146,880 results match your criteria acid mutagenesis


Current Advances in Covalent Stabilization of Macromolecular Complexes for Structural Biology.

Bioconjug Chem 2021 Apr 16. Epub 2021 Apr 16.

Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Technical University of Munich, 85747 Garching, Germany.

Structural characterization of macromolecular assemblies is often limited by the transient nature of the interactions. The development of specific chemical tools to covalently tether interacting proteins to each other has played a major role in various fundamental discoveries in recent years. To this end, protein engineering techniques such as mutagenesis, incorporation of unnatural amino acids, and methods using synthetic substrate/cosubstrate derivatives were employed. Read More

View Article and Full-Text PDF

The N-terminal Leu-Pro-Gln sequence of Rab34 is required for ciliogenesis in hTERT-RPE1 cells.

Small GTPases 2021 Apr 16:1-7. Epub 2021 Apr 16.

Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan.

We have previously shown that Rab34 is an important regulator of ciliogenesis and that its unique long N-terminal region (amino acids 1-49) is essential for ciliogenesis in certain cultured mammalian cells. In the present study, we performed an in-depth deletion analysis of the N-terminal region of Rab34 together with Ala-based site-directed mutagenesis to identify the essential amino acids that are required for serum-starvation-induced ciliogenesis in hTERT-RPE1 cells. The results showed that a Rab34 mutant lacking an N-terminal 18 amino acids and a Rab34 mutant carrying an LPQ-to-AAA mutation (amino acids 16-18) failed to rescue a Rab34-KO phenotype (i. Read More

View Article and Full-Text PDF

Alteration of cofactor specificity of the acrylyl-CoA reductase from Escherichia coli.

Biotechnol Lett 2021 Apr 16. Epub 2021 Apr 16.

Laboratory of Methylotrophy, Federal Research Center, "Pushchino Scientific Center for Biological Research, Russian Academy of Sciences" IBPM RAS, Pushchino, Moscow Region, Russia, 142290.

Objectives: Alteration of the cofactor specificity of acrylyl-CoA reductase (AcuI) catalyzing the NAD(P)H-dependent reduction of acrylyl-CoA to propionyl-CoA is often desirable for designing of artificial metabolic pathways of various appointments.

Results: Several variants of AcuIs from Escherichia coli K-12 with multiple amino acid substitutions to alter the cofactor preference were obtained by site directed mutagenesis and the modified enzymes as His-tagged proteins were characterized. The simultaneous substitutions of arginine-180, arginine-198 and serine-178 residues by alanine in the enzyme pocket sequence as well as other amino acid changes decreased both NADPH- and NADH-dependent activities in comparison to the wild-type enzyme. Read More

View Article and Full-Text PDF

The V framework region 1 as a target of efficient mutagenesis for generating a variety of affinity-matured scFv mutants.

Sci Rep 2021 Apr 15;11(1):8201. Epub 2021 Apr 15.

Kobe Pharmaceutical University, 4-19-1, Motoyama-Kitamachi, Higashinada-ku, Kobe, 658-8558, Japan.

In vitro affinity-maturation potentially generates antibody fragments with enhanced antigen-binding affinities that allow for developing more sensitive diagnostic systems and more effective therapeutic agents. Site-directed mutagenesis targeting "hot regions," i.e. Read More

View Article and Full-Text PDF

The antiviral sirtuin 3 bridges protein acetylation to mitochondrial integrity and metabolism during human cytomegalovirus infection.

PLoS Pathog 2021 Apr 15;17(4):e1009506. Epub 2021 Apr 15.

Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Princeton, NJ, United States of America.

Regulation of mitochondrial structure and function is a central component of infection with viruses, including human cytomegalovirus (HCMV), as a virus means to modulate cellular metabolism and immune responses. Here, we link the activity of the mitochondrial deacetylase SIRT3 and global mitochondrial acetylation status to host antiviral responses via regulation of both mitochondrial structural integrity and metabolism during HCMV infection. We establish that SIRT3 deacetylase activity is necessary for suppressing virus production, and that SIRT3 maintains mitochondrial pH and membrane potential during infection. Read More

View Article and Full-Text PDF

Impact of 3-deazapurine nucleobases on RNA properties.

