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Sample records for addition site-directed mutagenesis

  1. Site-directed mutagenesis of tobacco anionic peroxidase: Effect of additional aromatic amino acids on stability and activity.

    PubMed

    Poloznikov, A A; Zakharova, G S; Chubar, T A; Hushpulian, D M; Tishkov, V I; Gazaryan, I G

    2015-08-01

    Tobacco anionic peroxidase (TOP) is known to effectively catalyze luminol oxidation without enhancers, in contrast to horseradish peroxidase (HRP). To pursue structure-activity relationship studies for TOP, two amino acids have been chosen for mutation, namely Thr151, close to the heme plane, and Phe140 at the entrance to the active site pocket. Three mutant forms TOP F140Y, T151W and F140Y/T151W have been expressed in Escherichia coli, and reactivated to yield active enzymes. Single-point mutations introducing additional aromatic amino acid residues at the surface of TOP exhibit a significant effect on the enzyme catalytic activity and stability as judged by the results of steady-state and transient kinetics studies. TOP T151W is up to 4-fold more active towards a number of aromatic substrates including luminol, whereas TOP F140Y is 2-fold more stable against thermal inactivation and 8-fold more stable in the reaction course. These steady-state observations have been rationalized with the help of transient kinetic studies on the enzyme reaction with hydrogen peroxide in a single turnover regime. The stopped-flow data reveal (a) an increased stability of F140Y Compound I towards hydrogen peroxide, and thus, a higher operational stability as compared to the wild-type enzyme, and (b) a lesser leakage of oxidative equivalents from TOP T151W Compound I resulting in the increased catalytic activity. The results obtained show that TOP unique properties can be further improved for practical applications by site-directed mutagenesis. PMID:25957835

  2. Homemade Site Directed Mutagenesis of Whole Plasmids

    PubMed Central

    Laible, Mark; Boonrod, Kajohn

    2009-01-01

    Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers. PMID:19488024

  3. Protein engineering: single or multiple site-directed mutagenesis.

    PubMed

    Hsieh, Pei-Chung; Vaisvila, Romualdas

    2013-01-01

    Site-directed mutagenesis techniques are invaluable tools in molecular biology to study the structural and functional properties of a protein. To expedite the time required and simplify methods for mutagenesis, we recommend two protocols in this chapter. The first method for single site-directed mutagenesis, which includes point mutations, insertions, or deletions, can be achieved by an inverse PCR strategy with mutagenic primers and the high-fidelity Phusion(®) DNA Polymerase to introduce a site-directed mutation with exceptional efficiency. The second method is for engineering multiple mutations into a gene of interest. This can be completed in one step by PCR with mutagenic primers and by assembling all mutagenized PCR products using the Gibson Assembly™ Master Mix. This method allows multiple nucleotides to be changed simultaneously, which not only saves time but also reagents compared to traditional methods of mutagenesis. PMID:23423897

  4. REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering

    PubMed Central

    Trehan, Ashutosh; Kiełbus, Michał; Czapinski, Jakub; Stepulak, Andrzej; Huhtaniemi, Ilpo; Rivero-Müller, Adolfo

    2016-01-01

    Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient. PMID:26750263

  5. REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering.

    PubMed

    Trehan, Ashutosh; Kiełbus, Michał; Czapinski, Jakub; Stepulak, Andrzej; Huhtaniemi, Ilpo; Rivero-Müller, Adolfo

    2016-01-01

    Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient. PMID:26750263

  6. Efficient site-directed saturation mutagenesis using degenerate oligonucleotides.

    PubMed

    Steffens, David L; Williams, John G K

    2007-07-01

    We describe a reliable protocol for constructing single-site saturation mutagenesis libraries consisting of all 20 naturally occurring amino acids at a specific site within a protein. Such libraries are useful for structure-function studies and directed evolution. This protocol extends the utility of Stratagene's QuikChange Site-Directed Mutagenesis Kit, which is primarily recommended for single amino acid substitutions. Two complementary primers are synthesized, containing a degenerate mixture of the four bases at the three positions of the selected codon. These primers are added to starting plasmid template and thermal cycled to produce mutant DNA molecules, which are subsequently transformed into competent bacteria. The protocol does not require purification of mutagenic oligonucleotides or PCR products. This reduces both the cost and turnaround time in high-throughput directed evolution applications. We have utilized this protocol to generate over 200 site-saturation libraries in a DNA polymerase, with a success rate of greater than 95%. PMID:17595310

  7. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

    Crawford, Lisa; Karr, Laurel J.; Nadarajah, Arunan; Pusey, Marc

    1999-01-01

    A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 43 axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to (3)500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 (Registered) PHE or ALA and ASN 113 (Registered) ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 43 helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

  8. Tetragonal Lysozyme Interactions Studied by Site Directed Mutagenesis

    NASA Technical Reports Server (NTRS)

    Crawford, Lisa; Karr, Laurel; Pusey, Marc

    1998-01-01

    A number of recent experimental and theoretical studies have indicated that tetragonal lysozyme crystal growth proceeds by the addition of aggregates, formed by reversible self association of the solute molecules in the bulk'solution. Periodic bond chain and atomic force microscopy studies have indicated that the probable growth unit is at minimum a 43 tetramer, and most likely an octamer composed of two complete turns about the 4(sub 3) axis. If these results are correct, then there are intermolecular interactions which are only formed in the solution and others only formed at the joining of the growth unit to the crystal surface. We have set out to study these interactions, and the correctness of this hypothesis, using site directed mutagenesis of specific amino acid residues involved in the different bonds. We had initially expressed wild type lysozyme in S. cervasiae with yields of approximately 5 mg/L, which were eventually raised to approximately 40 mg/L. We are now moving the expression to the Pichia system, with anticipated yields of 300 to greater than 500 mg/L, comparable to what can be obtained from egg whites. An additional advantage of using recombinant protein is the greater genetic homogeneity of the material obtained and the absence of any other contaminating egg proteins. The first mutation experiments are TYR 23 yields PHE or ALA and ASN 113 yields ALA or ASP. Both TYR 23 and ASN 113 form part of the postulated dimerization intermolecular binding site which lead to the formation of the 4(sub 3) helix. Tyrosine also participates in an intermolecular hydrogen bond with ARG 114. The results of these and subsequent experiments will be discussed.

  9. [Rapid site-directed mutagenesis on full-length plasmid DNA by using designed restriction enzyme assisted mutagenesis].

    PubMed

    Zhang, Baozhong; Ran, Duoliang; Zhang, Xin; An, Xiaoping; Shan, Yunzhu; Zhou, Yusen; Tong, Yigang

    2009-02-01

    To use the designed restriction enzyme assisted mutagenesis technique to perform rapid site-directed mutagenesis on double-stranded plasmid DNA. The target amino acid sequence was reversely translated into DNA sequences with degenerate codons, resulting in large amount of silently mutated sequences containing various restriction endonucleases (REs). Certain mutated sequence with an appropriate RE was selected as the target DNA sequence for designing mutation primers. The full-length plasmid DNA was amplified with high-fidelity Phusion DNA polymerase and the amplified product was 5' phosphorylated by T4 polynucleotide kinase and then self-ligated. After transformation into an E. coli host the transformants were rapidly screened by cutting with the designed RE. With this strategy we successfully performed the site-directed mutagenesis on an 8 kb plasmid pcDNA3.1-pIgR and recovered the wild-type amino acid sequence of human polymeric immunoglobulin receptor (pIgR). A novel site-directed mutagenesis strategy based on DREAM was developed which exploited RE as a rapid screening measure. The highly efficient, high-fidelity Phusion DNA polymerase was applied to ensure the efficient and faithful amplification of the full-length sequence of a plasmid of up to 8 kb. This rapid mutagenesis strategy avoids using any commercial site-directed mutagenesis kits, special host strains or isotopes. PMID:19459340

  10. Site-directed mutagenesis and gene deletion using reverse genetics.

    PubMed

    Muhl, Daniela; Filloux, Alain

    2014-01-01

    Understanding gene function is far easier when tools are available to engineer a bacterial strain lacking a specific gene and phenotypically compare its behavior with the corresponding parental strain. Such mutants could be selected randomly, either by natural selection under particular stress conditions or by random mutagenesis using transposon delivery as described elsewhere in this book. However, with the advent of the genomic era there are now hundreds of bacterial genomes whose sequence is available, and thus, genes can be identified, chosen, and strategies designed to specifically inactivate them. This can be done by using suicide plasmids and is most convenient when the bacterium of interest is easily amenable to genetic manipulation. The method presented here will describe the use of a suicide vector, pKNG101, which allows the selection of a double-recombination event. The first event results in the integration of the pKNG101 derivative carrying the "mutator" fragment onto the chromosome, and could be selected on plates containing appropriate antibiotics. The pKNG101 carries the sacB gene, which induces death when cells are grown on sucrose. Growth on sucrose plates will thus select the second homologous recombination event, which results in removing the plasmid backbone and leaving behind the mutated target gene. This method has been widely used over the last 20 years to inactivate genes in a wide range of gram-negative bacteria and in particular in Pseudomonas aeruginosa. PMID:24818930

  11. Software-supported USER cloning strategies for site-directed mutagenesis and DNA assembly.

    PubMed

    Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen; Jespersen, Jakob Berg; Sommer, Morten O A; Wernersson, Rasmus; Olsen, Lars Rønn

    2015-03-20

    USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at http://www.cbs.dtu.dk/services/AMUSER/. PMID:24847672

  12. Improving the neutral phytase activity from Bacillus amyloliquefaciens DSM 1061 by site-directed mutagenesis.

    PubMed

    Xu, Wei; Shao, Rong; Wang, Zupeng; Yan, Xiuhua

    2015-03-01

    Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed. PMID:25613522

  13. Site-directed mutagenesis of a conserved Asn450 residue of Bacillus licheniformis γ-glutamyltranspeptidase.

    PubMed

    Lin, Min-Guan; Chi, Meng-Chun; Chen, Yu-Yi; Wang, Tzu-Fan; Lo, Hui-Fen; Lin, Long-Liu

    2016-10-01

    Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) belongs to N-terminal nucleophile hydrolase superfamily in which all inclusive members are synthetized as single-chain precursors, and then self-processed to form mature enzymes. Here we investigated the role of a conserved Asn450 residue in BlGGT through site-directed mutagenesis and molecular characterization of four relevant variants. Substitution of Asn450 by arginine resulted in a significant reduction in the catalytic activity of BlGGT. Conversely, N450A and N450D displayed an enhanced activity. The catalytic efficiency of BlGGT was calculated to be 16.04mM(-1)s(-1), but this value was either decreased to 8.93mM(-1)s(-1) in N450K or increased to more than 123.65mM(-1)s(-1) in N450A and N450D. In addition, the ratio of transpeptidation to hydrolysis was increased from 3.5 to more than 7.6 by the mutations. Structural analyses showed that fluorescence, circular dichroism spectra and thermal denaturation profiles of mutant proteins were essentially consistent with those of BlGGT. However, guanidine hydrochloride (GdnHCl)-induced transition was significantly reduced in comparison with the wild-type enzyme. Molecular modeling suggests that residue Asn450 of BlGGT is important to create suitable environments for both autoprocessing and catalytic reactions. PMID:27246377

  14. Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    PubMed

    Weng, Meizhi; Deng, Xiongwei; Bao, Wei; Zhu, Li; Wu, Jieyuan; Cai, Yongjun; Jia, Yan; Zheng, Zhongliang; Zou, Guolin

    2015-09-25

    Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent. PMID:26291268

  15. Crystal structure and site-directed mutagenesis of a nitroalkane oxidase from Streptomyces ansochromogenes.

    PubMed

    Li, Yanhua; Gao, Zengqiang; Hou, Haifeng; Li, Lei; Zhang, Jihui; Yang, Haihua; Dong, Yuhui; Tan, Huarong

    2011-02-18

    Nitroalkane oxidase (NAO) catalyzes neutral nitroalkanes to their corresponding aldehydes or ketones, hydrogen peroxide and nitrite. The crystal structure of NAO from Streptomyces ansochromogenes was determined; it consists of two domains, a TIM barrel domain bound to FMN and C-terminal domain with a novel folding pattern. Site-directed mutagenesis of His179, which is spatially adjacent to FMN, resulted in the loss of enzyme activity, demonstrating that this amino acid residue is important for catalysis. The crystal structure of mutant H179D-nitroethane was also analyzed. Interestingly, Sa-NAO shows the typical function as nitroalkane oxidase but its structure is similar to that of 2-nitropropane dioxygenase. Overall, these results suggest that Sa-NAO is a novel nitroalkane oxidase with TIM barrel structure. PMID:21147069

  16. Site-directed mutagenesis studies of acetylglutamate synthase delineate the site for the arginine inhibitor.

    PubMed

    Sancho-Vaello, Enea; Fernández-Murga, M Leonor; Rubio, Vicente

    2008-04-01

    N-acetyl-L-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain. However, the acetylglutamate kinase-like domain may modulate glutamate binding, since one mutation affecting this domain increases the K(m) for glutamate. The effects on PaNAGS of two mutations found in human NAGS deficiency support the similarity of bacterial and human NAGSs despite their low sequence identity. PMID:18319063

  17. Isolation of temperature-sensitive Abelson virus mutants by site-directed mutagenesis.

    PubMed Central

    Engelman, A; Rosenberg, N

    1987-01-01

    Mutants of Abelson virus encoding temperature-sensitive protein-tyrosine kinase (EC 2.7.1.112) were created by site-directed mutagenesis using sequence information from temperature-sensitive mutants of the related v-src oncogene. Expression of these two independent mutations in Escherichia coli resulted in reduced phosphorylation of the mutant proteins at high temperature. Viruses containing one of the mutations induced conditional transformation of both NIH 3T3 and lymphoid cells when expressed in the context of a truncated transforming protein. These results underscore the functional homology between protein-tyrosine kinases and suggest that transfer of mutations within a related gene family may provide a rapid method to create mutants. Images PMID:2825174

  18. Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System.

    PubMed

    Olshefsky, Audrey; Shehata, Laila; Kuldell, Natalie

    2016-01-01

    Two-component signaling (2CS) systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli's natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or "coliroids," rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by β-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein's local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system's performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling. PMID:26799494

  19. Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System

    PubMed Central

    Kuldell, Natalie

    2016-01-01

    Two-component signaling (2CS) systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli’s natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or “coliroids,” rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by β-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein’s local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system’s performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling. PMID:26799494

  20. Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis.

