671 results match your criteria ph-dependent mutagenesis

Light chain subunit of a poorly soluble human IgG2λ crystallizes in physiological pH environment both in cellulo and in vitro.

Biochim Biophys Acta Mol Cell Res 2021 Jun 10;1868(9):119078. Epub 2021 Jun 10.

Department of Therapeutic Discovery, Amgen Inc., Thousand Oaks, CA 91320, USA.

Prominent inclusion bodies can develop in the endoplasmic reticulum (ER) when overexpressed antibodies possess intrinsically high condensation propensities. These observations suggest that antibodies deemed to show notable solubility problems may reveal such characteristics preemptively in the form of ER-associated inclusion bodies during antibody overexpression. To define the relationships between solubility problems and inclusion body phenotypes, we investigated the biosynthesis of a model human IgG2λ that shows severe opalescence in an acidic formulation buffer yet retains high solubility at physiological pH. Read More

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The mutation of Thr315 to Asn of GH10 xylanase XynR increases the alkaliphily but decreases the alkaline resistance.

Biosci Biotechnol Biochem 2021 Jun 2. Epub 2021 Jun 2.

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

XynR is a thermophilic and alkaline GH10 xylanase, identified in the culture broth of alkaliphilic and thermophilic Bacillus sp. strain TAR-1. We previously selected S92E as a thermostable variant from a site saturation mutagenesis library. Read More

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Structure, interdomain dynamics and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes.

J Biol Chem 2021 Mar 18:100565. Epub 2021 Mar 18.

Department of Chemistry, Biochemistry Division, Johannes Gutenberg-University, Mainz, Germany; Centre for Biomolecular Magnetic Resonance (BMRZ), Goethe University, Frankfurt, Germany; current address: Faculty of Chemistry and Earth Sciences, Institute of Organic and Macromolecular Chemistry, Friedrich-Schiller-University, Jena, Germany; Cluster of Excellence 'Balance of the Microverse', Friedrich-Schiller-University, Jena, Germany. Electronic address:

Rhodesain is the lysosomal cathepsin L-like cysteine protease of T. brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Read More

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Modification of the pH Dependence of Assembly of Yeast Cargo Receptor Emp47p Coiled-Coil Domains: Computational Design and Experimental Mutagenesis.

J Phys Chem B 2021 03 1;125(9):2222-2230. Epub 2021 Mar 1.

Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya, Aichi 468-8503, Japan.

The coiled-coil domains of the putative yeast cargo receptors Emp46p and Emp47p (Emp46p and Emp47p) assemble into heterocomplexes at neutral pH. Upon lowering the pH, the complex dissociates and reassembles into homo-oligomers. A glutamate residue (E303) located on the hydrophobic surface of Emp46p serves as the pH-sensing switch for assembly and segregation, and we have suggested that its side chains are protonated in the heterocomplex, even at neutral pH. Read More

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pH-dependent and dynamic interactions of cystatin C with heparan sulfate.

Commun Biol 2021 Feb 12;4(1):198. Epub 2021 Feb 12.

Department of Oral Biology, The University at Buffalo, Buffalo, NY, USA.

Cystatin C (Cst-3) is a potent inhibitor of cysteine proteases with diverse biological functions. As a secreted protein, the potential interaction between Cst-3 and extracellular matrix components has not been well studied. Here we investigated the interaction between Cst-3 and heparan sulfate (HS), a major component of extracellular matrix. Read More

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February 2021

pH-dependence of signaling-state formation in Drosophila cryptochrome.

Arch Biochem Biophys 2021 03 3;700:108787. Epub 2021 Feb 3.

Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Albertstraße 21, 79104, Freiburg, Germany. Electronic address:

Cryptochromes, FAD-dependent blue light photoreceptors, undergo a series of electron transfer reactions after light excitation. Time-resolved optical spectroscopy was employed to investigate the pH dependence of all light-dependent reactions in the cryptochrome from fruit flies. Signal state formation experiments on a time scale of seconds were found to be strongly pH dependent, and formation of both anionic and neutral FAD radicals could be detected, with reaction rates increasing by a factor of ~2. Read More

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Mutational and structural analysis of an ancestral fungal dye-decolorizing peroxidase.

