s):  
Metal Ions ◽  
Active Site ◽  
Effect Of Temperature ◽  
Adsorption Parameters ◽  
X Ray ◽  
Solution Interface ◽  
Varied Temperature ◽  
Adsorption Temperature ◽  
Endothermic Process ◽  
Higher Temperature

Shale mineral in its raw form was collected, processed, calcinated and activated using tetraoxosulphate (VI) acid. The microstructural arrangement and chemical composition of the raw, calcinated and acid-activated shale was determined using x-ray fluorescence and scanning electron microscope to verify its ability for the removal of Pb2+, Cr3+ and Mn2+ from wastewater. Batch experimental method was used to study the effect of different adsorption parameters on the sorption efficiency of shale. The effect of temperature on the sorption of Pb2+, Cr3+ and Mn2+ on acid-activated shale was investigated at varied temperature of 15 – 40 . The calculated value of enthalpy () was 12.50 kJ/mol for Pb2+ adsorption, 5 kJ/mol for Cr3+ and 11 kJ/mol for Mn2+ adsorption. The calculated values of Gibbs free energy () varies from -6.576 kJ/mol to 1.358 kJ/mol for Pb2+ adsorptions, from -2.696 kJ/mol to 0.192 kJ/mol for Cr3+ adsorptions, and -4.994 kJ/mol to 1.870 kJ/mol for Mn2+ adsorptions. The entropy () range is 38.68 – 60.946 kJ/mol for Pb2+ adsorptions, 16.69 – 24.58 kJ/mol for Cr3+ adsorptions, and 31.70 – 51.10 kJ/mol for Mn2+ adsorptions. The positive value of  shows that the adsorption of Pb2+, Cr3+ and Mn2+ onto acid-activated shale was an endothermic process. The values of  are negative at temperature of 298 K and above for the three metal ions studied, which confirmed that the adsorption of Pb2+, Cr3+ and Mn2+ on acid-activated shale was a spontaneous process. The decline in  with increasing adsorption temperature showed that adsorptions of Pb2+, Cr3+ and Mn2+ onto acid-activated shale became better at higher temperature while the positive value of  for all metal ions studied showed the amplified arbitrariness at the solid-solution interface during the fixation of the adsorbate on the active site of acid-activated shale.

Nucleation of Disorder in Fe3Al Alloys

1967 ◽  
Vol 25 ◽  
pp. 312-313
Author(s):  
P. R. Swann ◽  
W. R. Duff ◽  
R. M. Fisher
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Annealing Temperature ◽  
Antiphase Domain ◽  
Effect Of Temperature ◽  
Domain Structures ◽  
Transmission Electron ◽  
Domain Boundaries ◽  
Higher Temperature ◽  
The One

Recently we have investigated the phase equilibria and antiphase domain structures of Fe-Al alloys containing from 18 to 50 at.% Al by transmission electron microscopy and Mössbauer techniques. This study has revealed that none of the published phase diagrams are correct, although the one proposed by Rimlinger agrees most closely with our results to be published separately. In this paper observations by transmission electron microscopy relating to the nucleation of disorder in Fe-24% Al will be described. Figure 1 shows the structure after heating this alloy to 776.6°C and quenching. The white areas are B2 micro-domains corresponding to regions of disorder which form at the annealing temperature and re-order during the quench. By examining specimens heated in a temperature gradient of 2°C/cm it is possible to determine the effect of temperature on the disordering reaction very precisely. It was found that disorder begins at existing antiphase domain boundaries but that at a slightly higher temperature (1°C) it also occurs by homogeneous nucleation within the domains. A small (∼ .01°C) further increase in temperature caused these micro-domains to completely fill the specimen.