Nucleic Acids Res 2021 Apr 15. Epub 2021 Apr 15.

Institute of Organic Chemistry, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria.

Deazapurine nucleosides such as 3-deazaadenosine (c3A) are crucial for atomic mutagenesis studies of functional RNAs. They were the key for our current mechanistic understanding of ribosomal peptide bond formation and of phosphodiester cleavage in recently discovered small ribozymes, such as twister and pistol RNAs. Here, we present a comprehensive study on the impact of c3A and the thus far underinvestigated 3-deazaguanosine (c3G) on RNA properties. Read More

View Article and Full-Text PDF

Enhancement of Pharmaceutical Urate Oxidase Thermostability by Rational Design of Disulfide Bridge.

Iran J Biotechnol 2020 Jul 1;18(3):e2662. Epub 2020 Jul 1.

Department of Medical Biotechnology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.

Background And Purpose: As a therapeutic enzyme, urate oxidase is utilized in the reduction of uric acid in various conditions such as gout or tumor syndrome lysis. However, even bearing kinetical advantage over other counterparts, it suffers from structural instability most likely due to its subcellular and fungal origin.

Objectives: In this research, by using rational design and introduction of disulfide bridge in urate oxidase structure, we designed and created a thermostable urate oxidase for the first time. Read More

View Article and Full-Text PDF

Cistrome analysis of YY1 uncovers a regulatory axis of YY1:BRD2/4-PFKP during tumorigenesis of advanced prostate cancer.

Nucleic Acids Res 2021 Apr 13. Epub 2021 Apr 13.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA.

Castration-resistant prostate cancer (CRPC) is a terminal disease and the molecular underpinnings of CRPC development need to be better understood in order to improve its treatment. Here, we report that a transcription factor Yin Yang 1 (YY1) is significantly overexpressed during prostate cancer progression. Functional and cistrome studies of YY1 uncover its roles in promoting prostate oncogenesis in vitro and in vivo, as well as sustaining tumor metabolism including the Warburg effect and mitochondria respiration. Read More

View Article and Full-Text PDF

The effect of flanking bases on direct and triplet sensitized cyclobutane pyrimidine dimer formation in DNA depends on the dipyrimidine, wavelength and the photosensitizer.

Nucleic Acids Res 2021 Apr 13. Epub 2021 Apr 13.

Department of Chemistry, Washington University, One Brookings Dr., St. Louis, MO 63130, USA.

Cyclobutane pyrimidine dimers (CPDs) are the major products of DNA produced by direct absorption of UV light, and result in C to T mutations linked to human skin cancers. Most recently a new pathway to CPDs in melanocytes has been discovered that has been proposed to arise from a chemisensitized pathway involving a triplet sensitizer that increases mutagenesis by increasing the percentage of C-containing CPDs. To investigate how triplet sensitization may differ from direct UV irradiation, CPD formation was quantified in a 129-mer DNA designed to contain all 64 possible NYYN sequences. Read More

View Article and Full-Text PDF

An anti-HER2 nanobody binds to its antigen HER2 via two independent paratopes.

Int J Biol Macromol 2021 Apr 14;182:502-511. Epub 2021 Apr 14.

Lab of Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, 5000 Rožna Dolina, Nova Gorica, Slovenia. Electronic address:

High-resolution structural data of complexes between antibodies and membrane receptors still represent a demanding task. In this study, we used complementary sets of experimental data to obtain a structural model of the complex formed by the human epidermal growth factor receptor 2 (HER2) and its specific nanobody A10. First we identified by NMR the residues that bind or rearrange as a consequence of the complex formation. Read More

View Article and Full-Text PDF

Structural comparison of Acinetobacter baumannii β-ketoacyl-acyl carrier protein reductases in fatty acid and aryl polyene biosynthesis.