FEBS J 2021 Jun 8;288(11):3602-3618. Epub 2021 Jan 8.

Molecular Enzymology Group, University of Groningen, The Netherlands.

Dye-decolorizing peroxidases (DyPs) constitute a superfamily of heme-containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. Read More

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Protonation status and control mechanism of flavin-oxygen intermediates in the reaction of bacterial luciferase.

FEBS J 2021 May 16;288(10):3246-3260. Epub 2020 Dec 16.

Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok, Thailand.

Bacterial luciferase catalyzes a bioluminescent reaction by oxidizing long-chain aldehydes to acids using reduced FMN and oxygen as co-substrates. Although a flavin C4a-peroxide anion is postulated to be the intermediate reacting with aldehyde prior to light liberation, no clear identification of the protonation status of this intermediate has been reported. Here, transient kinetics, pH variation, and site-directed mutagenesis were employed to probe the protonation state of the flavin C4a-hydroperoxide in bacterial luciferase. Read More

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Role of E270 in pH- and metal-sensitivities of firefly luciferases.

Photochem Photobiol Sci 2020 Nov;19(11):1548-1558

Graduate Program of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil.

Firefly luciferases display a typical change in bioluminescence color to red at acidic pH, high temperatures and in the presence of heavy metals. Recently, the proton and metal sensing site responsible for the pH-sensitivity of firefly luciferases, which involves the salt bridges between E311-R337 and H310-E354, was identified. However, it is unclear what other residues contribute to the distinct degrees of pH-sensitivity observed in other firefly luciferases. Read More

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November 2020

Studying the Role of a Single Mutation of a Family 11 Glycoside Hydrolase Using High-Resolution X-ray Crystallography.

Protein J 2020 12 31;39(6):671-680. Epub 2020 Oct 31.

College of Science, Nanjing Agricultural University, Nanjing, 210095, People's Republic of China.

XynII is a family 11 glycoside hydrolase that uses the retaining mechanism for catalysis. In the active site, E177 works as the acid/base and E86 works as the nucleophile. Mutating an uncharged residue (N44) to an acidic residue (D) near E177 decreases the enzyme's optimal pH by ~ 1. Read More

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December 2020

Visualizing a protonated RNA state that modulates microRNA-21 maturation.

Nat Chem Biol 2021 01 26;17(1):80-88. Epub 2020 Oct 26.

Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

MicroRNAs are evolutionarily conserved small, noncoding RNAs that regulate diverse biological processes. Due to their essential regulatory roles, microRNA biogenesis is tightly regulated, where protein factors are often found to interact with specific primary and precursor microRNAs for regulation. Here, using NMR relaxation dispersion spectroscopy and mutagenesis, we reveal that the precursor of oncogenic microRNA-21 exists as a pH-dependent ensemble that spontaneously reshuffles the secondary structure of the entire apical stem-loop region, including the Dicer cleavage site. Read More

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January 2021

Discovery and mechanism of a pH-dependent dual-binding-site switch in the interaction of a pair of protein modules.

Sci Adv 2020 Oct 23;6(43). Epub 2020 Oct 23.

CAS Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China.

Many important proteins undergo pH-dependent conformational changes resulting in "on-off" switches for protein function, which are essential for regulation of life processes and have wide application potential. Here, we report a pair of cellulosomal assembly modules, comprising a cohesin and a dockerin from , which interact together following a unique pH-dependent switch between two functional sites rather than on-off states. The two cohesin-binding sites on the dockerin are switched from one to the other at pH 4. Read More

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October 2020

Full-length three-dimensional structure of the influenza A virus M1 protein and its organization into a matrix layer.

PLoS Biol 2020 09 30;18(9):e3000827. Epub 2020 Sep 30.

Departments of Genetics Stanford University School of Medicine, Stanford, California, United States of America.