Exploring secondary interactions and the role of temperature in moisture-contaminated polymer networks through molecular simulations

Soft Matter ◽  
2021 ◽  
Vol 17 (10) ◽  
pp. 2942-2956
Author(s):  
Rishabh D. Guha ◽  
Ogheneovo Idolor ◽  
Katherine Berkowitz ◽  
Melissa Pasquinelli ◽  
Landon R. Grace
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Temperature Variation ◽  
Polymer Networks ◽  
Molecular Simulations ◽  
Effect Of Temperature ◽  
Secondary Interactions ◽  
Dynamics Simulations ◽  
Secondary Bonding

We investigated the effect of temperature variation on the secondary bonding interactions between absorbed moisture and epoxies with different morphologies using molecular dynamics simulations.

scholarly journals Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity

2021 ◽  
Vol 22 (9) ◽  
pp. 4769
Author(s):  
Pablo Maturana ◽  
María S. Orellana ◽  
Sixto M. Herrera ◽  
Ignacio Martínez ◽  
Maximiliano Figueroa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Conformational Dynamics ◽  
High Specificity ◽  
Arginine Decarboxylase ◽  
Space Groups ◽  
X Ray Crystallography ◽  
High Dynamics ◽  
Dynamics Simulations

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer’s, Parkinson’s, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

Discovery of natural product ovalicin sensitive type 1 methionine aminopeptidases: molecular and structural basis

2019 ◽  
Vol 476 (6) ◽  
pp. 991-1003 ◽  
Author(s):  
Vijaykumar Pillalamarri ◽  
Tarun Arya ◽  
Neshatul Haque ◽  
Sandeep Chowdary Bala ◽  
Anil Kumar Marapaka ◽  
...  
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Covalent Modification ◽  
Structural Basis ◽  
Wild Type ◽  
Methionine Aminopeptidases ◽  
Synthetic Derivative ◽  
Dynamics Simulations

Abstract Natural product ovalicin and its synthetic derivative TNP-470 have been extensively studied for their antiangiogenic property, and the later reached phase 3 clinical trials. They covalently modify the conserved histidine in Type 2 methionine aminopeptidases (MetAPs) at nanomolar concentrations. Even though a similar mechanism is possible in Type 1 human MetAP, it is inhibited only at millimolar concentration. In this study, we have discovered two Type 1 wild-type MetAPs (Streptococcus pneumoniae and Enterococcus faecalis) that are inhibited at low micromolar to nanomolar concentrations and established the molecular mechanism. F309 in the active site of Type 1 human MetAP (HsMetAP1b) seems to be the key to the resistance, while newly identified ovalicin sensitive Type 1 MetAPs have a methionine or isoleucine at this position. Type 2 human MetAP (HsMetAP2) also has isoleucine (I338) in the analogous position. Ovalicin inhibited F309M and F309I mutants of human MetAP1b at low micromolar concentration. Molecular dynamics simulations suggest that ovalicin is not stably placed in the active site of wild-type MetAP1b before the covalent modification. In the case of F309M mutant and human Type 2 MetAP, molecule spends more time in the active site providing time for covalent modification.

scholarly journals Structure and Molecular Recognition Mechanism of IMP-13 Metallo-β-Lactamase

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Charlotte A. Softley ◽  
Krzysztof M. Zak ◽  
Mark J. Bostock ◽  
Roberto Fino ◽  
Richard Xu Zhou ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Rational Drug Design ◽  
Tryptophan Residue ◽  
Resonance Spectroscopy ◽  
Global Public Health ◽  
Recognition Mechanism ◽  
Gram Negative ◽  
Content Type ◽  
Locking Mechanism

ABSTRACT Multidrug resistance among Gram-negative bacteria is a major global public health threat. Metallo-β-lactamases (MBLs) target the most widely used antibiotic class, the β-lactams, including the most recent generation of carbapenems. Interspecies spread renders these enzymes a serious clinical threat, and there are no clinically available inhibitors. We present the crystal structures of IMP-13, a structurally uncharacterized MBL from the Gram-negative bacterium Pseudomonas aeruginosa found in clinical outbreaks globally, and characterize the binding using solution nuclear magnetic resonance spectroscopy and molecular dynamics simulations. The crystal structures of apo IMP-13 and IMP-13 bound to four clinically relevant carbapenem antibiotics (doripenem, ertapenem, imipenem, and meropenem) are presented. Active-site plasticity and the active-site loop, where a tryptophan residue stabilizes the antibiotic core scaffold, are essential to the substrate-binding mechanism. The conserved carbapenem scaffold plays the most significant role in IMP-13 binding, explaining the broad substrate specificity. The observed plasticity and substrate-locking mechanism provide opportunities for rational drug design of novel metallo-β-lactamase inhibitors, essential in the fight against antibiotic resistance.