    PubMed

    Xie, Guangrong; Yang, Weizhen; Chen, Jing; Li, Miaomiao; Jiang, Nan; Zhao, Baixue; Chen, Si; Wang, Min; Chen, Jianhua

    2016-01-01

    The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more "human-like" uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine-human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU) gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1-2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7-8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H1-2P₃H₄P5-6H7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H1-2P₃H₄P5-6H7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H1-2P₃H₄P5-6H7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine-baboon chimera (PBC) was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5-11.0 and temperature range of 20-40 °C. PMID:27213357

  1. Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis

    PubMed Central

    Xie, Guangrong; Yang, Weizhen; Chen, Jing; Li, Miaomiao; Jiang, Nan; Zhao, Baixue; Chen, Si; Wang, Min; Chen, Jianhua

    2016-01-01

    The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more “human-like” uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine–human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU) gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1–2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7–8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H1-2P3H4P5-6H7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H1-2P3H4P5-6H7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H1-2P3H4P5-6H7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine–baboon chimera (PBC) was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5–11.0 and temperature range of 20–40 °C. PMID:27213357

  2. Improving the thermostability and catalytic efficiency of Bacillus deramificans pullulanase by site-directed mutagenesis.

    PubMed

    Duan, Xuguo; Chen, Jian; Wu, Jing

    2013-07-01

    Pullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Its instability and low catalytic efficiency are the major factors preventing its widespread application. To address these issues, Asp437 and Asp503 of the pullulanase from Bacillus deramificans were selected in this study as targets for site-directed mutagenesis based on a structure-guided consensus approach. Four mutants (carrying the mutations D503F, D437H, D503Y, and D437H/D503Y) were generated and characterized in detail. The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme. In addition, the D437H/D503Y double mutant displayed a larger shift in thermostability, with an optimal temperature of 60°C and a half-life at 60°C of more than 4.3-fold that of the wild-type enzyme. Kinetic studies showed that the Km values for the D503F, D437H, D503Y, and D437H/D503Y mutants decreased by 7.1%, 11.4%, 41.4%, and 45.7% and the Kcat/Km values increased by 10%, 20%, 140%, and 100%, respectively, compared to those of the wild-type enzyme. Mechanisms that could account for these enhancements were explored. Moreover, in conjunction with the enzyme glucoamylase, the D503Y and D437H/D503Y mutants exhibited an improved reaction rate and glucose yield during starch hydrolysis compared to those of the wild-type enzyme, confirming the enhanced properties of the mutants. The mutants generated in this study have potential applications in the starch industry. PMID:23624477

  3. Improving the Thermostability and Catalytic Efficiency of Bacillus deramificans Pullulanase by Site-Directed Mutagenesis

    PubMed Central

    Duan, Xuguo; Chen, Jian

    2013-01-01

    Pullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Its instability and low catalytic efficiency are the major factors preventing its widespread application. To address these issues, Asp437 and Asp503 of the pullulanase from Bacillus deramificans were selected in this study as targets for site-directed mutagenesis based on a structure-guided consensus approach. Four mutants (carrying the mutations D503F, D437H, D503Y, and D437H/D503Y) were generated and characterized in detail. The results showed that the D503F, D437H, and D503Y mutants had an optimum temperature of 55°C and a pH optimum of 4.5, similar to that of the wild-type enzyme. However, the half-lives of the mutants at 60°C were twice as long as that of the wild-type enzyme. In addition, the D437H/D503Y double mutant displayed a larger shift in thermostability, with an optimal temperature of 60°C and a half-life at 60°C of more than 4.3-fold that of the wild-type enzyme. Kinetic studies showed that the Km values for the D503F, D437H, D503Y, and D437H/D503Y mutants decreased by 7.1%, 11.4%, 41.4%, and 45.7% and the Kcat/Km values increased by 10%, 20%, 140%, and 100%, respectively, compared to those of the wild-type enzyme. Mechanisms that could account for these enhancements were explored. Moreover, in conjunction with the enzyme glucoamylase, the D503Y and D437H/D503Y mutants exhibited an improved reaction rate and glucose yield during starch hydrolysis compared to those of the wild-type enzyme, confirming the enhanced properties of the mutants. The mutants generated in this study have potential applications in the starch industry. PMID:23624477

  4. Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthesized oxygen-evolving system

    SciTech Connect

    Debus, R.J.; Barry, B.A.; Babcock, G.T.; McIntosh, L.

    1988-01-01

    Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P/sub 680//sup +/, while D /center dot//sup +/ gives rise to the dark-stable EPR spectrum known as signal II. D/center dot//sup +/ has recently been shown to be a tyrosine radical. Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and E2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, the authors have changed Tyr-160 of the D2 polypeptide to phenylalanine by site-directed mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D/center dot//sup +/. The authors conclude that D is Tyr-160 of the D2 polypeptide. They suggest that the C/sub 2/ symmetry in photosystem II extends beyond P/sub 680/ to its immediate electron donor and conclude that Z is Try-161 of the D1 polypeptide.

  5. [Enhancing glutamate decarboxylase activity by site-directed mutagenesis: an insight from Ramachandran plot].

    PubMed

    Ke, Piyu; Huang, Jun; Hu, Sheng; Zhao, Weirui; Lü, Changjiang; Yu, Kai; Lei, Yinlin; Wang, Jinbo; Mei, Lehe

    2016-01-01

    Glutamate decarboxylase (GAD) can catalyze the decarboxylation of glutamate into γ-aminobutyrate (GABA) and is the only enzyme of GABA biosynthesis. Improving GAD activity and thermostability will be helpful for the highly efficient biosynthesis of GABA. According to the Ramachandran plot information of GAD 1407 three-dimensional structure from Lactobacillus brevis CGMCC No. 1306, we identified the unstable site K413 as the mutation target, constructed the mutant GAD by site-directed mutagenesis and measured the thermostability and activity of the wide type and mutant GAD. Mutant K413A led to a remarkably slower inactivation rate, and its half-life at 50 °C reached 105 min which was 2.1-fold higher than the wild type GAD1407. Moreover, mutant K413I exhibited 1.6-fold higher activity in comparison with the wide type GAD1407, although it had little improvement in thermostability of GAD. Ramachandran plot can be considered as a potential approach to increase GAD thermostability and activity. PMID:27443004

  6. Metal binding 'finger' structures in the glucocorticoid receptor defined by site-directed mutagenesis.

    PubMed Central

    Severne, Y; Wieland, S; Schaffner, W; Rusconi, S

    1988-01-01

    The glucocorticoid receptor and the other members of the steroid receptor super-family share a highly conserved, cysteine-rich region which coincides with the DNA binding/transactivating domain. It has been postulated that this region is folded into two 'zinc finger' structures, similar to those originally reported for the transcription factor TFIIIA. The first potential finger domain contains four conserved cysteines and one conserved histidine, while the second contains five conserved cysteines. Using site-directed mutagenesis, we have analysed the consequences of altering the proposed finger-like structures. Our results show that most of the mutations affecting the conserved cysteines result in a total loss of glucocorticoid receptor function. In one important exception, however, a conserved cysteine (Cys500) is dispensable for glucocorticoid receptor activity and therefore cannot be involved in complexing a metal ion to form a finger structure. Moreover, the replacement of either Cys476 or Cys482 by His residues maintains partial in vivo activity of the glucocorticoid receptor, while their exchange for an alanine or serine residue, respectively, eliminates receptor function. These results support, at a genetic level, the involvement of cysteines of the glucocorticoid receptor DNA binding domain in metal ion complexation and define the candidate residues involved in such coordination. Images PMID:3191912

  7. Site-directed mutagenesis of the base recognition loop of ribonuclease from Bacillus intermedius (binase).

    PubMed

    Okorokov, A L; Panov, K I; Kolbanovskaya EYu; Karpeisky MYa; Polyakov, K M; Wilkinson, A J; Dodson, G G

    1996-04-15

    Members of the microbial guanyl-specific ribonuclease family show a high level of structural homology. The structural basis for guanyl base binding by microbial ribonucleases has been established for all members of the family and the existence of a guanine recognition loop was shown. However, bacillar RNases such as binase and barnase show far less specificity towards the guanyl base in hydrolysing oligonucleotides composed of more than 4 or 5 nucleotides. Using site-directed mutagenesis we introduced a number of amino acid substitutions into the base recognition loop of binase. The donor sequence originated from the guanyl specific ribonuclease Sa. Two single, two double and one triple (entire loop substitution) mutants were constructed and overproduced in E. coli. The kinetic properties of the mutant variants are different from the wild-type protein. Amino acid substitutions R61V, G60S, S56Q/R61V, G60S/R61V show 3-fold, 7-fold, 4-fold and 12-fold increased guanyl specificity respectively. However, all mutants retain the ability to catalyse the hydrolysis of a poly(A) substrate. PMID:8612811

  8. Coupled Site-Directed Mutagenesis/Transgenesis Identifies Important Functional Domains of the Mouse Agouti Protein

    PubMed Central

    Perry, W. L.; Nakamura, T.; Swing, D. A.; Secrest, L.; Eagleson, B.; Hustad, C. M.; Copeland, N. G.; Jenkins, N. A.

    1996-01-01

    The agouti locus encodes a novel paracrine signaling molecule containing a signal sequence, an N-linked glycosylation site, a central lysine-rich basic domain, and a C-terminal tail containing 10 cysteine (Cys) residues capable of forming five disulfide bonds. When overexpressed, agouti causes a number of pleiotropic effects including yellow coat and adult-onset obesity. Numerous studies suggest that agouti causes yellow coat color by antagonizing the binding of α-melanocyte-stimulating hormone (α-MSH) to the α-MSH-(melanocortin-1) receptor. With the goal of identifying functional domains of agouti important for its diverse biological activities, we have generated 14 agouti mutations by in vitro site-directed mutagenesis and analyzed these mutations in transgenic mice for their effects on coat color and obesity. These studies demonstrate that the signal sequence, the N-linked glycosylation site, and the C-terminal Cys residues are important for full biological activity, while at least a portion of the lysine-rich basic domain is dispensable for normal function. They also show that the same functional domains of agouti important in coat color determination are important for inducing obesity, consistent with the hypothesis that agouti induces obesity by antagonizing melanocortin binding to other melanocortin receptors. PMID:8878691

  9. Site-directed mutagenesis and metal ion studies with E. coli L-threonine dehydrogenase (TDH)

    SciTech Connect

    Yenwen Chen; Epperly, B.R.; Dekker, E.E. )

    1991-03-11

    TDH, which catalyzes L-threonine + NAD{sup +}{yields}2-amino-3-ketobutyrate + NADH + H{sup +}, initiates the primary route for threonine utilization in eukaryotes and prokaryotes. E. coli TDH is a homotetramer of mass {congruent} 50 kD. Chemical inactivation/peptide sequencing studies showed that Cys-38 and 1 Arg residue/subunit are required for catalytic activity. The primary structure of E. coli TDH has been deduced by DNA sequencing of the tdh gene; homology comparisons with other dehydrogenases place it with the zinc-containing long-chain alcohol/polyol dehydrogenase family. Neutron activation and atomic absorption analyses of pure E. coli TDH as isolated now show it contains 1 Zn{sup 2+}/enzyme subunit. Timed removal of Zn{sup 2+} with 1,10-phenanthroline gives a good correlation between remaining activity and zinc content; inactive enzyme has no Zn{sup 2+}. The native metal ion can be exchanged with either {sup 65}Zn{sup 2+}, Co{sup 2+}, or Cd{sup 2+} with no change in sp. act. Exchange studies with {sup 65}Zn{sup 2+} frequently yields 1.9 to 1.0 Zn{sup 2+}/TDH subunit with no increase in sp. act. above 1 Zn{sup 2+}/subunit, suggesting a possible second Zn{sup 2+}-binding site. Cys-38 was projected to be one possible zinc ligand. Cys-38 was changed to Ser by site-directed mutagenesis. The pure mutant protein as isolated contains 1 Zn{sup 2+}/subunit but shows no TDH activity; very low activity is seen in the presence of high (Zn{sup 2+}).

  10. Site-directed mutagenesis of Arg60 and Cys271 in ornithine transcarbamylase from rat liver.

    PubMed

    McDowall, S; van Heeswijck, R; Hoogenraad, N

    1990-10-01

    We have investigated the putative carbamylphosphate- and ornithine-binding domains in ornithine transcarbamylase from rat liver using site-directed mutagenesis. Arg60, present in the phosphate-binding motif X-Ser-X-Arg-X and therefore implicated in the binding of the phosphate moiety of carbamylphosphate has been replaced with a leucine. This results in a dramatic reduction of catalytic activity, although the enzyme is synthesized in cells stably transfected with the mutant clone and imported, correctly processed and assembled into a homotrimer in mitochondria. The sole cysteine residue (Cys271) has been implicated in ornithine binding by the chemical modification studies of Marshall and Cohen in 1972 and 1980 (J. Biol. Chem., 247, 1654-1668, 1669-1682; 255, 7291-7295, 7296-7300). Replacement of this residue with serine did not eliminate enzyme activity but affected the Michaelis constant for ornithine (Kb), increasing it 5-fold from 0.71 to 3.7 mM and reduced the kcat at pH 8.5 by 20-fold. These changes represent a loss in apparent binding energy for the enzyme--ornithine complex of 2.9 kcal/mol, suggesting that Cys271 is normally involved in hydrogen bonding to the substrate, ornithine. The cysteine to serine substitution also caused the dissociation constant (Kii) for the competitive inhibitor, L-norvaline to be increased 10-fold, from 12 to 120 microM. The small loss in binding energy and relatively high residual catalytic activity of the mutant strongly suggests that a number of other residues are involved in the binding of ornithine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2290837

  11. One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections

    PubMed Central

    Mingo, Janire; Erramuzpe, Asier; Luna, Sandra; Aurtenetxe, Olaia; Amo, Laura; Diez, Ibai; Schepens, Jan T. G.; Hendriks, Wiljan J. A. J.; Cortés, Jesús M.; Pulido, Rafael

    2016-01-01

    Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis. PMID:27548698

  12. One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections.

    PubMed

    Mingo, Janire; Erramuzpe, Asier; Luna, Sandra; Aurtenetxe, Olaia; Amo, Laura; Diez, Ibai; Schepens, Jan T G; Hendriks, Wiljan J A J; Cortés, Jesús M; Pulido, Rafael

    2016-01-01

    Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis. PMID:27548698

  13. New system based on site-directed mutagenesis for highly accurate comparison of resistance levels conferred by SHV beta-lactamases.