Matrix proteins are encoded by many enveloped viruses, including influenza viruses, herpes viruses, and coronaviruses. Underneath the viral envelope of influenza virus, matrix protein 1 (M1) forms an oligomeric layer critical for particle stability and pH-dependent RNA genome release. However, high-resolution structures of full-length monomeric M1 and the matrix layer have not been available, impeding antiviral targeting and understanding of the pH-dependent transitions involved in cell entry. Read More

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September 2020

Hsp90 chaperones have an energetic hot-spot for binding inhibitors.

Protein Sci 2020 10 8;29(10):2101-2111. Epub 2020 Sep 8.

Department of Biochemistry, Brandeis University, Waltham, Massachusetts, USA.

Although Hsp90-family chaperones have been extensively targeted with ATP-competitive inhibitors, it is unknown whether high affinity is achieved from a few highly stabilizing contacts or from many weaker contacts within the ATP-binding pocket. A large-scale analysis of Hsp90α:inhibitor structures shows that inhibitor hydrogen-bonding to a conserved aspartate (D93 in Hsp90α) stands out as most universal among Hsp90 inhibitors. Here we show that the D93 region makes a dominant energetic contribution to inhibitor binding for both cytosolic and organelle-specific Hsp90 paralogs. Read More

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October 2020

Structural Basis of Oligomerization of N-Terminal Domain of Spider Aciniform Silk Protein.

Int J Mol Sci 2020 Jun 23;21(12). Epub 2020 Jun 23.

Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.

Spider silk is self-assembled from water-soluble silk proteins through changes in the environment, including pH, salt concentrations, and shear force. The N-terminal domains of major and minor ampullate silk proteins have been found to play an important role in the assembly process through salt- and pH-dependent dimerization. Here, we identified the sequences of the N-terminal domains of aciniform silk protein (AcSpN) and major ampullate silk protein (MaSpN) from (). Read More

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Structure-based design approach to rational site-directed mutagenesis of β-lactoglobulin.

J Struct Biol 2020 05 10;210(2):107493. Epub 2020 Mar 10.

Jagiellonian University, Faculty of Chemistry, Gronostajowa 2, 30-387 Kraków, Poland. Electronic address:

Recombinant proteins play an important role in medicine and have diverse applications in industrial biotechnology. Lactoglobulin has shown great potential for use in targeted drug delivery and body fluid detoxification because of its ability to bind a variety of molecules. In order to modify the biophysical properties of β-lactoglobulin, a series of single-site mutations were designed using a structure-based approach. Read More

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Design of Protein Logic Gate System Operating on Lipid Membranes.

ACS Synth Biol 2020 02 11;9(2):316-328. Epub 2020 Feb 11.

Department of Molecular Biology and Nanobiotechnology , National Institute of Chemistry , Hajdrihova ulica 19 , 1001 Ljubljana , Slovenia.

Lipid membranes are becoming increasingly popular in synthetic biology due to their biophysical properties and crucial role in communication between different compartments. Several alluring protein-membrane sensors have already been developed, whereas protein logic gates designs on membrane-embedded proteins are very limited. Here we demonstrate the construction of a two-level protein-membrane logic gate with an OR-AND logic. Read More

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February 2020

A subtle structural change in the distal haem pocket has a remarkable effect on tuning hydrogen peroxide reactivity in dye decolourising peroxidases from Streptomyces lividans.

Dalton Trans 2020 Feb 16;49(5):1620-1636. Epub 2020 Jan 16.

School of Life Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, UK.

Dye decolourising peroxidases (DyPs) are oxidative haem containing enzymes that can oxidise organic substrates by first reacting with hydrogen peroxide. Herein, we have focused on two DyP homologs, DtpAa and DtpA, from the soil-dwelling bacterium Streptomyces lividans. By using X-ray crystallography, stopped-flow kinetics, deuterium kinetic isotope studies and EPR spectroscopy, we show that both DyPs react with peroxide to form compound I (a Fe[double bond, length as m-dash]O species and a porphyrin π-cation radical), via a common mechanism, but the reactivity and rate limits that define the mechanism are markedly different between the two homologs (DtpA forms compound I rapidly, no kinetic isotope effect; DtpAa 100-fold slower compound I formation and a distinct kinetic isotope effect). Read More

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February 2020

Structure-based engineering of pH-dependent antibody binding for selective targeting of solid-tumor microenvironment.