Sci Rep 2021 Apr 12;11(1):7945. Epub 2021 Apr 12.

Department of Bioscience and Biotechnology, Konkuk University, Seoul, 05029, Republic of Korea.

Some Gram-negative bacteria harbor lipids with aryl polyene (APE) moieties. Biosynthesis gene clusters (BGCs) for APE biosynthesis exhibit striking similarities with fatty acid synthase (FAS) genes. Despite their broad distribution among pathogenic and symbiotic bacteria, the detailed roles of the metabolic products of APE gene clusters are unclear. Read More

View Article and Full-Text PDF

Optimal pH shift of the NADH oxidase from Lactobacillus rhamnosus with a single mutation.

Biotechnol Lett 2021 Apr 12. Epub 2021 Apr 12.

School of Pharmacy, Jiangsu University, Zhenjiang, 212013, People's Republic of China.

Objective: To improve the activity of a water-forming NADH oxidase from Lactobacillus rhamnosus under neutral or alkaline pH for coupling NAD-dependent dehydrogenases with an alkaline optimal pH.

Results: The water-forming NADH oxidase from Lactobacillus rhamnosus was engineered by replacing the aspartic acid or glutamic acid with arginine on the surface. The mutant D251R improved the activity with a 112%, 111%, and 244% relative activity to the wild-type at pH 6. Read More

View Article and Full-Text PDF

Structural basis for substrate specificity of the peroxisomal acyl-CoA hydrolase MpaH' involved in mycophenolic acid biosynthesis.

FEBS J 2021 Apr 12. Epub 2021 Apr 12.

State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong, 266237, China.

Mycophenolic acid (MPA) is a fungal natural product and first-line immunosuppressive drug for organ transplantations and autoimmune diseases. In the compartmentalized biosynthesis of MPA, the acyl-coenzyme A (CoA) hydrolase MpaH' located in peroxisomes catalyzes the highly specific hydrolysis of MPA-CoA to produce the final product MPA. The strict substrate specificity of MpaH' not only averts undesired hydrolysis of various cellular acyl-CoAs, but also prevents MPA-CoA from further peroxisomal β-oxidation catabolism. Read More

View Article and Full-Text PDF

ThermoScan: Semi-automatic Identification of Protein Stability Data From PubMed.

Front Mol Biosci 2021 25;8:620475. Epub 2021 Mar 25.

Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, Bologna, Italy.

During the last years, the increasing number of DNA sequencing and protein mutagenesis studies has generated a large amount of variation data published in the biomedical literature. The collection of such data has been essential for the development and assessment of tools predicting the impact of protein variants at functional and structural levels. Nevertheless, the collection of manually curated data from literature is a highly time consuming and costly process that requires domain experts. Read More

View Article and Full-Text PDF

Identification of Critical Residues in the Carboxy Terminus of the Dopamine Transporter Involved in the G Protein βγ-Induced Dopamine Efflux.

Front Pharmacol 2021 24;12:642881. Epub 2021 Mar 24.

Department of Molecular, Cellular and Biomedical Sciences, CUNY School of Medicine at City College of New York, New York, NY, United States.

The dopamine transporter (DAT) plays a crucial role in the regulation of brain dopamine (DA) homeostasis through the re-uptake of DA back into the presynaptic terminal. In addition to re-uptake, DAT is also able to release DA through a process referred to as DAT-mediated DA efflux. This is the mechanism by which potent and highly addictive psychostimulants, such as amphetamine (AMPH) and its analogues, increase extracellular DA levels in motivational and reward areas of the brain. Read More

View Article and Full-Text PDF

Surfactant cocamide monoethanolamide causes eye irritation by activating nociceptor TRPV1.

Br J Pharmacol 2021 Apr 10. Epub 2021 Apr 10.