    PubMed Central

    Nüesch-Inderbinen, M T; Hächler, H; Kayser, F H

    1995-01-01

    We developed a system based on site-directed mutagenesis that allows a precise comparison of SHV enzymes under isogenic conditions. In addition, the influences of two different, naturally occurring promoters were examined for each SHV derivative. The system comprised two separately cloned DNA fragments, each the size of 3.6 kb. Both fragments encoded an SHV gene originating from clinical isolates but with different promoters. The structural genes were made identical by site-directed mutagenesis. Other mutations were then introduced into both fragments by means of site-directed mutagenesis, resulting in the SHV derivatives SHV-1, SHV-2, SHV-2a, SHV-3, and SHV-5. The amino acid exchange of glutamic acid at position 235 for lysine in SHV-5 resulted in the highest resistance levels. SHV-3, differing from SHV-2 by the exchange of arginine at position 201 for leucine and previously described as indistinguishable from SHV-2, was shown to cause slightly higher resistance to ceftazidime and lower resistance to ceftriaxone, cefotaxime, and cefepime than SHV-2. The point mutation in SHV-2a, with the leucine-to-glutamine replacement at the unusual position 31, previously considered almost insignificant, proved to increase resistance to ceftazidime but reduced the MICs of all other cephalosporins tested when compared with those for SHV-2. For all clones harboring SHV derivatives, resistance was increased by a stronger promoter, in some cases masking the effect of the point mutation itself and demonstrating the importance of regulatory mechanisms of resistance. PMID:7486909

  14. New insights into the QuikChange™ process guide the use of Phusion DNA polymerase for site-directed mutagenesis.

    PubMed

    Xia, Yongzhen; Chu, Wenqiao; Qi, Qingsheng; Xun, Luying

    2015-01-01

    The QuikChange™ site-directed mutagenesis method is popular but imperfect. An improvement by using partially overlapping primers has been reported several times; however, it is incompatible with the proposed mechanism. The QuikChange™ method using complementary primers is proposed to linearly amplify a target plasmid with the products annealing to produce double-stranded DNA molecules with 5'-overhangs. The overhang annealing is supposed to form circular plasmids with staggered breaks, which can be repaired in Escherichia coli after transformation. Here, we demonstrated that the PCR enzyme fills the 5'-overhangs in the early cycles, and the product is then used as the template for exponential amplification. The linear DNA molecules with homologous ends are joined to generate the plasmid with the desired mutations through homologous recombination in E. coli. The correct understanding is important to method improvements, guiding us to use partially overlapping primers and Phusion DNA polymerase for site-directed mutagenesis. Phusion did not amplify a plasmid with complementary primers but used partially overlapping primers to amplify the plasmid, producing linear DNA molecules with homologous ends for site-directed mutagenesis. PMID:25399421

  15. SDM-Assist software to design site-directed mutagenesis primers introducing “silent” restriction sites

    PubMed Central

    2013-01-01

    Background Over the past decades site-directed mutagenesis (SDM) has become an indispensable tool for biological structure-function studies. In principle, SDM uses modified primer pairs in a PCR reaction to introduce a mutation in a cDNA insert. DpnI digestion of the reaction mixture is used to eliminate template copies before amplification in E. coli; however, this process is inefficient resulting in un-mutated clones which can only be distinguished from mutant clones by sequencing. Results We have developed a program – ‘SDM-Assist’ which creates SDM primers adding a specific identifier: through additional silent mutations a restriction site is included or a previous one removed which allows for highly efficient identification of ‘mutated clones’ by a simple restriction digest. Conclusions The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the primers based on factors such as Tm, GC content and secondary structure allowing for simplified selection of optimal primer pairs. PMID:23522286

  16. Tailor-Made Protein Tyrosine Phosphatases: In Vitro Site-Directed Mutagenesis of PTEN and PTPRZ-B.

    PubMed

    Luna, Sandra; Mingo, Janire; Aurtenetxe, Olaia; Blanco, Lorena; Amo, Laura; Schepens, Jan; Hendriks, Wiljan J; Pulido, Rafael

    2016-01-01

    In vitro site-directed mutagenesis (SDM) of protein tyrosine phosphatases (PTPs) is a commonly used approach to experimentally analyze PTP functions at the molecular and cellular level and to establish functional correlations with PTP alterations found in human disease. Here, using the tumor-suppressor PTEN and the receptor-type PTPRZ-B (short isoform from PTPRZ1 gene) phosphatases as examples, we provide a brief insight into the utility of specific mutations in the experimental analysis of PTP functions. We describe a standardized, rapid, and simple method of mutagenesis to perform single and multiple amino acid substitutions, as well as deletions of short nucleotide sequences, based on one-step inverse PCR and DpnI restriction enzyme treatment. This method of SDM is generally applicable to any other protein of interest. PMID:27514801

  17. QM/MM model of the mouse olfactory receptor MOR244-3 validated by site-directed mutagenesis experiments.

    PubMed

    Sekharan, Sivakumar; Ertem, Mehmed Z; Zhuang, Hanyi; Block, Eric; Matsunami, Hiroaki; Zhang, Ruina; Wei, Jennifer N; Pan, Yi; Batista, Victor S

    2014-09-01

    Understanding structure/function relationships of olfactory receptors is challenging due to the lack of x-ray structural models. Here, we introduce a QM/MM model of the mouse olfactory receptor MOR244-3, responsive to organosulfur odorants such as (methylthio)methanethiol. The binding site consists of a copper ion bound to the heteroatoms of amino-acid residues H105, C109, and N202. The model is consistent with site-directed mutagenesis experiments and biochemical measurements of the receptor activation, and thus provides a valuable framework for further studies of the sense of smell at the molecular level. PMID:25185561

  18. QM/MM Model of the Mouse Olfactory Receptor MOR244-3 Validated by Site-Directed Mutagenesis Experiments

    PubMed Central

    Sekharan, Sivakumar; Ertem, Mehmed Z.; Zhuang, Hanyi; Block, Eric; Matsunami, Hiroaki; Zhang, Ruina; Wei, Jennifer N.; Pan, Yi; Batista, Victor S.

    2014-01-01

    Understanding structure/function relationships of olfactory receptors is challenging due to the lack of x-ray structural models. Here, we introduce a QM/MM model of the mouse olfactory receptor MOR244-3, responsive to organosulfur odorants such as (methylthio)methanethiol. The binding site consists of a copper ion bound to the heteroatoms of amino-acid residues H105, C109, and N202. The model is consistent with site-directed mutagenesis experiments and biochemical measurements of the receptor activation, and thus provides a valuable framework for further studies of the sense of smell at the molecular level. PMID:25185561

  19. Characterization of cyclo-Acetoacetyl-L-Tryptophan Dimethylallyltransferase in Cyclopiazonic Acid Biosynthesis: Substrate Promiscuity and Site Directed Mutagenesis Studies

    PubMed Central

    Liu, Xinyu; Walsh, Christopher T.

    2009-01-01

    The fungal neurotoxin α-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca2+-ATPase with a unique pentacyclic indole tetramic acid scaffold is assembled by a three enzyme pathway CpaS, CpaD and CpaO in Aspergillus sp. We recently characterized the first pathway-specific enzyme CpaS, a hybrid two module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) that generates cyclo-acetoacetyl-L-tryptophan (cAATrp). Here we report the characterization of the second pathway-specific enzyme CpaD that regiospecifically dimethylallylates cAATrp to form β-cyclopiazonic acid. By exploring the tryptophan and tetramate moieties of cAATrp, we demonstrate that CpaD discriminates against free Trp but accepts tryptophan-containing thiohydantoins, diketopiperazines and linear peptides as substrates for C4-prenylation and also acts as regiospecific O-dimethylallyltransferase (DMAT) on a tyrosine-derived tetramic acid. Comparative evaluation of CpaDs from A. oryzae RIB40 and A. flavus NRRL3357 indicated the importance of the N-terminal region for its activity. Sequence alignment of CpaD with eleven homologous fungal Trp-DMATs revealed five regions of conservation suggesting the presense of critical motifs that could be diagonostic for discovering additional Trp-DMATs. Subsequent site-directed mutagenesis studies identified five polar/charged residues and five tyrosine residues within these motifs that are critical for CpaD activity. This motif characerization will enable a gene probe-based approach to discover additional biosynthetic Trp-DMATs. PMID:19877600

  20. Elucidation of strain-specific interaction of a GII-4 norovirus with HBGA receptors by site-directed mutagenesis study

    SciTech Connect

    Tan Ming |; Xia Ming; Cao Sheng; Huang Pengwei; Farkas, Tibor |; Meller, Jarek |; Hegde, Rashmi S. |; Li Xuemei; Rao Zihe; Jiang Xi |

    2008-09-30

    Noroviruses interact with histo-blood group antigen (HBGA) receptors in a strain-specific manner probably detecting subtle structural differences in the carbohydrate receptors. The specific recognition of types A and B antigens by various norovirus strains is a typical example. The only difference between the types A and B antigens is the acetamide linked to the terminal galactose of the A but not to the B antigen. The crystal structure of the P dimer of a GII-4 norovirus (VA387) bound to types A and B trisaccharides has elucidated the A/B binding site on the capsid but did not explain the binding specificity of the two antigens. In this study, using site-directed mutagenesis, we have identified three residues on the VA387 capsid that are sterically close to the acetamide and are required for binding to A but not B antigens, indicating that the acetamide determines the binding specificity between the A and B antigens. Further mutational analysis showed that a nearby open cavity may also be involved in binding specificity to HBGAs. In addition, a systematic mutational analysis of residues in and around the binding interface has identified a group of amino acids that are required for binding but do not have direct contact with the carbohydrate antigens, implying that these residues may be involved in the structural integrity of the receptor binding interface. Taken together, our study provides new insights into the carbohydrate/capsid interactions which are a valuable complement to the atomic structures in understanding the virus/host interaction and in the future design of antiviral agents.

  1. The Roles of Cytochrome b559 in Assembly and Photoprotection of Photosystem II Revealed by Site-Directed Mutagenesis Studies

    PubMed Central

    Chu, Hsiu-An; Chiu, Yi-Fang

    2016-01-01

    Cytochrome b559 (Cyt b559) is one of the essential components of the Photosystem II reaction center (PSII). Despite recent accomplishments in understanding the structure and function of PSII, the exact physiological function of Cyt b559 remains unclear. Cyt b559 is not involved in the primary electron transfer pathway in PSII but may participate in secondary electron transfer pathways that protect PSII against photoinhibition. Site-directed mutagenesis studies combined with spectroscopic and functional analysis have been used to characterize Cyt b559 mutant strains and their mutant PSII complex in higher plants, green algae, and cyanobacteria. These integrated studies have provided important in vivo evidence for possible physiological roles of Cyt b559 in the assembly and stability of PSII, protecting PSII against photoinhibition, and modulating photosynthetic light harvesting. This mini-review presents an overview of recent important progress in site-directed mutagenesis studies of Cyt b559 and implications for revealing the physiological functions of Cyt b559 in PSII. PMID:26793230

  2. Specificity of site directed psoralen addition to RNA.

    PubMed Central

    Teare, J; Wollenzien, P

    1989-01-01

    We describe the attachment of a psoralen derivative (site specific psoralen, SSP) to the 5' end of a DNA oligonucleotide and the hybridization and the photoreaction of this reagent with a complementary target site on an RNA molecule. SSP was coupled to a variety of DNA oligonucleotides to investigate the structural requirements for addition to the RNA. Efficient SSP photoadducts were made on specific uridines by designing an intercalation site at an unpaired nucleotide in the RNA strand within the heteroduplex region. The optimal location for this site was five nucleotides from the oligonucleotide 5' end and just 5' to the target uridine residue. Because the attachment of the SSP to the oligonucleotide is through a disulfide bond, the DNA oligonucleotide can be removed with reduction to leave SSP attached to the RNA strand. The SSP adduct made in this way will be useful for subsequent biochemical and biophysical experiments. Images PMID:2471154

  3. The catalytic triad of the influenza C virus glycoprotein HEF esterase: characterization by site-directed mutagenesis and functional analysis.

    PubMed

    Pleschka, S; Klenk, H D; Herrler, G

    1995-10-01

    Influenza C virus is able to inactivate its own cellular receptors by virtue of a sialate 9-O-acetylesterase that releases the acetyl residue at position C-9 of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). The receptor-destroying enzyme activity is a function of the surface glycoprotein HEF and this esterase belongs to the class of serine hydrolases. In their active site, these enzymes contain a catalytic triad made up of a serine, a histidine and an aspartic acid residue. Sequence comparison with other serine esterases has indicated that, in addition to serine-71 (S71), the amino acids histidine-368 or -369 (H368/369) and aspartic acid 261 (D261) are the most likely candidates to form the catalytic triad of the influenza C virus glycoprotein. By site-directed mutagenesis, mutants were generated in which alanine substituted for either of these amino acids. Using a phagemid expression vector, pSP1D-HEF the HEF gene was expressed in both COS 7 and MDCK I cells. The glycoprotein was obtained in a functional form only in the latter cells, as indicated by its transport to the cell surface and measurable enzyme activity. The low level of expression could be increased by stimulating the NF-KB-binding activity of the cytomegalovirus immediate-early promoter/enhancer element of the vector. The esterase activity of the mutant proteins was compared with that of the wild-type glycoprotein. With Neu5,9Ac2 as the substrate, the esterase specific activities of the S71/A mutant and the H368,369/A mutant were reduced by more than 90%. In the case of the D261/A mutant the specific activity was reduced by 64%. From this data we conclude that S71, H368/369 and D261 are likely to represent the catalytic triad of the influenza C virus glycoprotein HEF. In addition, N280 is proposed to stabilize the oxyanion of the presumptive transition state intermediate formed by the enzyme-substrate complex. PMID:7595356

  4. Development and characterization of cyclodextrin glucanotransferase as a maltoheptaose-producing enzyme using site-directed mutagenesis.

    PubMed

    Koo, Ye-Seul; Lee, Hye-Won; Jeon, Hye-Yeon; Choi, Hye-Jeong; Choung, Woo-Jae; Shim, Jae-Hoon

    2015-11-01

    Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) mainly produces cyclodextrins (CDs) using linear maltooligosaccharides. We performed site-directed saturation mutagenesis on the +1 substrate-binding residue, H233, of CGTase from alkalophilic Bacillus sp. I-5 to prepare specific-length oligosaccharides. The obtained mutant CGTase, H233Y, primarily produced maltoheptaose (G7) using β-CD via a hydrolysis reaction. A kinetic study of H233Y showed that the kcat/Km value of β-CD was 7-fold greater than that of G7, which accounts for the accumulation of G7 during the H233Y enzyme reaction. A structure comparison of CGTases with H233Y modeling suggests that the substitution of H233Y may alter the position of the +1 subsite and slow the further hydrolysis of G7 after the ring-opening reaction. PMID:26359254

  5. Enhancement of oxidative stability of the subtilisin nattokinase by site-directed mutagenesis expressed in Escherichia coli.