MAbs 2020 Jan-Dec;12(1):1682866

Human Health Therapeutics Research Centre, National Research Council Canada, Montreal, Quebec, Canada.

Recent development of monoclonal antibodies as mainstream anticancer agents demands further optimization of their safety for use in humans. Potent targeting and/or effector activities on normal tissues is an obvious toxicity concern. Optimization of specific tumor targeting could be achieved by taking advantage of the extracellular acidity of solid tumors relative to normal tissues. Read More

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January 2021

Tuning Radical Relay Residues by Proton Management Rescues Protein Electron Hopping.

J Am Chem Soc 2019 11 28;141(44):17571-17587. Epub 2019 Oct 28.

Department of Chemistry and Chemical Biology , Cornell University , Ithaca , New York 14853 , United States.

Transient tyrosine and tryptophan radicals play key roles in the electron transfer (ET) reactions of photosystem (PS) II, ribonucleotide reductase (RNR), photolyase, and many other proteins. However, Tyr and Trp are not functionally interchangeable, and the factors controlling their reactivity are often unclear. Cytochrome peroxidase (CcP) employs a Trp191 radical to oxidize reduced cytochrome c (). Read More

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November 2019

Extracellular lysine 38 plays a crucial role in pH-dependent transport via human monocarboxylate transporter 1.

Biochim Biophys Acta Biomembr 2020 02 5;1862(2):183068. Epub 2019 Oct 5.

Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12-jo, Nishi-6-chome, Kita-ku, Sapporo 060-0812, Japan. Electronic address:

Human monocarboxylate transporters (hMCTs) are expressed in many tissues and mediate the transport of various substrates across the plasma membrane. Among hMCTs, hMCT1-4 cotransport H with monocarboxylates such as pyruvate and l-lactate, implying that these proteins recognize both substrate and H. However, the mechanism of translocation, and particularly that of hMCT1 pH-dependent transport, remains largely unknown. Read More

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February 2020

Structural basis of the pH-dependent conformational change of the N-terminal region of human mannose receptor/CD206.

J Struct Biol 2019 12 3;208(3):107384. Epub 2019 Sep 3.

National Center for Protein Science Shanghai, Shanghai Science Research Center; CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China. Electronic address:

Mannose receptor (MR, CD206) is an immune receptor highly expressed on macrophages and plays important roles in glycoprotein clearance, immune response and matrix turnover. Previous studies have shown that MR recognizes multiple ligands and recycles between cell surface and endosomes, and the conformation and ligand binding of MR are regulated by environmental pH. However, due to the lack of high-resolution details, the mechanisms of the pH-dependent properties of MR have not been fully understood. Read More

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December 2019

Molecular adaptations of NADP-malic enzyme for its function in C photosynthesis in grasses.

Nat Plants 2019 07 24;5(7):755-765. Epub 2019 Jun 24.

Plant Molecular Physiology and Biotechnology Group, Institute of Developmental and Molecular Biology of Plants, Heinrich Heine University Düsseldorf, Düsseldorf, Germany.

In C grasses of agronomical interest, malate shuttled into the bundle sheath cells is decarboxylated mainly by nicotinamide adenine dinucleotide phosphate (NADP)-malic enzyme (C-NADP-ME). The activity of C-NADP-ME was optimized by natural selection to efficiently deliver CO to Rubisco. During its evolution from a plastidic non-photosynthetic NADP-ME, C-NADP-ME acquired increased catalytic efficiency, tetrameric structure and pH-dependent inhibition by its substrate malate. Read More

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Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life.

MAbs 2019 10 18;11(7):1276-1288. Epub 2019 Jul 18.

Biologics Research, Sanofi , Framingham , MA , USA.

The neonatal Fc receptor (FcRn) promotes antibody recycling through rescue from normal lysosomal degradation. The binding interaction is pH-dependent with high affinity at low pH, but not under physiological pH conditions. Here, we combined rational design and saturation mutagenesis to generate novel antibody variants with prolonged half-life and acceptable development profiles. Read More

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October 2019

Functional Analysis of the Chemosensory Protein GmolCSP8 From the Oriental Fruit Moth, (Busck) (Lepidoptera: Tortricidae).

