scholarly journals Tuning the formation of a covalent haem–protein link by selection of reductive or oxidative conditions as exemplified by ascorbate peroxidase

2007 ◽  
Vol 408 (3) ◽  
pp. 355-361 ◽  
Author(s):  
Clive L. Metcalfe ◽  
Oliver Daltrop ◽  
Stuart J. Ferguson ◽  
Emma Lloyd Raven
Keyword(s):  
Ascorbate Peroxidase ◽  
Covalent Bond ◽  
Cysteine Residue ◽  
Covalent Attachment ◽  
Methionine Residue ◽  
Reducing Conditions ◽  
Mass Spectrometric Evidence ◽  
Covalent Bond Formation ◽  
Haem Protein ◽  
Vinyl Group

Previous work [Metcalfe, Ott, Patel, Singh, Mistry, Goff and Raven (2004) J. Am. Chem. Soc. 126, 16242–16248] has shown that the introduction of a methionine residue (S160M variant) close to the 2-vinyl group of the haem in ascorbate peroxidase leads to the formation of a covalent haem–methionine linkage under oxidative conditions (i.e. on reaction with H2O2). In the present study, spectroscopic, HPLC and mass spectrometric evidence is presented to show that covalent attachment of the haem to an engineered cysteine residue can also occur in the S160C variant, but, in this case, under reducing conditions analogous to those used in the formation of covalent links in cytochrome c. The data add an extra dimension to our understanding of haem to protein covalent bond formation because they show that different types of covalent attachment (one requiring an oxidative mechanism, the other a reductive pathway) are both accessible within same protein architecture.

scholarly journals Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yusaku Hontani ◽  
Mikhail Baloban ◽  
Francisco Velazquez Escobar ◽  
Swetta A. Jansen ◽  
Daria M. Shcherbakova ◽  
...  
Keyword(s):  
Real Time ◽  
Rational Design ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Covalent Bond ◽  
Binding Pocket ◽  
Tissue Imaging ◽  
Covalent Attachment ◽  
Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

scholarly journals Physical and chemical imaging of adhesive interfaces with soft X-rays

2021 ◽  
Vol 2 (1) ◽  
Author(s):  
Hiroyuki Yamane ◽  
Masaki Oura ◽  
Osamu Takahashi ◽  
Tomoko Ishihara ◽  
Noriko Yamazaki ◽  
...  
Keyword(s):  
Molecular Level ◽  
Covalent Bond ◽  
Chemical Imaging ◽  
Diglycidyl Ether ◽  
X Rays ◽  
X Ray ◽  
Covalent Bond Formation ◽  
Chemical States ◽  
The Individual ◽  
Physical And Chemical

AbstractAdhesion is an interfacial phenomenon that is critical for assembling carbon structural composites for next-generation aircraft and automobiles. However, there is limited understanding of adhesion on the molecular level because of the difficulty in revealing the individual bonding factors. Here, using soft X-ray spectromicroscopy we show the physical and chemical states of an adhesive interface composed of a thermosetting polymer of 4,4’-diaminodiphenylsulfone-cured bisphenol A diglycidyl ether adhered to a thermoplastic polymer of plasma-treated polyetheretherketone. We observe multiscale phenomena in the adhesion mechanisms, including sub-mm complex interface structure, sub-μm distribution of the functional groups, and molecular-level covalent-bond formation. These results provide a benchmark for further research to examine how physical and chemical states correlate with adhesion, and demonstrate that soft X-ray imaging is a promising approach for visualizing the physical and chemical states at adhesive interfaces from the sub-mm level to the molecular level.

scholarly journals Chemical Investigations Into the Biosynthesis of the Gymnastatin and Dankastatin Alkaloids

2021 ◽  
Author(s):  
Bingqi Tong ◽  
Bridget Belcher ◽  
Daniel Nomura ◽  
Thomas Maimone
Keyword(s):  
Natural Products ◽  
Protein Function ◽  
Bond Formation ◽  
Covalent Bond ◽  
Fungal Strain ◽  
Fertile Ground ◽  
Covalent Bond Formation

Electrophilic natural products have provided fertile ground for understanding how nature inhibits protein function using covalent bond formation. The fungal strain Gymnascella dankaliensis has provided an especially interesting collection of...