    PubMed

    Weng, MeiZhi; Zheng, ZhongLiang; Bao, Wei; Cai, YongJun; Yin, Yan; Zou, GuoLin; Zou, GouLin

    2009-11-01

    Nattokinase (subtilisin NAT, NK) is a bacterial serine protease with strong fibrinolytic activity and it is a potent cardiovascular drug. In medical and commercial applications, however, it is susceptible to chemical oxidation, and subsequent inactivation or denaturation. Here we show that the oxidative stability of NK was substantially increased by optimizing the amino acid residues Thr(220) and Met(222), which were in the vicinity of the catalytic residue Ser(221) of the enzyme. Two nonoxidative amino acids (Ser and Ala) were introduced at these sites using site-directed mutagenesis. Active enzymes were successfully expressed in Escherichia coli with periplasmic secretion and enzymes were purified to homogeneity. The purified enzymes were analyzed with respect to oxidative stability, kinetic parameters, fibrinolytic activity and thermal stability. M222A mutant was found to have a greatly increased oxidative stability compared with wild-type enzyme and it was resistant to inactivation by more than 1 M H(2)O(2), whereas the wild-type enzyme was inactivated by 0.1 M H(2)O(2) (t(1/2) approximately 11.6 min). The other mutant (T220S) also showed an obvious increase in antioxidative ability. Molecular dynamic simulations on wild-type and T220S mutant proteins suggested that a hydrogen bond was formed between Ser(220) and Asn(155), and the spatial structure of Met(222) was changed compared with the wild-type. The present study demonstrates the feasibility of improving oxidative stability of NK by site-directed mutagenesis and shows successful protein engineering cases to improve stability of NK as a potent therapeutic agent. PMID:19631297

  6. Novel structure--function information on biogenic amine transporters revealed by site-directed mutagenesis and alkylation.

    PubMed

    Reith, Maarten E A

    2013-07-01

    The study reported by Wenge and Bönisch in this issue provides critical structural information regarding extracellular loop 2 (EL2) of the human norepinephrine transporter (NET). A systematic search among all 10 cysteine and 13 histidine residues in NET led to His222 in EL2 as the target for N-ethylmaleimide: its alkylation interferes with [(3)H]nisoxetine binding, indicating the part of EL2 containing His 222 reaches back into the protein interior where it prevents access by nisoxetine to its binding site. Thus, EL2 in human NET does much more than conformationally assisting substrate translocation. The present study underscores the importance of site-directed mutagenesis approaches to elucidate structural features that cannot be deduced from crystals of homolog proteins. In the case of NET, the closest crystal structure is that of the homolog LeuT, but EL2 is difficult to align with 22 less loop residues in LeuT than in NET. The present results could only be achieved by the systematic mutagenesis study of all cysteines and all histidines in NET. PMID:23532308

  7. Site-directed Mutagenesis Switching a Dimethylallyl Tryptophan Synthase to a Specific Tyrosine C3-Prenylating Enzyme*

    PubMed Central

    Fan, Aili; Zocher, Georg; Stec, Edyta; Stehle, Thilo; Li, Shu-Ming

    2015-01-01

    The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C4- and C7-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C3-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis. PMID:25477507

  8. Virus-based Photo-Responsive Nanowires Formed By Linking Site-Directed Mutagenesis and Chemical Reaction

    PubMed Central

    Murugesan, Murali; Abbineni, Gopal; Nimmo, Susan L.; Cao, Binrui; Mao, Chuanbin

    2013-01-01

    Owing to the genetic flexibility and error-free bulk production, bio-nanostructures such as filamentous phage showed great potential in materials synthesis, however, their photo-responsive behaviour is neither explored nor unveiled. Here we show M13 phage genetically engineered with tyrosine residues precisely fused to the major coat protein is converted into a photo-responsive organic nanowire by a site-specific chemical reaction with an aromatic amine to form an azo dye structure on the surface. The resulting azo-M13-phage nanowire exhibits reversible photo-responsive properties due to the photo-switchable cis-trans isomerisation of the azo unit formed on the phage. This result shows that site-specific display of a peptide on bio-nanostructures through site-directed genetic mutagenesis can be translated into site-directed chemical reaction for developing advanced materials. The photo-responsive properties of the azo-M13-phage nanowires may open the door for the development of light controllable smart devices for use in non-linear optics, holography data storage, molecular antenna, and actuators. PMID:23673356

  9. Virus-based photo-responsive nanowires formed by linking site-directed mutagenesis and chemical reaction.

    PubMed

    Murugesan, Murali; Abbineni, Gopal; Nimmo, Susan L; Cao, Binrui; Mao, Chuanbin

    2013-01-01

    Owing to the genetic flexibility and error-free bulk production, bio-nanostructures such as filamentous phage showed great potential in materials synthesis, however, their photo-responsive behaviour is neither explored nor unveiled. Here we show M13 phage genetically engineered with tyrosine residues precisely fused to the major coat protein is converted into a photo-responsive organic nanowire by a site-specific chemical reaction with an aromatic amine to form an azo dye structure on the surface. The resulting azo-M13-phage nanowire exhibits reversible photo-responsive properties due to the photo-switchable cis-trans isomerisation of the azo unit formed on the phage. This result shows that site-specific display of a peptide on bio-nanostructures through site-directed genetic mutagenesis can be translated into site-directed chemical reaction for developing advanced materials. The photo-responsive properties of the azo-M13-phage nanowires may open the door for the development of light controllable smart devices for use in non-linear optics, holography data storage, molecular antenna, and actuators. PMID:23673356

  10. Virus-based Photo-Responsive Nanowires Formed By Linking Site-Directed Mutagenesis and Chemical Reaction

    NASA Astrophysics Data System (ADS)

    Murugesan, Murali; Abbineni, Gopal; Nimmo, Susan L.; Cao, Binrui; Mao, Chuanbin

    2013-05-01

    Owing to the genetic flexibility and error-free bulk production, bio-nanostructures such as filamentous phage showed great potential in materials synthesis, however, their photo-responsive behaviour is neither explored nor unveiled. Here we show M13 phage genetically engineered with tyrosine residues precisely fused to the major coat protein is converted into a photo-responsive organic nanowire by a site-specific chemical reaction with an aromatic amine to form an azo dye structure on the surface. The resulting azo-M13-phage nanowire exhibits reversible photo-responsive properties due to the photo-switchable cis-trans isomerisation of the azo unit formed on the phage. This result shows that site-specific display of a peptide on bio-nanostructures through site-directed genetic mutagenesis can be translated into site-directed chemical reaction for developing advanced materials. The photo-responsive properties of the azo-M13-phage nanowires may open the door for the development of light controllable smart devices for use in non-linear optics, holography data storage, molecular antenna, and actuators.

  11. Site-directed mutagenesis and molecular modelling studies show the role of Asp82 and cysteines in rat acylase 1, a member of the M20 family

    SciTech Connect

    Herga, Sameh; Brutus, Alexandre; Vitale, Rosa Maria; Miche, Helene; Perrier, Josette; Puigserver, Antoine; Scaloni, Andrea; Giardina, Thierry . E-mail: thierry.giardina@univ.u-3mrs.fr

    2005-05-06

    Acylase 1 from rat kidney catalyzes the hydrolysis of acyl-amino acids. Sequence alignment has shown that this enzyme belongs to the metalloprotein family M20. Site-directed mutagenesis experiments led to the identification of one functionally important amino acid residue located near one of the zinc coordinating residues, which play a critical role in the enzymatic activity. The D82N- and D82E-substituted forms showed no significant activity and very low activity, respectively, along with a loss of zinc coordination. Molecular modelling investigations indicated a putative role of D82 in ensuring a proper protonation of catalytic histidine. In addition, none of the five cysteine residues present in the rat kidney acylase 1 sequence seemed involved in the catalytic process: the loss of activity induced by the C294A substitution was probably due to a conformational change in the 3D structure.

  12. Site-Directed Mutagenesis of a Hyperthermophilic Endoglucanase Cel12B from Thermotoga maritima Based on Rational Design

    PubMed Central

    Zhang, Jinfeng; Shi, Hao; Xu, Linyu; Zhu, Xiaoyan; Li, Xiangqian

    2015-01-01

    To meet the demand for the application of high activity and thermostable cellulases in the production of new-generation bioethanol from nongrain-cellulose sources, a hyperthermostable β-1,4-endoglucase Cel12B from Thermotoga maritima was selected for further modification by gene site-directed mutagenesis method in the present study, based on homology modeling and rational design. As a result, two recombinant enzymes showed significant improvement in enzyme activity by 77% and 87%, respectively, higher than the parental enzyme TmCel12B. Furthermore, the two mutants could retain 80% and 90.5% of their initial activity after incubation at 80°C for 8 h, while only 45% for 5 h to TmCel12B. The Km and Vmax of the two recombinant enzymes were 1.97±0.05 mM, 4.23±0.15 μmol·mg-1·min-1 of TmCel12B-E225H-K207G-D37V, and 2.97±0.12 mM, 3.15±0.21 μmol·mg-1·min-1 of TmCel12B-E225H-K207G, respectively, when using CMC-Na as the substrate. The roles of the mutation sites were also analyzed and evaluated in terms of electron density, hydrophobicity of the modeled protein structures. The recombinant enzymes may be used in the hydrolysis of cellulose at higher temperature in the future. It was concluded that the gene mutagenesis approach of a certain active residues may effectively improve the performance of cellulases for the industrial applications and contribute to the study the thermostable mechanism of thermophilic enzymes. PMID:26218520

  13. Molecular analysis of sialoside binding to sialoadhesin by NMR and site-directed mutagenesis.

    PubMed Central

    Crocker, P R; Vinson, M; Kelm, S; Drickamer, K

    1999-01-01

    The molecular interactions between sialoadhesin and sialylated ligands have been investigated by using proton NMR. Addition of ligands to the 12 kDa N-terminal immunoglobulin-like domain of sialoadhesin result in resonance shifts in the protein spectrum that have been used to determine the affinities of sialoadhesin for several sialosides. The results indicate that alpha2, 3-sialyl-lactose and alpha2,6-sialyl-lactose bind respectively 2- and 1.5-fold more strongly than does alpha-methyl-N-acetylneuraminic acid (alpha-Me-NeuAc). The resonances corresponding to the methyl protons within the N-acetyl moiety of sialic acid undergo upfield shifting and broadening during titrations, reflecting an interaction of this group with Trp2 in sialoadhesin as observed in co-crystals of the terminal domain with bound ligand. This resonance shift was used to measure the affinities of mutant and wild-type forms of sialoadhesin in which the first three domains are fused to the Fc region of human IgG1. Substitution of Arg97 by alanine completely abrogated measurable interaction with alpha-Me-NeuAc, whereas a conservative substitution with lysine resulted in a 10-fold decrease in affinity. These results provide the first direct measurement of the affinity of sialoadhesin for sialosides and confirm the critical importance of the conserved arginine in interactions between sialosides and members of the siglec family of sialic acid-binding, immunoglobulin-like lectins. PMID:10393093

  14. Enhancement of thermostability and kinetic efficiency of Aspergillus niger PhyA phytase by site-directed mutagenesis.

    PubMed

    Hesampour, Ardeshir; Siadat, Seyed Ehsan Ranaei; Malboobi, Mohammad Ali; Mohandesi, Nooshin; Arab, Seyed Shahriar; Ghahremanpour, Mohammad Mehdi

    2015-03-01

    Phytase efficiently catalyzes the hydrolysis of phytate to phosphate; it can be utilized as an animal supplement to provide animals their nutrient requirements for phosphate and to mitigate environmental pollution caused by unutilized feed phosphate. Owing to animal feed being commonly pelleted at 70 to 90 °C, phytase with a sufficiently high thermal stability is desirable. Based on the crystal structure of PhyA and bioinformatics analysis at variant heat treatments, 12 single and multiple mutants were introduced by site-directed mutagenesis in order to improve phytase thermostability. Mutated constructs were expressed in Pichia pastoris. The manipulated phytases were purified; their biochemical and kinetic investigation revealed that while the thermostability of six mutants was improved, P9 (T314S Q315R V62N) and P12 (S205N S206A T151A T314S Q315R) showed the highest heat stability (P < 0.05) with 24 and 22.6 % greater retention, respectively, compared with the PhyA of the wild type at 80 °C. The K m value of the improved thermostable P9 and P12 mutant enzymes for sodium phytate were 35 and 20 % lower (P < 0.05) with respect to the wild-type enzyme. In conclusion, it is feasible to simultaneously improve the thermostability and the catalytic efficiency of phytase to be used as an animal feed supplement. PMID:25527139

  15. Investigation of the active site and the conformational stability of nucleoside diphosphate kinase by site-directed mutagenesis.

    PubMed

    Tepper, A D; Dammann, H; Bominaar, A A; Véron, M

    1994-12-23

    Nucleoside-diphosphate kinase (EC 2.7.4.6) catalyzes phosphate exchange between nucleoside triphosphates and nucleoside diphosphates. Its 17 kDa subunits are highly conserved throughout evolution in both sequence and tertiary structure. Using site-directed mutagenesis we investigated the function of 8 amino acids (Lys16, Tyr56, Arg92, Thr98, Arg109, Asn119, Ser124, and Glu133) that are totally conserved among all nucleoside diphosphate kinases known to date. The mutant proteins all show decreased specific activity and support roles for these residues in catalysis, substrate binding, or both, as was previously proposed on the basis of the x-ray structure (Moréra, S., Lascu, I., Dumas, C., LeBras, G., Briozzo, P., Véron, M., and Janin, J. (1994) Biochemistry 33, 459-467). Furthermore, residues Lys16, Arg109, and Asn 119 were identified to play important roles in conformational stability or subunit interactions. We show that Lys16 and Asn119 form a rigid structure that is important for enzymatic function and that Arg109, known to interact with the phosphate moiety of the substrate, also plays an important role in subunit association. The dual roles of Lys16, Arg109, and Asn119 in both substrate binding and subunit assembly provide further evidence for a functional coupling between catalytic activity and quaternary structure in nucleoside diphosphate kinase. PMID:7798215

  16. Heparin binding to platelet factor-4. An NMR and site-directed mutagenesis study: arginine residues are crucial for binding.

    PubMed Central

    Mayo, K H; Ilyina, E; Roongta, V; Dundas, M; Joseph, J; Lai, C K; Maione, T; Daly, T J

    1995-01-01

    Native platelet factor-4 (PF4) is an asymmetrically associated, homo-tetrameric protein (70 residues/subunit) known for binding polysulphated glycosaminoglycans like heparin. PF4 N-terminal chimeric mutant M2 (PF4-M2), on the other hand, forms symmetric tetramers [Mayo, Roongta, Ilyina, Milius, Barker, Quinlan, La Rosa and Daly (1995) Biochemistry 34, 11399-11409] making NMR studies with this 32 kDa protein tractable. PF4-M2, moreover, binds heparin with a similar affinity to that of native PF4. NMR data presented here indicate that heparin (9000 Da cut-off) binding to PF4-M2, while not perturbing the overall structure of the protein, does perturb specific side-chain proton resonances which map to spatially related residues within a ring of positively charged side chains on the surface of tetrameric PF4-M2. Contrary to PF4-heparin binding models which centre around C-terminal alpha-helix lysines, this study indicates that a loop containing Arg-20, Arg-22, His-23 and Thr-25, as well as Lys-46 and Arg-49, are even more affected by heparin binding. Site-directed mutagenesis and heparin binding data support these NMR findings by indicating that arginines more than C-terminal lysines, are crucial to the heparin binding process. Images Figure 4 PMID:8526843

  17. Functional evaluation of residues in the herbicide-binding site of Mycobacterium tuberculosis acetohydroxyacid synthase by site-directed mutagenesis.