Front Physiol 2019 7;10:552. Epub 2019 May 7.

Key Laboratory of Plant Protection Resources and Pest Management (Northwest A&F University), Ministry of Education, Yangling, China.

Chemosensory proteins (CSPs) belong to a family of small water-soluble proteins that can selectively bind and transport odorant molecules for olfactory communication in insects. To date, their definite physiological functions in olfaction remain controversial when compared with odorant binding proteins (OBPs). To investigate the functions of CSPs in the oriental fruit moth , we determined the tissue expression patterns and binding properties of the CSP, GmolCSP8. Read More

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Structural and functional characterization of an otopetrin family proton channel.

Elife 2019 04 11;8. Epub 2019 Apr 11.

Department of Physiology, University of Texas Southwestern Medical Center, Dallas, United States.

The otopetrin (OTOP) proteins were recently characterized as proton channels. Here we present the cryo-EM structure of OTOP3 from (XtOTOP3) along with functional characterization of the channel. XtOTOP3 forms a homodimer with each subunit containing 12 transmembrane helices that can be divided into two structurally homologous halves; each half assembles as an α-helical barrel that could potentially serve as a proton conduction pore. Read More

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Genetically Encoded Biotin Analogues: Incorporation and Application in Bacterial and Mammalian Cells.

Chembiochem 2019 07 26;20(14):1795-1798. Epub 2019 Jun 26.

Division of Physical Sciences and Engineering, KAUST Catalysis Center (KCC), King Abdullah University of Science and Technology, Thuwal, 23955, Saudi Arabia.

The biotin-streptavidin interaction is among the strongest known in nature. Herein, the site-directed incorporation of biotin and 2-iminobiotin composed of noncanonical amino acids (ncAAs) into proteins is reported. 2-Iminobiotin lysine was employed for protein purification based on the pH-dependent dissociation constant to streptavidin. Read More

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Identification of mNeonGreen as a pH-Dependent, Turn-On Fluorescent Protein Sensor for Chloride.

Chembiochem 2019 07 8;20(14):1759-1765. Epub 2019 May 8.

Department of Chemistry and Biochemistry, The University of Texas at Dallas, 800 West Campbell Road, Richardson, TX, 75080, USA.

Chloride-sensitive fluorescent proteins generated from laboratory evolution have a characteristic tyrosine residue that interacts with a chloride ion and π-stacks with the chromophore. However, the engineered yellow-green fluorescent protein mNeonGreen lacks this interaction but still binds chloride, as seen in a recently reported crystal structure. Based on its unique coordination sphere, we were curious if chloride could influence the optical properties of mNeonGreen. Read More

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Structural elements of a pH-sensitive inhibitor binding site in NMDA receptors.

Nat Commun 2019 01 18;10(1):321. Epub 2019 Jan 18.

WM Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 11724, USA.

Context-dependent inhibition of N-methyl-D-aspartate (NMDA) receptors has important therapeutic implications for the treatment of neurological diseases that are associated with altered neuronal firing and signaling. This is especially true in stroke, where the proton concentration in the afflicted area can increase by an order of magnitude. A class of allosteric inhibitors, the 93-series, shows greater potency against GluN1-GluN2B NMDA receptors in such low pH environments, allowing targeted therapy only within the ischemic region. Read More

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January 2019

Antibody engineering to generate SKY59, a long-acting anti-C5 recycling antibody.

PLoS One 2018 28;13(12):e0209509. Epub 2018 Dec 28.

Research Division, Chugai Pharmaceutical Co., Ltd., Kamakura, Kanagawa, Japan.

Modulating the complement system is a promising strategy in drug discovery for disorders with uncontrolled complement activation. Although some of these disorders can be effectively treated with an antibody that inhibits complement C5, the high plasma concentration of C5 requires a huge dosage and frequent intravenous administration. Moreover, a conventional anti-C5 antibody can cause C5 to accumulate in plasma by reducing C5 clearance when C5 forms an immune complex (IC) with the antibody, which can be salvaged from endosomal vesicles by neonatal Fc receptor (FcRn)-mediated recycling. Read More

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