WHICH IS THE PROTON-SHUTTLE IN ISOXANTHOPTERIN DEAMINASE? QM/MM MD UNDERSTANDING

2013 ◽  
Vol 12 (08) ◽  
pp. 1341002 ◽  
Author(s):  
XIN ZHANG ◽  
MING LEI
Keyword(s):  
Crystal Structure ◽  
Molecular Dynamics ◽  
Hydrogen Bonds ◽  
Proton Transfer ◽  
Glutamic Acid ◽  
Active Site ◽  
Nucleophilic Attack ◽  
Zinc Ions ◽  
Initial Model ◽  
Dynamics Simulations

The deamination process of isoxanthopterin catalyzed by isoxanthopterin deaminase was determined using the combined QM(PM3)/MM molecular dynamics simulations. In this paper, the updated PM3 parameters were employed for zinc ions and the initial model was built up based on the crystal structure. Proton transfer and following steps have been investigated in two paths: Asp336 and His285 serve as the proton shuttle, respectively. Our simulations showed that His285 is more effective than Aap336 in proton transfer for deamination of isoxanthopterin. As hydrogen bonds between the substrate and surrounding residues play a key role in nucleophilic attack, we suggested mutating Thr195 to glutamic acid, which could enhance the hydrogen bonds and help isoxanthopterin get close to the active site. The simulations which change the substrate to pterin 6-carboxylate also performed for comparison. Our results provide reference for understanding of the mechanism of deaminase and for enhancing the deamination rate of isoxanthopterin deaminase.

Effect of Temperature on Column Bioleaching of a Refractory Gold Ore

2013 ◽  
Vol 825 ◽  
pp. 352-355 ◽  
Author(s):  
Zeng Ling Wu ◽  
Zhong Sheng Huang ◽  
Ren Man Ruan ◽  
Shui Ping Zhong ◽  
Brenda K.C. Chan
Keyword(s):  
Sulfide Oxidation ◽  
Effect Of Temperature ◽  
Low Grade ◽  
Key Factors ◽  
Gold Extraction ◽  
Gold Ores ◽  
Fraction Distribution ◽  
Higher Temperature ◽  
Liberation Analysis ◽  
Main Factor

Low-grade, finely disseminated refractory sulfide gold ores associated with high arsenic are ubiquitous resources all over the world. Since heap bio-oxidation is an economic and promising biotechnology to recover gold, low grade, high organic carbon and arsenic bearing gold ores from Zhesang Mines in China were chosen for this purpose to study the key factors that would affect biooxidation. Pyrite and arsenopyrite (particle size 0.002-0.22 mm) were the main minerals from Mineral Liberation Analysis (MLA). Column biooxidation and cyanidation of mineral size < 10 mm were evaluated for its potential for gold extraction. Results showed that temperature was the main factor influencing sulfide oxidation. 58-67 % of sulfide was oxidized at 35-45°C after > 240 days of biooxidation with mixed mesophiles, while higher sulfide-S dissolution (77%) was obtained at 60°C. Sulfide-S fraction distribution revealed higher mineral decomposition, finer fractions and eventually higher sulfide oxidation at 60°C. Jarosite and scorodite were found from the residues at 60°C by SEM and EDX, which implies higher temperature accelerated arsenic precipitation. No elemental sulfur was detected during the biooxidation at 35-60°C. After bio-oxidation, column cyanidation was successfully demonstrated recovery of gold from the residues, with gold extraction rate reaching 66%.

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