    PubMed

    Jung, In-Pil; Cho, Jun-Haeng; Koo, Bon-Sung; Yoon, Moon-Young

    2015-10-01

    Mycobacterium tuberculosis acetohydroxyacid synthase (M. tuberculosis AHAS) has been proposed to bean essential target for novel herbicide- and chemical-based antibacterial agents. Therefore, here we investigated the roles of multiple conserved herbicide-binding site residues (R318, A146, Q148, M512, and V513) in M. tuberculosis AHAS through site-directed mutagenesis by characterizing the kinetic parameters and herbicide sensitivities of various point mutants. Interestingly, all mutant enzymes showed significantly altered kinetic parameters, specifically reduced affinity towards both the substrate and cofactor. Importantly, mutation of R318 led to a complete loss of AHAS activity, indicating a key role for this residue in substrate binding. Furthermore, all mutants demonstrated significant herbicide resistance against chlorimuron ethyl (CE), with several-fold higher IC50 than that of wild type AHAS. Docking analysis also indicated that binding of CE was slightly affected upon mutation of these residues. Taken together, these data suggest that the residues examined here mediate CE binding and may also be important for the catalytic activity of AHAS. This study will pave the way for future structure-function studies of CE and will also aid the development of novel anti-tuberculosis agents based on this chemical scaffold. PMID:26215340

  18. Modifying the photoelectric behavior of bacteriorhodopsin by site-directed mutagenesis: electrochemical and genetic engineering approaches to molecular devices

    NASA Astrophysics Data System (ADS)

    Hong, F. T.; Hong, F. H.; Needleman, R. B.; Ni, B.; Chang, M.

    1992-07-01

    Bacteriorhodopsins (bR's) modified by substitution of the chromophore with synthetic vitamin A analogues or by spontaneous mutation have been reported as successful examples of using biomaterials to construct molecular optoelectronic devices. The operation of these devices depends on desirable optical properties derived from molecular engineering. This report examines the effect of site-directed mutagenesis on the photoelectric behavior of bR thin films with an emphasis on their application to the construction of molecular devices based on their unique photoelectric behavior. We examine the photoelectric signals induced by a microsecond light pulse in thin films which contain reconstituted oriented purple membrane sheets isolated from several mutant strains of Halobacterium halobium. A recently developed expression system is used to synthesize mutant bR's in their natural host, H. halobium. We then use a unique analytical method (tunable voltage clamp method) to investigate the effect of pH on the relaxation of two components of the photoelectric signals, B1 and B2. We found that for the four mutant bR's examined, the pH dependence of the B2 component varies significantly. Our results suggest that genetic engineering approaches can produce mutant bR's with altered photoelectric characteristics that can be exploited in the construction of devices.

  19. Crystal structure and site-directed mutagenesis analyses of haloalkane dehalogenase LinB from Sphingobium sp. strain MI1205.

    PubMed

    Okai, Masahiko; Ohtsuka, Jun; Imai, Lica Fabiana; Mase, Tomoko; Moriuchi, Ryota; Tsuda, Masataka; Nagata, Koji; Nagata, Yuji; Tanokura, Masaru

    2013-06-01

    The enzymes LinB(UT) and LinB(MI) (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of β-hexachlorocyclohexane (β-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinB(MI) and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinB(MI) were categorized into three groups based on the efficiency of the first-step (from β-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinB(MI) and its seven point mutants indicated how each mutated residue contributed to the first- and second-step conversions by LinB(MI). The dynamics simulation analyses of wild-type LinB(MI) and LinB(UT) revealed that the entrance of the substrate access tunnel of LinB(UT) was more flexible than that of LinB(MI), which could lead to the different efficiencies of dehalogenation activity between these dehalogenases. PMID:23564170

  20. Crystal Structure and Site-Directed Mutagenesis Analyses of Haloalkane Dehalogenase LinB from Sphingobium sp. Strain MI1205

    PubMed Central

    Okai, Masahiko; Ohtsuka, Jun; Imai, Lica Fabiana; Mase, Tomoko; Moriuchi, Ryota; Tsuda, Masataka; Nagata, Koji; Nagata, Yuji

    2013-01-01

    The enzymes LinBUT and LinBMI (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of β-hexachlorocyclohexane (β-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinBMI and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinBMI were categorized into three groups based on the efficiency of the first-step (from β-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinBMI and its seven point mutants indicated how each mutated residue contributed to the first- and second-step conversions by LinBMI. The dynamics simulation analyses of wild-type LinBMI and LinBUT revealed that the entrance of the substrate access tunnel of LinBUT was more flexible than that of LinBMI, which could lead to the different efficiencies of dehalogenation activity between these dehalogenases. PMID:23564170

  1. Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis.

    PubMed

    Pizza, M; Domenighini, M; Hol, W; Giannelli, V; Fontana, M R; Giuliani, M M; Magagnoli, C; Peppoloni, S; Manetti, R; Rappuoli, R

    1994-10-01

    Computer analysis of the crystallographic structure of the A subunit of Escherichia coli heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53-->Glu or Asp, Ser-63-->Lys, Val-97-->Lys, Tyr-104-->Lys or Asp, and Ser-114-->Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41-->Phe, Ala-45-->Tyr or Glu, Val-53-->Tyr, Val-60-->Gly, Ser-68-->Pro, His-70-->Pro, Val-97-->Tyr and Ser-114-->Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54-->Lys or Ala, Tyr-59-->Met, Ser-68-->Lys, Ala-72-->Arg, His or Asp and Arg-192-->Asn. The results provide a further understanding of the structure-function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases. PMID:7830560

  2. High-resolution mapping of the HyHEL-10 epitope of chicken lysozyme by site-directed mutagenesis

    SciTech Connect

    Kam-Morgan, L.N.; Taylor, M.G.; Kirsch, J.F. ); Smith-Gill, S.J. ); Wilson, A.C.

    1993-05-01

    The complex formed between hen egg white lysozyme (HEL) and the monoclonal antibody HyHEL-10 Fab fragment has an interface composed of van der Waals interactions, hydrogen bonds, and a single ion pair. The antibody overlaps part of the active site cleft. Putative critical residues within the epitope region of HEL, identified from the x-ray crystallographic structure of the complex, were replaced by site-directed mutagenesis to probe their relative importance in determining affinity of the antibody for HEL. Twenty single mutations of HEL at three contact residues (Arg-21[sub HEL], Asp-101[sub HEL], and Gly-102[sub HEL]) and at a partially buried residue (Asn-19[sub HEL]) in the epitope were made, and the effects on the free energies of dissociation were measured. A correlation between increased amino acid side-chain volume and reduced affinity for HELs with mutations at position 101 was observed. The D101G[sub HEL] mutant is bound to HyHEL-10 as tightly as wild-type enzyme, but the [delta][delta]G[sub dissoc] is increased by about 2.2 kcal (9.2 kJ)/mol for the larger residues in this position. HEL variants with lysine or histidine replacements for arginine at position 21 are bound 1.4-2.7 times more tightly than those with neutral or negatively charged amino acids in this position. These exhibit 1/40 the affinity for HyHEL-10 Fab compared with wild type. There is no side-chain volume correlation with [delta][delta]G[sub dissoc] at position 21. Although Gly-102[sub HEL] and Asn-19[sub HEL] are in the epitope, replacements at these positions have no effect on the affinity of HEL for the antibody. 34 refs., 2 figs., 3 tabs.

  3. Alteration of the substrate specificity of cytochrome P450 CYP199A2 by site-directed mutagenesis.

    PubMed

    Furuya, Toshiki; Shitashima, Yoh; Kino, Kuniki

    2015-01-01

    CYP199A2, a member of the cytochrome P450 family, is a monooxygenase that specializes in the oxidation of aromatic carboxylic acids. The crystal structure of CYP199A2 determined by Bell et al. (J. Mol. Biol., 383, 561-574, 2008) suggested that the S97 and S247 residues conferred the substrate specificity on this enzyme through interaction between the hydroxy side chains of these Ser residues and the carboxy group of the substrates. In this study, we attempted to design and construct CYP199A2 mutants that recognize hydroxy aromatic compounds as substrates by protein engineering. We speculated that substitution of the S97 and S247 residues with acidic amino acids Asp and Glu, which have carboxy side chains, would provide CYP199A2 mutants that recognize hydroxy aromatic compounds instead of aromatic carboxylic acids. The S97 and S247 residues were substituted with Asp and Glu using site-directed mutagenesis. In whole-cell assays with p-methylbenzylalcohol and phenol as hydroxy aromatic substrates, the S247D mutant regioselectively oxidized these compounds to 1,4-benzenedimethanol and hydroquinone, respectively, although the wild-type enzyme exhibited no oxidation activity for these compounds. Furthermore, the S97D, S247D, and S247E mutants acquired oxidation activity for p-cresol. Especially, the S247D mutant rapidly oxidized p-cresol; the whole cells expressing the S247D mutant completely converted 1 mM p-cresol to p-hydroxybenzylalcohol in only 30 min. These results also clearly demonstrate that S97 and S247 are important residues that control the substrate specificity of CYP199A2. PMID:24982017

  4. Dissecting the Catalytic Mechanism of Betaine-Homocysteine S-Methyltransferase Using Intrinsic Tryptophan Fluorescence and Site-Directed Mutagenesis

    SciTech Connect

    Castro, C.; Gratson, A.A.; Evans, J.C.; Jiracek, J.; Collinsova, M.; Ludwig, M.L.; Garrow, T.A.

    2010-03-05

    Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-({delta}-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind and Met is the last product released. Hcy, Met, and CBHcy bind to the enzyme to form binary complexes with K{sub d} values of 7.9, 6.9, and 0.28 {micro}M, respectively. Binary complexes with Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly to BHMT-Hcy to form ternary complexes with K{sub d} values of 1.1 and 0.73 {micro}M, respectively. Mutation of each of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoes two distinct conformational changes that are reflected in the fluorescence of the enzyme. The first is induced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT, the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacing these residues by site-directed mutagenesis significantly reduces the catalytic efficiency (V{sub max}/K{sub m}) of the enzyme. Replacing Tyr77 with Phe abolishes enzyme activity.

  5. Site-directed mutagenesis of the heterotrimeric killer toxin zymocin identifies residues required for early steps in toxin action.

    PubMed

    Wemhoff, Sabrina; Klassen, Roland; Meinhardt, Friedhelm

    2014-10-01

    Zymocin is a Kluyveromyces lactis protein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activity in vitro that might be important during γ import. Saccharomyces cerevisiae strains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase from Pichia acaciae (P. acaciae Orf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells. PMID:25128337

  6. Proton transfers in a channelrhodopsin-1 studied by Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis.

    PubMed

    Ogren, John I; Yi, Adrian; Mamaev, Sergey; Li, Hai; Spudich, John L; Rothschild, Kenneth J

    2015-05-15

    Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2 (380) state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2 (380) formation. The unusual charge neutrality of both Schiff base counterions in the P2 (380) conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs. PMID:25802337

  7. Proton Transfers in a Channelrhodopsin-1 Studied by Fourier Transform Infrared (FTIR) Difference Spectroscopy and Site-directed Mutagenesis*

    PubMed Central

    Ogren, John I.; Yi, Adrian; Mamaev, Sergey; Li, Hai; Spudich, John L.; Rothschild, Kenneth J.

    2015-01-01

    Channelrhodopsin-1 from the alga Chlamydomonas augustae (CaChR1) is a low-efficiency light-activated cation channel that exhibits properties useful for optogenetic applications such as a slow light inactivation and a red-shifted visible absorption maximum as compared with the more extensively studied channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2). Previously, both resonance Raman and low-temperature FTIR difference spectroscopy revealed that unlike CrChR2, CaChR1 under our conditions exhibits an almost pure all-trans retinal composition in the unphotolyzed ground state and undergoes an all-trans to 13-cis isomerization during the primary phototransition typical of other microbial rhodopsins such as bacteriorhodopsin (BR). Here, we apply static and rapid-scan FTIR difference spectroscopy along with site-directed mutagenesis to characterize the proton transfer events occurring upon the formation of the long-lived conducting P2380 state of CaChR1. Assignment of carboxylic C=O stretch bands indicates that Asp-299 (homolog to Asp-212 in BR) becomes protonated and Asp-169 (homolog to Asp-85 in BR) undergoes a net change in hydrogen bonding relative to the unphotolyzed ground state of CaChR1. These data along with earlier FTIR measurements on the CaChR1 → P1 transition are consistent with a two-step proton relay mechanism that transfers a proton from Glu-169 to Asp-299 during the primary phototransition and from the Schiff base to Glu-169 during P2380 formation. The unusual charge neutrality of both Schiff base counterions in the P2380 conducting state suggests that these residues may function as part of a cation selective filter in the open channel state of CaChR1 as well as other low-efficiency ChRs. PMID:25802337

  8. Directed optimization of a newly identified squalene synthase from Mortierella alpine based on sequence truncation and site-directed mutagenesis.

    PubMed

    Huang, Di; Yao, Yongpeng; Zhang, Hang; Mei, Zhu; Wang, Ru; Feng, Lu; Liu, Bin

    2015-10-01

    Terpenoids, a class of isoprenoids usually isolated from plants, are always used as commercial flavor and anticancer drugs. As a key precursor for triterpenes and sterols, biosynthesis of squalene (SQ) can be catalyzed by squalene synthase (SQS) from two farnesyl diphosphate molecules. In this work, the key SQS gene involved in sterols synthesis by Mortierella alpine, an industrial strain often used to produce unsaturated fatty acid such as γ-linolenic acid and arachidonic acid, was identified and characterized. Bioinformatic analysis indicated that MaSQS contained 416 amino acid residues involved in four highly conserved regions. Phylogenetic analysis revealed the closest relationship of MaSQS with Ganoderma lucidum and Aspergillus, which also belonged to the member of the fungus. Subsequently, the recombinant protein was expressed in Escherichia coli BL21(DE3) and detected by SDS-PAGE. To improve the expression and solubility of protein, 17 or 27 amino acids in the C-terminal were deleted. In vitro activity investigation based on gas chromatography-mass spectrometry revealed that both the truncated enzymes could functionally catalyze the reaction from FPP to SQ and the enzymatic activity was optimal at 37 °C, pH 7.2. Moreover, based on the site-directed mutagenesis, the mutant enzyme mMaSQSΔC17 (E186K) displayed a 3.4-fold improvement in catalytic efficiency (k(cat)/K(m)) compared to the control. It was the first report of characterization and modification of SQS from M. alpine, which facilitated the investigation of isoprenoid biosynthesis in the fungus. The engineered mMaSQSΔC17 (E186K) can be a potential candidate of the terpenes and steroids synthesis employed for synthetic biology. PMID:26275528

  9. Site-directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose-phosphate synthase modulation

    NASA Technical Reports Server (NTRS)

    Toroser, D.; McMichael, R. Jr; Krause, K. P.; Kurreck, J.; Sonnewald, U.; Stitt, M.; Huber, S. C.; Davies, E. (Principal Investigator)

    1999-01-01

    Site-directed mutagenesis of spinach sucrose-phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild-type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated in vitro. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS-kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach-specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre-incubation with leaf PP2A or ATP plus SPS-kinase (the PKIII enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPS in vivo, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.