Material Adhesion through Direct Covalent Bond Formation Assisted by Noncovalent Interactions

2021 ◽  
Author(s):  
Motofumi Osaki ◽  
Tomoko Sekine ◽  
Hiroyasu Yamaguchi ◽  
Yoshinori Takashima ◽  
Akira Harada
Keyword(s):  
Noncovalent Interactions ◽  
Bond Formation ◽  
Covalent Bond ◽  
Covalent Bond Formation

scholarly journals Labile disulfide bonds are common at the leucocyte cell surface

Open Biology ◽  
2011 ◽  
Vol 1 (3) ◽  
pp. 110010 ◽  
Author(s):  
Clive Metcalfe ◽  
Peter Cresswell ◽  
Laura Ciaccia ◽  
Benjamin Thomas ◽  
A. Neil Barclay
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Cell Surface ◽  
Disulfide Bonds ◽  
Reduction Process ◽  
Cysteine Residue ◽  
Surface Proteins ◽  
Protein Activity ◽  
Functional Changes ◽  
Reducing Conditions

Redox conditions change in events such as immune and platelet activation, and during viral infection, but the biochemical consequences are not well characterized. There is evidence that some disulfide bonds in membrane proteins are labile while others that are probably structurally important are not exposed at the protein surface. We have developed a proteomic/mass spectrometry method to screen for and identify non-structural, redox-labile disulfide bonds in leucocyte cell-surface proteins. These labile disulfide bonds are common, with several classes of proteins being identified and around 30 membrane proteins regularly identified under different reducing conditions including using enzymes such as thioredoxin. The proteins identified include integrins, receptors, transporters and cell–cell recognition proteins. In many cases, at least one cysteine residue was identified by mass spectrometry as being modified by the reduction process. In some cases, functional changes are predicted (e.g. in integrins and cytokine receptors) but the scale of molecular changes in membrane proteins observed suggests that widespread effects are likely on many different types of proteins including enzymes, adhesion proteins and transporters. The results imply that membrane protein activity is being modulated by a ‘redox regulator’ mechanism.

scholarly journals Molecular recognition of organic ammonium ions in solution using synthetic receptors

2010 ◽  
Vol 6 ◽  
Author(s):  
Andreas Späth ◽  
Burkhard König
Keyword(s):  
Molecular Recognition ◽  
Hydrophobic Interactions ◽  
Covalent Bond ◽  
Ammonium Ion ◽  
Synthetic Receptors ◽  
Ammonium Ions ◽  
Wide Range ◽  
Covalent Bond Formation ◽  
Reversible Covalent ◽  
Ion Receptors

Ammonium ions are ubiquitous in chemistry and molecular biology. Considerable efforts have been undertaken to develop synthetic receptors for their selective molecular recognition. The type of host compounds for organic ammonium ion binding span a wide range from crown ethers to calixarenes to metal complexes. Typical intermolecular interactions are hydrogen bonds, electrostatic and cation–π interactions, hydrophobic interactions or reversible covalent bond formation. In this review we discuss the different classes of synthetic receptors for organic ammonium ion recognition and illustrate the scope and limitations of each class with selected examples from the recent literature. The molecular recognition of ammonium ions in amino acids is included and the enantioselective binding of chiral ammonium ions by synthetic receptors is also covered. In our conclusion we compare the strengths and weaknesses of the different types of ammonium ion receptors which may help to select the best approach for specific applications.

scholarly journals Mechanism of Covalent Binding of Ibrutinib to Bruton’s Tyrosine Kinase revealed by QM/MM Calculations