State Key Laboratory of Natural Medicines and Department of TCM pharmacology, School of Traditional Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu, China, 211198.

Background And Purpose: Cocamide monoethanolamide (CMEA) is commonly used as a surfactant-foam booster in cosmetic formulations. Upon contact with the eye or other sensitive skin areas, CMEA elicits sting and lasting irritation. We hypothesized a specific molecular interaction with TRPV1 by which CMEA caused eye irritation. Read More

View Article and Full-Text PDF

Comprehensive mutagenesis identifies the peptide repertoire of a p53 T-cell receptor mimic antibody that displays no toxicity in mice transgenic for human HLA-A*0201.

PLoS One 2021 9;16(4):e0249967. Epub 2021 Apr 9.

Nuffield Division of Clinical Laboratory Science, Radcliffe Department of Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom.

T-cell receptor mimic (TCRm) antibodies have expanded the repertoire of antigens targetable by monoclonal antibodies, to include peptides derived from intracellular proteins that are presented by major histocompatibility complex class I (MHC-I) molecules on the cell surface. We have previously used this approach to target p53, which represents a valuable target for cancer immunotherapy because of the high frequency of its deregulation by mutation or other mechanisms. The T1-116C TCRm antibody targets the wild type p5365-73 peptide (RMPEAAPPV) presented by HLA-A*0201 (HLA-A2) and exhibited in vivo efficacy against triple receptor negative breast cancer xenografts. Read More

View Article and Full-Text PDF

[Deletion polymorphism of GSTT1 gene and lipid peroxidation hyperactivation in menopausal insomnia].

Zh Nevrol Psikhiatr Im S S Korsakova 2021 ;121(3):93-97

Scientific Centre for Family Health and Human Reproduction Problems, Irkutsk, Russia.

Objective: A comparative analysis of the parameters of lipid peroxidation - antioxidant defense system in menopausal women with- and without insomnia, depending on the polymorphism.

Material And Methods: The study included 181 menopausal Caucasian women (ethnic group - Russians), who according to the results of the clinical-anamnestic examinations were divided into insomnia group (=120, age 53.68±4. Read More

View Article and Full-Text PDF

Surface charge engineering of Thermomyces lanuginosus lipase improves enzymatic activity and biodiesel synthesis.

Biotechnol Lett 2021 Apr 8. Epub 2021 Apr 8.

School of Life Sciences, Yunnan Normal University, Kunming, China.

Objectives: This study was aimed at engineering charged residues on the surface of Thermomyces lanuginosus lipase (TLL) to obtain TLL variant with elevated performance for industrial applications.

Results: Site-directed mutagenesis of eight charged amino acids on the TLL surface were conducted and substitutions on the negatively charged residues D111, D158, D165, and E239 were identified with elevated specific activities and biodiesel yields. Synergistic effect was not discovered in the double mutants, D111E/D165E and D165E/E239R, when compared with the corresponding single mutants. Read More

View Article and Full-Text PDF

Identification of critical amino acid residues in the regulatory N-terminal domain of PMEL.

Sci Rep 2021 Apr 8;11(1):7730. Epub 2021 Apr 8.

Department of Immunobiology, Yale University School of Medicine, 300 Cedar Street, New Haven, CT, 06519, USA.

The pigment cell-specific protein PMEL forms a functional amyloid matrix in melanosomes onto which the pigment melanin is deposited. The amyloid core consists of a short proteolytic fragment, which we have termed the core-amyloid fragment (CAF) and perhaps additional parts of the protein, such as the PKD domain. A highly O-glycosylated repeat (RPT) domain also derived from PMEL proteolysis associates with the amyloid and is necessary to establish the sheet-like morphology of the assemblies. Read More

View Article and Full-Text PDF

Site-directed mutagenesis of the succinate dehydrogenase subunits B and D from Corynespora cassiicola reveals different fitness costs and sensitivities to SDHI fungicides.

Environ Microbiol 2021 Apr 8. Epub 2021 Apr 8.

Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China.

Carboxamide fungicides target succinate dehydrogenase (SDH). Recently published monitoring studies have shown that Corynespora cassiicola isolates are resistant to one or several SDH inhibitors (SDHIs) with amino acid substitutions in the SDH B and D subunits. We confirmed, by site-directed mutagenesis of the sdhB and sdhD genes, that each of the mutations identified in the field strains of C. Read More

View Article and Full-Text PDF

Identifying metal binding amino acids based on backbone geometries as a tool for metalloprotein engineering.

Protein Sci 2021 Apr 7. Epub 2021 Apr 7.

Messiah University, Department of Chemistry and Biochemistry, One University Ave. Mechanicsburg, PA, USA.

Metal cofactors within proteins perform a versatile set of essential cellular functions. In order to take advantage of the diverse functionality of metalloproteins, researchers have been working to design or modify metal binding sites in proteins to rationally tune the function or activity of the metal cofactor. This study has performed an analysis on the backbone atom geometries of metal-binding amino acids among ten different metal binding sites within the entire protein data bank. Read More

View Article and Full-Text PDF

Cotranscriptional R-loop formation by Mfd involves topological partitioning of DNA.

Proc Natl Acad Sci U S A 2021 Apr;118(15)

Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Ecole Normale Supérieure, PSL University, INSERM, CNRS, Paris 75005, France;

R-loops are nucleic acid hybrids which form when an RNA invades duplex DNA to pair with its template sequence. Although they are implicated in a growing number of gene regulatory processes, their mechanistic origins remain unclear. We here report real-time observations of cotranscriptional R-loop formation at single-molecule resolution and propose a mechanism for their formation. Read More

View Article and Full-Text PDF

TRPV1 Ion Channel: Structural Features, Activity Modulators, and Therapeutic Potential.

Biochemistry (Mosc) 2021 Jan;86(Suppl 1):S50-S70

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.

Although TRPV1 ion channel has been attracting researchers' attention for many years, its functions in animal organisms, the principles of regulation, and the involvement in pathological processes have not yet been fully clarified. Mutagenesis experiments and structural studies have identified the structural features of the channel and binding sites for its numerous ligands; however, these studies are far from conclusion. This review summarizes recent achievements in the TRPV1 research with special focus on structural and functional studies of the channel and on its ligands, which are extremely diverse in their nature and interaction specificity to TRPV1. Read More

View Article and Full-Text PDF
January 2021

A conserved BAH module within mammalian BAHD1 connects H3K27me3 to Polycomb gene silencing.

Nucleic Acids Res 2021 Apr 6. Epub 2021 Apr 6.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA.

Trimethylation of histone H3 lysine 27 (H3K27me3) is important for gene silencing and imprinting, (epi)genome organization and organismal development. In a prevalent model, the functional readout of H3K27me3 in mammalian cells is achieved through the H3K27me3-recognizing chromodomain harbored within the chromobox (CBX) component of canonical Polycomb repressive complex 1 (cPRC1), which induces chromatin compaction and gene repression. Here, we report that binding of H3K27me3 by a Bromo Adjacent Homology (BAH) domain harbored within BAH domain-containing protein 1 (BAHD1) is required for overall BAHD1 targeting to chromatin and for optimal repression of the H3K27me3-demarcated genes in mammalian cells. Read More

View Article and Full-Text PDF

The Pif1 helicase is actively inhibited during meiotic recombination which restrains gene conversion tract length.

Nucleic Acids Res 2021 Apr 6. Epub 2021 Apr 6.

Institut Curie, Université PSL, Sorbonne Université, CNRS UMR3244, Dynamics of Genetic Information, Paris, France.

Meiotic recombination ensures proper chromosome segregation to form viable gametes and results in gene conversions events between homologs. Conversion tracts are shorter in meiosis than in mitotically dividing cells. This results at least in part from the binding of a complex, containing the Mer3 helicase and the MutLβ heterodimer, to meiotic recombination intermediates. Read More

View Article and Full-Text PDF

The crystal structure of yeast regulatory subunit reveals key evolutionary insights into Protein Kinase A oligomerization.