  10. Insight into the mechanism of phosphoenolpyruvate mutase catalysis derived from site-directed mutagenesis studies of active site residues.

    PubMed

    Jia, Y; Lu, Z; Huang, K; Herzberg, O; Dunaway-Mariano, D

    1999-10-26

    PEP mutase catalyzes the conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate in biosynthetic pathways leading to phosphonate secondary metabolites. A recent X-ray structure [Huang, K., Li, Z., Jia, Y., Dunaway-Mariano, D., and Herzberg, O. (1999) Structure (in press)] of the Mytilus edulis enzyme complexed with the Mg(II) cofactor and oxalate inhibitor reveals an alpha/beta-barrel backbone-fold housing an active site in which Mg(II) is bound by the two carboxylate groups of the oxalate ligand and the side chain of D85 and, via bridging water molecules, by the side chains of D58, D85, D87, and E114. The oxalate ligand, in turn, interacts with the side chains of R159, W44, and S46 and the backbone amide NHs of G47 and L48. Modeling studies identified two feasible PEP binding modes: model A in which PEP replaces oxalate with its carboxylate group interacting with R159 and its phosphoryl group positioned close to D58 and Mg(II) shifting slightly from its original position in the crystal structure, and model B in which PEP replaces oxalate with its phosphoryl group interacting with R159 and Mg(II) retaining its original position. Site-directed mutagenesis studies of the key mutase active site residues (R159, D58, D85, D87, and E114) were carried out in order to evaluate the catalytic roles predicted by the two models. The observed retention of low catalytic activity in the mutants R159A, D85A, D87A, and E114A, coupled with the absence of detectable catalytic activity in D58A, was interpreted as evidence for model A in which D58 functions in nucleophilic catalysis (phosphoryl transfer), R159 functions in PEP carboxylate group binding, and the carboxylates of D85, D87 and E114 function in Mg(II) binding. These results also provide evidence against model B in which R159 serves to mediate the phosphoryl transfer. A catalytic motif, which could serve both the phosphoryl transfer and the C-C cleavage enzymes of the PEP mutase superfamily, is proposed. PMID:10571990

  11. Identification of active site lysyl residues of phenylalanine dehydrogenase by chemical modification with methyl acetyl phosphate combined with site-directed mutagenesis.

    PubMed

    Kataoka, K; Tanizawa, K; Fukui, T; Ueno, H; Yoshimura, T; Esaki, N; Soda, K

    1994-12-01

    A monoanionic acetylation reagent, methyl acetyl phosphate, was used to acetylate lysyl residues of the recombinant thermostable phenylalanine dehydrogenase from Thermoactinomyces intermedius. The enzyme was irreversibly inactivated with the reagent in a time- and dose-dependent manner. Simultaneous addition of substrate and coenzyme markedly protected the enzyme from inactivation. Acetylated lysyl residues presumably occurring at the active site were determined by differential modification; the enzyme was first modified with a cold reagent in the presence of both substrate and coenzyme and, after removal of the added substances by gel filtration, was then labeled with a radioactive reagent. At least 7 lysyl residues per enzyme subunit were radiolabeled by this method. To further specify the lysyl residue(s) whose modification results in inactivation of the enzyme, 5 lysyl residues highly conserved in various amino acid dehydrogenase sequences were replaced with Ala by site-directed mutagenesis. Although all of the single mutant enzymes were inactivated with the reagent as effectively as the wild-type enzyme, a double mutant enzyme in which both Lys-69 and Lys-81 were replaced with Ala was found to be inactivated very slowly. These results suggest that the reagent can acetylate both of these lysyl residues and inactivate the enzyme. Kinetic analyses of the single Lys-69 and Lys-81 mutant enzymes revealed that they are involved in substrate binding and catalysis, respectively, like the corresponding residues in the homologous leucine dehydrogenase. PMID:7706231

  12. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    PubMed Central

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea

    2015-01-01

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin. PMID:25724962

  13. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    SciTech Connect

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea; Wall, Judy D.

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.

  14. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    DOE PAGESBeta

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea; Wall, Judy D.

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential formore » mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.« less

  15. Identification of active-site residues in protease 3C of hepatitis A virus by site-directed mutagenesis.

    PubMed Central

    Gosert, R; Dollenmaier, G; Weitz, M

    1997-01-01

    Picornavirus 3C proteases (3Cpro) are cysteine proteases related by amino acid sequence to trypsin-like serine proteases. Comparisons of 3Cpro of hepatitis A virus (HAV) to those of other picornaviruses have resulted in prediction of active-site residues: histidine at position 44 (H44), aspartic acid (D98), and cysteine (C172). To test whether these residues are key members of a putative catalytic triad, oligonucleotide-directed mutagenesis was targeted to 3Cpro in the context of natural polypeptide precursor P3. Autocatalytic processing of the polyprotein containing wild-type or variant 3Cpro was tested by in vivo expression of vaccinia virus-HAV chimeras in an animal cell-T7 hybrid system and by in vitro translation of corresponding RNAs. Comparison with proteins present in HAV-infected cells showed that both expression systems mimicked authentic polyprotein processing. Individual substitutions of H44 by tyrosine and of C172 by glycine or serine resulted in complete loss of the virus-specific proteolytic cascade. In contrast, a P3 polyprotein in which D98 was substituted by asparagine underwent only slightly delayed processing, while an additional substitution of valine (V47) by glycine within putative protein 3A caused a more pronounced loss of processing. Therefore, apparently H44 and C172 are active-site constituents whereas D98 is not. The results, furthermore, suggest that substitution of amino acid residues distant from polyprotein cleavage sites may reduce proteolytic activity, presumably by altering substrate conformation. PMID:9060667

  16. From Green to Blue: Site-Directed Mutagenesis of the Green Fluorescent Protein to Teach Protein Structure-Function Relationships

    ERIC Educational Resources Information Center

    Giron, Maria D.; Salto, Rafael

    2011-01-01

    Structure-function relationship studies in proteins are essential in modern Cell Biology. Laboratory exercises that allow students to familiarize themselves with basic mutagenesis techniques are essential in all Genetic Engineering courses to teach the relevance of protein structure. We have implemented a laboratory course based on the…

  17. Site-directed mutagenesis of amino acid residues of D1 protein interacting with phosphatidylglycerol affects the function of plastoquinone QB in photosystem II.

    PubMed

    Endo, Kaichiro; Mizusawa, Naoki; Shen, Jian-Ren; Yamada, Masato; Tomo, Tatsuya; Komatsu, Hirohisa; Kobayashi, Masami; Kobayashi, Koichi; Wada, Hajime

    2015-12-01

    Recent X-ray crystallographic analysis of photosystem (PS) II at 1.9-Å resolution identified 20 lipid molecules in the complex, five of which are phosphatidylglycerol (PG). In this study, we mutagenized amino acid residues S232 and N234 of D1, which interact with two of the PG molecules (PG664 and PG694), by site-directed mutagenesis in Synechocystis sp. PCC 6803 to investigate the role of the interaction in PSII. The serine and asparagine residues at positions 232 and 234 from the N-terminus were mutagenized to alanine and aspartic acid, respectively, and a mutant carrying both amino acid substitutions was also produced. Although the obtained mutants, S232A, N234D, and S232AN234D, exhibited normal growth, they showed decreased photosynthetic activities and slower electron transport from QA to QB than the control strain. Thermoluminescence analysis suggested that this slower electron transfer in the mutants was caused by more negative redox potential of QB, but not in those of QA and S2. In addition, the levels of extrinsic proteins, PsbV and PsbU, were decreased in PSII monomer purified from the S232AN234D mutant, while that of Psb28 was increased. In the S232AN234D mutant, the content of PG in PSII was slightly decreased, whereas that of monogalactosyldiacylglycerol was increased compared with the control strain. These results suggest that the interactions of S232 and N234 with PG664 and PG694 are important to maintain the function of QB and to stabilize the binding of extrinsic proteins to PSII. PMID:25921208

  18. Lysine-Based Site-Directed Mutagenesis Increased Rigid β-Sheet Structure and Thermostability of Mesophilic 1,3-1,4-β-Glucanase.

    PubMed

    Niu, Chengtuo; Zhu, Linjiang; Zhu, Pei; Li, Qi

    2015-06-01

    1,3-1,4-β-Glucanase is widely applied in the food industry, while its low thermostability often reduces its performance. In a previous study, chemical modification of surface lysine residues was proved to increase the thermostability of β-glucanase. To improve the thermostability, the mesophilic β-glucanase from Bacillus terquilensis was rationally engineered through site-directed mutagenesis of the 12 lysines into serines. The results showed that the K20S, K117S, and K165S mutants could both enhance the specific activities and thermostability of β-glucanase. The triple mutant (K20S/K117S/K165S) could increase the optimal temperature and T50 value by 15 and 14 °C, respectively. Five percent more structured residues were observed in the mutant, which formed new β-sheet structures in the concave side. Molecular dynamics simulation analysis showed that the flexibility in the mutation regions was decreased, which resulted in the overall rigidity of the β-glucanase. Therefore, the lysine-based site-directed mutagenesis is a simple and effective method for improving the thermostability of β-glucanase. PMID:25953154

  19. Delineation of the structural and functional role of Arg111 in GSTU4-4 from Glycine max by chemical modification and site-directed mutagenesis.

    PubMed

    Labrou, Nikolaos E; Muharram, Magdy Mohamed; Abdelkader, Maged Saad

    2016-10-01

    The structural and functional role of Arg111 in GSTU4-4 from Glycine max (GmGSTU4-4) was studied by chemical modification followed by site-directed mutagenesis. The arginine-specific reagent 2,3-butanedione (BTD) inactivates the enzyme in borate buffer at pH8.0, with pseudo-first-order saturation kinetics. The rate of inactivation exhibited a non-linear dependence on the concentration of BTD which can be described by reversible binding of reagent to the enzyme (KD 81.2±9.2mM) prior to the irreversible reaction, with maximum rate constants of 0.18±0.01min(-1). Protection from inactivation was afforded by substrate analogues demonstrating the specificity of the reaction. Structural analysis suggested that the modified residue is Arg111, which was confirmed by protein chemistry experiments. Site-directed mutagenesis was used in dissecting the role of Arg111 in substrate binding, specificity and catalytic mechanism. The mutant Arg111Ala enzyme exhibited unchanged Km value for GSH but showed reduced affinity for the xenobiotic substrates, higher kcat and specific activities towards aromatic substrates and lower specific activities towards aliphatic substrates. The biological significance of the specific modification of Arg111 by dicarbonyl compounds and the role of Arg111 as a target for engineering xenobiotic substrate specificity were discussed. PMID:27375050

  20. Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

    PubMed Central

    Rubinstein, M; Mogil, J S; Japón, M; Chan, E C; Allen, R G; Low, M J

    1996-01-01

    A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms. Images Fig. 1 Fig. 2 PMID:8633004

  1. Site-directed mutagenesis of conserved cysteine residues in NqrD and NqrE subunits of Na+-translocating NADH:quinone oxidoreductase.

    PubMed

    Fadeeva, M S; Bertsova, Y V; Verkhovsky, M I; Bogachev, A V

    2008-02-01

    Each of two hydrophobic subunits of Na+-translocating NADH:quinone oxidoreductase (NQR), NqrD and NqrE, contain a pair of strictly conserved cysteine residues within their transmembrane alpha-helices. Site-directed mutagenesis showed that substitutions of these residues in NQR of Vibrio harveyi blocked the Na+-dependent and 2-n-heptyl-4-hydroxyquinoline N-oxide-sensitive quinone reductase activity of the enzyme. However, these mutations did not affect the interaction of NQR with NADH and menadione. It was demonstrated that these conserved cysteine residues are necessary for the correct folding and/or the stability of the NQR complex. Mass and EPR spectroscopy showed that NQR from V. harveyi bears only a 2Fe-2S cluster as a metal-containing prosthetic group. PMID:18298367

  2. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

    SciTech Connect

    Scott, A.I.; Roessner, C.A.; Stolowich, N.J.; Karuso, P.; Williams, H.J.; Grant, S.K.; Gonzalez, M.D.; Hoshino, T. )

    1988-10-18

    The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-(2,11-{sup 13}C{sub 2})PBG reveals that the aminomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the {alpha}-free pyrrole. NMR spectroscopy of the ES{sub 2} complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the {alpha}-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

  3. Site-directed mutagenesis of Lysine{sup 382}, the activator-binding site, of ADP-Glucose pyrophosphorylase from Anabaena PCC 6120

    SciTech Connect

    Sheng, Jun; Charng, Yee-yung; Preiss, J.

    1996-03-05

    Previous studies have shown that a highly conserved lysyl residue (Lys{sup 419}) near the C-terminus of Anabaena ADP-glucose pyrophosphorylase is involved in the binding of 3-P-glycerate, the allosteric activator. Phosphopyridoxylation of the K419R mutant enzyme modified another conserved lysyl residue (Lys{sup 382}), suggesting that this residue might be also located within the activator-binding site. Site-directed mutagenesis of Lys{sup 382} of the Anabaena enzyme was performed to determine the role of this residue. Replacing Lys{sup 382} with either arginine, alanine, or glutamine produced mutant enzymes with apparent affinities for 3-P-glycerate 10-160-fold lower than that of the wild-type enzyme. The glutamic acid mutant enzyme was inhibited by 3-P-glycerate. These mutations had lesser impact on the kinetic constants for the substrates and inhibitor, P{sub i}, and on the thermal stability. These results indicate that both the charge and size of the residue at position 382 influence the binding of 3-P-glycerate. Site-directed mutagenesis was also performed to obtain a K382R-K419R double mutant. The apparent affinity for 3-P-glycerate of this double-mutant enzyme was 104-fold lower than that of the wild-type enzyme, and the specificity for activator of this mutant enzyme was altered. The K382R-K419R enzyme could not be phosphopyridoxylated, suggesting that other lysine residues are not involved in the binding of 3-P-glycerate. 32 refs., 2 figs., 3 tabs.