2020 ◽  
Author(s):  
Angus Voice ◽  
Gary Tresadern ◽  
Rebecca Twidale ◽  
Herman Van Vlijmen ◽  
Adrian Mulholland
Keyword(s):  
Tyrosine Kinase ◽  
Covalent Binding ◽  
Bond Formation ◽  
Covalent Bond ◽  
Covalent Modification ◽  
Mechanistic Study ◽  
Bruton’S Tyrosine Kinase ◽  
Bruton's Tyrosine Kinase ◽  
Covalent Inhibitors ◽  
Covalent Bond Formation

<p>Ibrutinib is the first covalent inhibitor of Bruton’s tyrosine kinase (BTK) to be used in the treatment of B-cell cancers. Understanding the mechanism of covalent inhibition is crucial for the design of safer and more selective covalent inhibitors that target BTK. There are questions surrounding the precise mechanism of covalent bond formation in BTK as there is no appropriate active site residue that can act as a base to deprotonate the cysteine thiol prior to covalent bond formation. To address this, we have investigated several mechanistic pathways of covalent modification of C481 in BTK by ibrutinib using QM/MM reaction simulations. The lowest energy pathway we identified involves a direct proton transfer from C481 to the acrylamide warhead in ibrutinib, followed by covalent bond formation to form an enol intermediate. There is a subsequent rate-limiting keto-enol tautomerisation step (DG<sup>‡</sup>=10.5 kcal mol<sup>-1</sup>) to reach the inactivated BTK/ibrutinib complex. Our results represent the first mechanistic study of BTK inactivation by ibrutinib to consider multiple mechanistic pathways. These findings should aid in the design of covalent drugs that target BTK and related proteins. </p>

scholarly journals The 8-Pyrrole-Benzothiazinones Are Noncovalent Inhibitors of DprE1 from Mycobacterium tuberculosis

2015 ◽  
Vol 59 (8) ◽  
pp. 4446-4452 ◽  
Author(s):  
Vadim Makarov ◽  
João Neres ◽  
Ruben C. Hartkoorn ◽  
Olga B. Ryabova ◽  
Elena Kazakova ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nitro Group ◽  
Covalent Bond ◽  
Antimycobacterial Activity ◽  
Content Type ◽  
Sar Studies ◽  
Covalent Bond Formation

ABSTRACT8-Nitro-benzothiazinones (BTZs), such as BTZ043 and PBTZ169, inhibit decaprenylphosphoryl-β-d-ribose 2′-oxidase (DprE1) and display nanomolar bactericidal activity againstMycobacterium tuberculosisin vitro. Structure-activity relationship (SAR) studies revealed the 8-nitro group of the BTZ scaffold to be crucial for the mechanism of action, which involves formation of a semimercaptal bond with Cys387 in the active site of DprE1. To date, substitution of the 8-nitro group has led to extensive loss of antimycobacterial activity. Here, we report the synthesis and characterization of the pyrrole-benzothiazinones PyrBTZ01 and PyrBTZ02, non-nitro-benzothiazinones that retain significant antimycobacterial activity, with MICs of 0.16 μg/ml againstM. tuberculosis. These compounds inhibit DprE1 with 50% inhibitory concentration (IC50) values of <8 μM and present favorablein vitroabsorption-distribution-metabolism-excretion/toxicity (ADME/T) andin vivopharmacokinetic profiles. The most promising compound, PyrBTZ01, did not show efficacy in a mouse model of acute tuberculosis, suggesting that BTZ-mediated killing through DprE1 inhibition requires a combination of both covalent bond formation and compound potency.

Supramolecular Assembly-Enabled Homochiral Polymerization of Short (dA)n Oligonucleotides

2021 ◽  
Author(s):  
Sreejith Mangalath ◽  
Suneesh C Karunakaran ◽  
Gary Newnam ◽  
Gary Schuster ◽  
Nicholas Hud
Keyword(s):  
Supramolecular Chemistry ◽  
Bond Formation ◽  
Supramolecular Assembly ◽  
Covalent Bond ◽  
Complex Mixtures ◽  
Covalent Bond Formation

A goal of supramolecular chemistry is to create covalent polymers of precise composition and stereochemistry from complex mixtures by the reversible assembly of specific monomers prior to covalent bond formation....

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