J Struct Biol 2021 Apr 2:107732. Epub 2021 Apr 2.

Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, C1428EHA, Argentina; Instituto de Química Biológica, Facultad de Ciencias Exactas y Naturales (IQUIBICEN-CONICET), Buenos Aires, C1428EHA, Argentina. Electronic address:

Protein kinase A (PKA) is a widespread enzyme that plays a key role in many signaling pathways from lower eukaryotes to metazoans. In mammals, the regulatory (R) subunits sequester and target the catalytic (C) subunits to proper subcellular locations. This targeting is accomplished by the dimerization and docking (D/D) domain of the R subunits. Read More

View Article and Full-Text PDF

Epithelia-sensory neuron crosstalk underlies cholestatic itch induced by lysophosphatidylcholine.

Gastroenterology 2021 Apr 2. Epub 2021 Apr 2.

Department of Neurology, Duke University, Durham, NC 27710, USA; Department of Anesthesiology, Duke University, Durham, NC 27710, USA; Department of Neurobiology, Duke University, Durham, NC 27710, USA; Neurology Clinics for Headache, Head-Pain and Trigeminal Sensory Disorders, Duke University, Durham NC 27705, USA; Clinics for Innovative Pain Therapy, Department of Anesthesiology, Duke University, Raleigh NC 27512, USA. Electronic address:

Background & Aims: Limited understanding of pruritus mechanisms in cholestatic liver diseases hinders development of anti-pruritic treatments. Previous studies implicated lysophosphatidic acid (LPA) as a potential mediator of cholestatic pruritus.

Methods: Pruritogenicity of LPC, LPA's precursor, was examined in naïve mice, cholestatic mice, and nonhuman primates. Read More

View Article and Full-Text PDF

Two Key Amino Acids Variant of α-l-arabinofuranosidase from Str. 168 with Altered Activity for Selective Conversion Ginsenoside Rc to Rd.

Molecules 2021 Mar 19;26(6). Epub 2021 Mar 19.

Molecular Biology Research Center, School of Life Sciences, Central South University, Changsha 410078, China.

α-l-arabinofuranosidase is a subfamily of glycosidases involved in the hydrolysis of l-arabinofuranosidic bonds, especially in those of the terminal non-reducing arabinofuranosyl residues of glycosides, from which efficient glycoside hydrolases can be screened for the transformation of ginsenosides. In this study, the ginsenoside Rc-hydrolyzing α-l-arabinofuranosidase gene, was cloned from and its codons were optimized for efficient expression in BL21 (DE3). The recombinant protein BsAbfA fused with an N-terminal His-tag was overexpressed and purified, and then subjected to enzymatic characterization. Read More

View Article and Full-Text PDF

Conformational Heterogeneity and Cooperative Effects of Mammalian ALOX15.

Int J Mol Sci 2021 Mar 23;22(6). Epub 2021 Mar 23.

Institute of Biochemistry, Charite-University Medicine Berlin, Corporate member of Free University Berlin, Humboldt University Berlin and Berlin Institute of Health, Charitéplatz 1, D-10117 Berlin, Germany.

Arachidonic acid lipoxygenases (ALOXs) have been suggested to function as monomeric enzymes, but more recent data on rabbit ALOX15 indicated that there is a dynamic monomer-dimer equilibrium in aqueous solution. In the presence of an active site ligand (the ALOX15 inhibitor RS7) rabbit ALOX15 was crystalized as heterodimer and the X-ray coordinates of the two monomers within the dimer exhibit subtle structural differences. Using native polyacrylamide electrophoresis, we here observed that highly purified and predominantly monomeric rabbit ALOX15 and human ALOX15B are present in two conformers with distinct electrophoretic mobilities. Read More

View Article and Full-Text PDF