  4. Site-directed mutagenesis of Gln103 reveals the influence of this residue on the redox properties and stability of MauG.

    PubMed

    Shin, Sooim; Yukl, Erik T; Sehanobish, Esha; Wilmot, Carrie M; Davidson, Victor L

    2014-03-01

    The diheme enzyme MauG catalyzes a six-electron oxidation that is required for the posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived cofactor, tryptophan tryptophylquinone (TTQ). Crystallographic and computational studies have implicated Gln103 in stabilizing the Fe(IV)═O moiety of the bis-Fe(IV) state by hydrogen bonding. The role of Gln103 was probed by site-directed mutagenesis. Q103L and Q103E mutations resulted in no expression and very little expression of the protein, respectively. Q103A MauG exhibited oxidative damage when isolated. Q103N MauG was isolated at levels comparable to that of wild-type MauG and exhibited normal activity in catalyzing the biosynthesis of TTQ from preMADH. The crystal structure of the Q103N MauG-preMADH complex suggests that a water may mediate hydrogen bonding between the shorter Asn103 side chain and the Fe(IV)═O moiety. The Q103N mutation caused the two redox potentials associated with the diferric/diferrous redox couple to become less negative, although the redox cooperativity of the hemes of MauG was retained. Upon addition of H2O2, Q103N MauG exhibits changes in the absorbance spectrum in the Soret and near-IR regions consistent with formation of the bis-Fe(IV) redox state. However, the rate of spontaneous return of the spectrum in the Soret region was 4.5-fold greater for Q103N MauG than for wild-type MauG. In contrast, the rate of spontaneous decay of the absorbance at 950 nm, which is associated with charge-resonance stabilization of the high-valence state, was similar for wild-type MauG and Q103N MauG. This suggests that as a consequence of the mutation a different distribution of resonance structures stabilizes the bis-Fe(IV) state. These results demonstrate that subtle changes in the structure of the side chain of residue 103 can significantly affect the overall protein stability of MauG and alter the redox properties of the

  5. A saxitoxin-binding aptamer with higher affinity and inhibitory activity optimized by rational site-directed mutagenesis and truncation.

    PubMed

    Zheng, X; Hu, B; Gao, S X; Liu, D J; Sun, M J; Jiao, B H; Wang, L H

    2015-07-01

    Saxitoxin (STX), a member of the family of paralytic shellfish poisoning toxins, poses toxicological and ecotoxicological risks. To develop an analytical recognition element for STX, a DNA aptamer (APT(STX1)) was previously discovered via an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by Handy et al. Our study focused on generating an improved aptamer based on APT(STX1) through rational site-directed mutation and truncation. In this study, we generated the aptamer, M-30f, with a 30-fold higher affinity for STX compared with APT(STX1). The Kd value for M-30f was 133 nM, which was calculated by Bio-Layer Interferometry. After optimization, we detected and compared the interaction of STX with aptamers (APT(STX1) or M-30f) through several techniques (ELISA, cell bioassay, and mouse bioassay). Both aptamers' STX-binding ability was demonstrated in all three methods. Moreover, M-30f performs better than its parent sequence with higher suppressive activity against STX. As a molecular recognition element, M-30f has good prospects for practical application. PMID:25937337

  6. Improvement of the amides forming capacity of the arylacetonitrilase from Pseudomonas fluorescens EBC191 by site-directed mutagenesis.

    PubMed

    Sosedov, Olga; Stolz, Andreas

    2015-03-01

    The influence of different amino acid substitutions in the nitrilase from Pseudomonas fluorescens EBC191 (NitA) on the catalytical activity and the ability to form amides was investigated. The enzyme variant Glu137Ala was constructed because glutamate residues homologous to Glu137 are highly conserved among different members of the nitrilase superfamily and it has been suggested that these residues are indispensable for the hydrolysis of amides by enzymes belonging to the nitrilase superfamily. The enzyme variant Glu137Ala demonstrated less than 1 % of the wild-type activity but was still enzymatically competent to convert mandelonitrile to mandelic acid and mandeloamide. The tryptophan residue at position 188, which was previously identified as important for the amide forming capacity of the nitrilase, was exchanged by saturation mutagenesis for all other proteinogenic amino acids. Surprisingly, 18 of these 19 exchanges resulted in an increased formation of mandeloamide from (R,S)-mandelonitrile and three of these variants converted (R,S)-mandelonitrile to more than 90 % of mandeloamide. Furthermore, these modifications also resulted in a reversal of stereoselectivity and these variants formed in contrast to the wild-type enzyme and almost all other known nitrilases preferentially (S)-mandelic acid. The synthetic potential of one of these variants was demonstrated by the construction of recombinant E. coli clones which simultaneously expressed the nitrilase variant and the (S)-hydroxynitrile lyase (oxynitrilase) from the cassava plant (Manihot esculenta). These "bienzymatic catalysts" converted benzaldehyde plus cyanide almost exclusively to (S)-mandeloamide and did not show any inhibition in the presence of cyanide in concentrations up to 200 mM. PMID:25248440

  7. Enzyme catalysis via control of activation entropy: site-directed mutagenesis of 6,7-dimethyl-8-ribityllumazine synthase.

    PubMed

    Fischer, Markus; Haase, Ilka; Kis, Klaus; Meining, Winfried; Ladenstein, Rudolf; Cushman, Mark; Schramek, Nicholas; Huber, Robert; Bacher, Adelbert

    2003-02-21

    6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyses the penultimate step in the biosynthesis of riboflavin. In Bacillus subtilis, 60 lumazine synthase subunits form an icosahedral capsid enclosing a homotrimeric riboflavin synthase unit. The ribH gene specifying the lumazine synthase subunit can be expressed in high yield. All amino acid residues exposed at the surface of the active site cavity were modified by PCR assisted mutagenesis. Polar amino acid residues in direct contact with the enzyme substrates, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, could be replaced with relative impunity with regard to the catalytic properties. Only the replacement of Arg127, which forms a salt bridge with the phosphate group of 3,4-dihydroxy-2-butanone 4-phosphate, reduced the catalytic rate by more than one order of magnitude. Replacement of His88, which is believed to assist in proton transfer reactions, reduced the catalytic activity by about one order of magnitude. Surprisingly, the activation enthalpy deltaH of the lumazine synthase reaction exceeds that of the uncatalysed reaction. On the other hand, the free energy of activation deltaG of the uncatalysed reaction is characterised by a large entropic term (TdeltaS) of -37.8 kJmol(-1), whereas the entropy of activation (TdeltaS) of the enzyme-catalysed reaction is -6.7 kJmol(-1). This suggests that the rate enhancement by the enzyme is predominantly achieved by establishing a favourable topological relation of the two substrates, whereas acid/base catalysis may play a secondary role. PMID:12581640

  8. Site-directed mutagenesis studies on the L-arginine-binding sites of feedback inhibition in N-acetyl-L-glutamate kinase (NAGK) from Corynebacterium glutamicum.

    PubMed

    Xu, Meijuan; Rao, Zhiming; Dou, Wenfang; Jin, Jian; Xu, Zhenghong

    2012-02-01

    Arginine biosynthesis in Corynebacterium glutamicum proceeds via a pathway that is controlled by arginine through feedback inhibition of NAGK, the enzyme that converts N-acetyl-L-glutamate (NAG) to N-acety-L-glutamy-L-phosphate. In this study, the gene argB encoding NAGK from C. glutamicum ATCC 13032 was site-directed, and the L-arginine-binding sites of feedback inhibition in Cglu_NAGK are described. The N-helix and C-terminal residues were first deleted, and the results indicated that they are both necessary for Cglu_NAGK, whereas, the complete N-helix deletion (the front 28 residues) abolished the L-arginine inhibition. Further, we study here the impact on these functions of 12 site-directed mutations affecting seven residues of Cglu_NAGK, chosen on the basis of homology structural alignment. The E19R, H26E, and H268N variants could increase the I₀.₅ (R) 50-60 fold, and the G287D and R209A mutants could increase the I₀.₅ (R) 30-40 fold. The E281A mutagenesis resulted in the substrate kinetics being greatly influenced. The W23A variant had a lower specific enzyme activity. These results explained that the five amino acid residues (E19, H26, R209, H268, and G287) located in or near N-helix are all essential for the formation of arginine inhibition. PMID:22101454

  9. The peptide agonist-binding site of the glucagon-like peptide-1 (GLP-1) receptor based on site-directed mutagenesis and knowledge-based modelling

    PubMed Central

    Dods, Rachel L.; Donnelly, Dan

    2015-01-01

    Glucagon-like peptide-1 (7–36)amide (GLP-1) plays a central role in regulating blood sugar levels and its receptor, GLP-1R, is a target for anti-diabetic agents such as the peptide agonist drugs exenatide and liraglutide. In order to understand the molecular nature of the peptide–receptor interaction, we used site-directed mutagenesis and pharmacological profiling to highlight nine sites as being important for peptide agonist binding and/or activation. Using a knowledge-based approach, we constructed a 3D model of agonist-bound GLP-1R, basing the conformation of the N-terminal region on that of the receptor-bound NMR structure of the related peptide pituitary adenylate cyclase-activating protein (PACAP21). The relative position of the extracellular to the transmembrane (TM) domain, as well as the molecular details of the agonist-binding site itself, were found to be different from the model that was published alongside the crystal structure of the TM domain of the glucagon receptor, but were nevertheless more compatible with published mutagenesis data. Furthermore, the NMR-determined structure of a high-potency cyclic conformationally-constrained 11-residue analogue of GLP-1 was also docked into the receptor-binding site. Despite having a different main chain conformation to that seen in the PACAP21 structure, four conserved residues (equivalent to His-7, Glu-9, Ser-14 and Asp-15 in GLP-1) could be structurally aligned and made similar interactions with the receptor as their equivalents in the GLP-1-docked model, suggesting the basis of a pharmacophore for GLP-1R peptide agonists. In this way, the model not only explains current mutagenesis and molecular pharmacological data but also provides a basis for further experimental design. PMID:26598711

  10. Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

  11. Site-directed mutagenesis maps interactions that enhance cognate and limit promiscuous catalysis by an alkaline phosphatase superfamily phosphodiesterase.

    PubMed

    Wiersma-Koch, Helen; Sunden, Fanny; Herschlag, Daniel

    2013-12-23

    Catalytic promiscuity, an evolutionary concept, also provides a powerful tool for gaining mechanistic insights into enzymatic reactions. Members of the alkaline phosphatase (AP) superfamily are highly amenable to such investigation, with several members having been shown to exhibit promiscuous activity for the cognate reactions of other superfamily members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase (NPP) exhibits a >10⁶-fold preference for the hydrolysis of phosphate diesters over phosphate monoesters, and that the reaction specificity is reduced 10³-fold when the size of the substituent on the transferred phosphoryl group of phosphate diester substrates is reduced to a methyl group. Here we show additional specificity contributions from the binding pocket for this substituent (herein termed the R' substituent) that account for an additional ~250-fold differential specificity with the minimal methyl substituent. Removal of four hydrophobic side chains suggested on the basis of structural inspection to interact favorably with R' substituents decreases phosphate diester reactivity 10⁴-fold with an optimal diester substrate (R' = 5'-deoxythymidine) and 50-fold with a minimal diester substrate (R' = CH₃). These mutations also enhance the enzyme's promiscuous phosphate monoesterase activity by nearly an order of magnitude, an effect that is traced by mutation to the reduction of unfavorable interactions with the two residues closest to the nonbridging phosphoryl oxygen atoms. The quadruple R' pocket mutant exhibits the same activity toward phosphate diester and phosphate monoester substrates that have identical leaving groups, with substantial rate enhancements of ~10¹¹-fold. This observation suggests that the Zn²⁺ bimetallo core of AP superfamily enzymes, which is equipotent in phosphate monoester and diester catalysis, has the potential to become specialized for the hydrolysis of each class of phosphate esters via addition

  12. Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis.

    PubMed

    Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2014-01-01

    We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170 kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50 kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl2, and HgCl2 functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl2 was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition. PMID:25126984

  13. Biological and Biochemical Characterization of Variant A Subunits of Cholera Toxin Constructed by Site-Directed Mutagenesis

    PubMed Central

    Jobling, Michael G.; Holmes, Randall K.

    2001-01-01

    Cholera toxin (CT) is the prototype for the Vibrio cholerae-Escherichia coli family of heat-labile enterotoxins having an AB5 structure. By substituting amino acids in the enzymatic A subunit that are highly conserved in all members of this family, we constructed 23 variants of CT that exhibited decreased or undetectable toxicity and we characterized their biological and biochemical properties. Many variants exhibited previously undescribed temperature-sensitive assembly of holotoxin and/or increased sensitivity to proteolysis, which in all cases correlated with exposure of epitopes of CT-A that are normally hidden in native CT holotoxin. Substitutions within and deletion of the entire active-site-occluding loop demonstrated a prominent role for His-44 and this loop in the structure and activity of CT. Several novel variants with wild-type assembly and stability showed significantly decreased toxicity and enzymatic activity (e.g., variants at positions R11, I16, R25, E29, and S68+V72). In most variants the reduction in toxicity was proportional to the decrease in enzymatic activity. For substitutions or insertions at E29 and Y30 the decrease in toxicity was 10- and 5-fold more than the reduction in enzymatic activity, but for variants with R25G, E110D, or E112D substitutions the decrease in enzymatic activity was 12- to 50-fold more than the reduction in toxicity. These variants may be useful as tools for additional studies on the cell biology of toxin action and/or as attenuated toxins for adjuvant or vaccine use. PMID:11395467

  14. Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains*

    PubMed Central

    Villalba, Miryam I.; Canul-Tec, Juan C.; Luna-Martínez, Oscar D.; Sánchez-Alcalá, Rosalba; Olamendi-Portugal, Timoteo; Rudiño-Piñera, Enrique; Rojas, Sonia; Sánchez-López, Rosana; Fernández-Velasco, Daniel A.; Becerril, Baltazar

    2015-01-01

    Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this work, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40–60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. This mutagenic approach helped to identify key regions implicated in λ3 AL. PMID:25505244

  15. Aldolase A Ins(1,4,5)P3-binding domains as determined by site-directed mutagenesis.

    PubMed Central

    Baron, C B; Tolan, D R; Choi, K H; Coburn, R F

    1999-01-01

    We substituted neutral amino acids for some positively charged residues (R42, K107, K146, R148 and K229) that line the active site of aldolase A in an effort to determine binding sites for inositol 1, 4,5-trisphosphate. In addition, D33 (involved in carbon-carbon bond cleavage) was mutated. K229A and D33S aldolases showed almost no catalytic activity, but Ins(1,4,5)P(3) binding was similar to that determined with the use of wild-type aldolase A. R42A, K107A, K146R and R148A had markedly decreased affinities for Ins(1,4,5)P(3) binding, increased EC(50) values for Fru(1,6)P(2)-evoked release of bound Ins(1,4,5)P(3) and increased K(i) values for Ins(1,4, 5)P(3)-evoked inhibition of aldolase activity. K146Q (positive charge removal) had essentially no catalytic activity and could not bind Ins(1,4,5)P(3). Computer-simulated docking of Ins(1,4,5)P(3) in the aldolase A structure was consistent with electrostatic binding of Ins(1,4,5)P(3) to K107, K146, R148, R42, R303 and backbone nitrogens, as has been reported for Fru(1,6)P(2) binding. Results indicate that Ins(1,4,5)P(3) binding occurs at the active site and is not dependent on having a catalytically active enzyme; they also suggest that there is competition between Ins(1,4,5)P(3) and Fru(1, 6)P(2) for binding. Although Ins(1,4,5)P(3) binding to aldolase involved electrostatic interactions, the aldolase A Ins(1,4, 5)P(3)-binding domain did not show other similarities to pleckstrin homology domains or phosphotyrosine-binding domains known to bind Ins(1,4,5)P(3) in other proteins. PMID:10417347

  16. A systematic survey of conserved histidines in the core subunits of Photosystem I by site-directed mutagenesis reveals the likely axial ligands of P700.

    PubMed

    Redding, K; MacMillan, F; Leibl, W; Brettel, K; Hanley, J; Rutherford, A W; Breton, J; Rochaix, J D

    1998-01-01

    The Photosystem I complex catalyses the transfer of an electron from lumenal plastocyanin to stromal ferredoxin, using the energy of an absorbed photon. The initial photochemical event is the transfer of an electron from the excited state of P700, a pair of chlorophylls, to a monomer chlorophyll serving as the primary electron acceptor. We have performed a systematic survey of conserved histidines in the last six transmembrane segments of the related polytopic membrane proteins PsaA and PsaB in the green alga Chlamydomonas reinhardtii. These histidines, which are present in analogous positions in both proteins, were changed to glutamine or leucine by site-directed mutagenesis. Double mutants in which both histidines had been changed to glutamine were screened for changes in the characteristics of P700 using electron paramagnetic resonance, Fourier transform infrared and visible spectroscopy. Only mutations in the histidines of helix 10 (PsaA-His676 and PsaB-His656) resulted in changes in spectroscopic properties of P700, leading us to conclude that these histidines are most likely the axial ligands to the P700 chlorophylls. PMID:9427740

  17. Key substrate recognition residues in the active site of a plant cytochrome P450, CYP73A1. Homology guided site-directed mutagenesis.

    PubMed

    Schoch, Guillaume A; Attias, Roger; Le Ret, Monique; Werck-Reichhart, Danièle

    2003-09-01

    CYP73 enzymes are highly conserved cytochromes P450 in plant species that catalyse the regiospecific 4-hydroxylation of cinnamic acid to form precursors of lignin and many other phenolic compounds. A CYP73A1 homology model based on P450 experimentally solved structures was used to identify active site residues likely to govern substrate binding and regio-specific catalysis. The functional significance of these residues was assessed using site-directed mutagenesis. Active site modelling predicted that N302 and I371 form a hydrogen bond and hydrophobic contacts with the anionic site or aromatic ring of the substrate. Modification of these residues led to a drastic decrease in substrate binding and metabolism without major perturbation of protein structure. Changes to residue K484, which is located too far in the active site model to form a direct contact with cinnamic acid in the oxidized enzyme, did not influence initial substrate binding. However, the K484M substitution led to a 50% loss in catalytic activity. K484 may affect positioning of the substrate in the reduced enzyme during the catalytic cycle, or product release. Catalytic analysis of the mutants with structural analogues of cinnamic acid, in particular indole-2-carboxylic acid that can be hydroxylated with different regioselectivities, supports the involvement of N302, I371 and K484 in substrate docking and orientation. PMID:12950252

  18. Enhancement of the catalytic activity of ferulic acid decarboxylase from Enterobacter sp. Px6-4 through random and site-directed mutagenesis.

    PubMed

    Lee, Hyunji; Park, Jiyoung; Jung, Chaewon; Han, Dongfei; Seo, Jiyoung; Ahn, Joong-Hoon; Chong, Youhoon; Hur, Hor-Gil

    2015-11-01

    The enzyme ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4 catalyzes the decarboxylation reaction of lignin monomers and phenolic compounds such as p-coumaric acid, caffeic acid, and ferulic acid into their corresponding 4-vinyl derivatives, that is, 4-vinylphenol, 4-vinylcatechol, and 4-vinylguaiacol, respectively. Among various ferulic acid decarboxylase enzymes, we chose the FADase from Enterobacter sp. Px6-4, whose crystal structure is known, and produced mutants to enhance its catalytic activity by random and site-directed mutagenesis. After three rounds of sequential mutations, FADase(F95L/D112N/V151I) showed approximately 34-fold higher catalytic activity than wild-type for the production of 4-vinylguaiacol from ferulic acid. Docking analyses suggested that the increased activity of FADase(F95L/D112N/V151I) could be due to formation of compact active site compared with that of the wild-type FADase. Considering the amount of phenolic compounds such as lignin monomers in the biomass components, successfully bioengineered FADase(F95L/D112N/V151I) from Enterobacter sp. Px6-4 could provide an ecofriendly biocatalytic tool for producing diverse styrene derivatives from biomass. PMID:26059194

  19. Adaptation of Very Virulent Infectious Bursal Disease Virus to Chicken Embryonic Fibroblasts by Site-Directed Mutagenesis of Residues 279 and 284 of Viral Coat Protein VP2

    PubMed Central

    Lim, Boon-Leong; Cao, Yongchang; Yu, Tiffany; Mo, Chi-Wai

    1999-01-01

    The full-length RNA genomes of a chicken embryonic fibroblast (CEF)-nonpermissive, very virulent infectious bursal disease virus (IBDV) (strain HK46) were amplified into cDNAs by reverse transcription-PCR. The full-length cDNAs were sequenced and subcloned into a eukaryotic expression vector, from which point mutations were introduced into the VP2 region by site-directed mutagenesis. The wild-type and mutated plasmids were transfected directly into CEFs to examine their ability to generate CEF-permissive recombinant viruses. Substitution of amino acid residues 279 (Asp→Asn) and 284 (Ala→Thr) of the VP2 protein yielded a recombinant virus which was able to be passaged in CEFs, whereas the wild-type cDNAs and an amino acid substitution at residue 330 (Ser→Arg) of the VP2 protein alone did not yield viable virus. The results indicated that mutation of other viral proteins, including VP1, VP3, VP4, and VP5, was not required for CEF adaptation of the virus. The same approach may be used to produce CEF-adapted strains from newly evolved IBDVs or to manipulate the antigenicity of the virus. PMID:10074133

  20. Structural evolution of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase) through site-directed mutagenesis of the luciferin binding site.

    PubMed

    Prado, R A; Barbosa, J A; Ohmiya, Y; Viviani, V R

    2011-07-01

    The structural origin and evolution of bioluminescent activity of beetle luciferases from AMP/CoA ligases remains a mystery. Previously we cloned the luciferase-like enzyme from Zophobas morio mealworm, a reasonable protoluciferase model that could shine light on this mystery. Kinetic characterization and studies with D- and L-luciferin and their adenylates showed that stereoselectivity constitutes a critical feature for the origin of luciferase activity in AMP/CoA ligases. Comparison of the primary structures and modeling studies of this protoluciferase and the three main families of beetle luciferases showed that the carboxylic acid substrate binding site of this enzyme is smaller and more hydrophobic than the luciferin binding site of beetle luciferases, showing several substitutions of otherwise conserved residues. Thus, here we performed a site-directed mutagenesis survey of the carboxylic binding site motifs of the protoluciferase by replacing their residues by the respective conserved ones found in beetle luciferases in order to identify the structural determinants of luciferase/oxygenase activity. Although most of the substitutions had negative impact on the luminescence activity of the protoluciferase, only the substitution I327T improved the luminescence activity, resulting in a broad and 15 nm blue-shifted luminescence spectrum. Such substitution indicates the importance of the loop motif 322YGMSEI327 (341YGLTETT347 in Photinus pyralis luciferase) for luciferase activity, and indicates a possible route for the evolution of bioluminescence function of beetle luciferases. PMID:21505686

  1. Toward an Understanding of Agonist Binding to Human Orexin-1 and Orexin-2 Receptors with G-Protein-Coupled Receptor Modeling and Site-Directed Mutagenesis

    PubMed Central

    2013-01-01

    The class A G-protein-coupled receptors (GPCRs) Orexin-1 (OX1) and Orexin-2 (OX2) are located predominantly in the brain and are linked to a range of different physiological functions, including the control of feeding, energy metabolism, modulation of neuro-endocrine function, and regulation of the sleep–wake cycle. The natural agonists for OX1 and OX2 are two neuropeptides, Orexin-A and Orexin-B, which have activity at both receptors. Site-directed mutagenesis (SDM) has been reported on both the receptors and the peptides and has provided important insight into key features responsible for agonist activity. However, the structural interpretation of how these data are linked together is still lacking. In this work, we produced and used SDM data, homology modeling followed by MD simulation, and ensemble-flexible docking to generate binding poses of the Orexin peptides in the OX receptors to rationalize the SDM data. We also developed a protein pairwise similarity comparing method (ProS) and a GPCR-likeness assessment score (GLAS) to explore the structural data generated within a molecular dynamics simulation and to help distinguish between different GPCR substates. The results demonstrate how these newly developed methods of structural assessment for GPCRs can be used to provide a working model of neuropeptide–Orexin receptor interaction. PMID:24144388

  2. Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    PubMed

    Zheng, Zhong-liang; Ye, Mao-qing; Zuo, Zhen-yu; Liu, Zhi-gang; Tai, Keng-chang; Zou, Guo-lin

    2006-05-01

    Hydrogen bonds occurring in the catalytic triad (Asp32, His64 and Ser221) and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Ser33, Asp60, Ser62 and Thr220. In order to remove the effect of these hydrogen bonds, four mutants (Ser33-->Ala33, Asp60-->Ala60, Ser62-->Ala62, and Thr220-->Ala220) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (DeltaDeltaG(T)). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the kcat but not the K(m) values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Asp32, His64 and Asn155 so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-p-nitroanilide), was used for FEP calculations and MD simulations. PMID:16411898

  3. Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation

    PubMed Central

    Zheng, Zhong-liang; Ye, Mao-qing; Zuo, Zhen-yu; Liu, Zhi-gang; Tai, Keng-chang; Zou, Guo-lin

    2006-01-01

    Hydrogen bonds occurring in the catalytic triad (Asp32, His64 and Ser221) and the oxyanion hole (Asn155) are very important to the catalysis of peptide bond hydrolysis by serine proteases. For the subtilisin NK (nattokinase), a bacterial serine protease, construction and analysis of a three-dimensional structural model suggested that several hydrogen bonds formed by four residues function to stabilize the transition state of the hydrolysis reaction. These four residues are Ser33, Asp60, Ser62 and Thr220. In order to remove the effect of these hydrogen bonds, four mutants (Ser33→Ala33, Asp60→Ala60, Ser62→Ala62, and Thr220→Ala220) were constructed by site-directed mutagenesis. The results of enzyme kinetics indicated that removal of these hydrogen bonds increases the free-energy of the transition state (ΔΔGT). We concluded that these hydrogen bonds are more important for catalysis than for binding the substrate, because removal of these bonds mainly affects the kcat but not the Km values. A substrate, SUB1 (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide), was used during enzyme kinetics experiments. In the present study we have also shown the results of FEP (free-energy perturbation) calculations with regard to the binding and catalysis reactions for these mutant subtilisins. The calculated difference in FEP also suggested that these four residues are more important for catalysis than binding of the substrate, and the simulated values compared well with the experimental values from enzyme kinetics. The results of MD (molecular dynamics) simulations further demonstrated that removal of these hydrogen bonds partially releases Asp32, His64 and Asn155 so that the stability of the transition state decreases. Another substrate, SUB2 (H-D-Val-Leu-Lys-p-nitroanilide), was used for FEP calculations and MD simulations. PMID:16411898

  4. Catalytic site of human protein-glucosylgalactosylhydroxylysine glucosidase: Three crucial carboxyl residues were determined by cloning and site-directed mutagenesis.

    PubMed

    Hamazaki, Hideaki; Hamazaki, Michiko Horikawa

    2016-01-15

    Protein-glucosylgalactosylhydroxylysine glucosidase (PGGHG; EC3.2.1.107) cleaves glucose from disaccharide unit (Glc-α1,2-Gal) linked to hydroxylysine residues of collagen. In the present paper we first show that PGGHG is the product of ATHL1 gene as follows. (1) PGGHG was purified from chick embryos and digested with trypsin. LC-MS/MS analysis suggested the tryptic-peptides were from the ATHL1 gene product. (2) Chick embryo ATHL1 cDNA was cloned to a cloning and expression vector and two plasmid clones with different ATHL1 CDS insert were obtained. (3) Each plasmid DNA was transformed into Escherichia coli cells for expression and two isoforms of chicken PGGHG were obtained. (4) Both isoforms effectively released glucose from type IV collagen. Next, we searched for carboxyl residues crucial for catalytic activity as follows; human ATHL1 cDNA was cloned into a cloning and expression vector and 18 mutants were obtained by site-directed mutagenesis for 15 carboxyl residues conserved in ATHL1 of jawed vertebrates. The expression analysis indicated that substitutions of Asp301, Glu430 and Glu574 with sterically conservative (D301N, E430Q, E574Q) or functionally conservative (D301E, E430D, E574D) residues led to the complete elimination of enzyme activity. These findings lead us to the conclusion that PGGHG is encoded by ATHL1 and three carboxyl residues (corresponding to Asp301, Glu430 and Glu574 of human PGGHG) might be involved in the catalytic site of PGGHG. PMID:26682924

  5. Site-directed mutagenesis of porcine pepsin: Possible role of Asp32, Thr33, Asp215 and Gly217 in maintaining the nuclease activity of pepsin.

    PubMed

    Zhang, Yanfang; Liu, Yu; Guo, Hui; Jiang, Wei; Dong, Ping; Liang, Xingguo

    2016-07-01

    Site-directed mutagenesis of porcine pepsin was performed to identify its active sites that regulate nucleic acid (NA) digestion activity and to analyze the mechanism pepsin-mediated NA digestion. The mutation sites were distributed at the catalytic center of the enzyme (T33A, G34A, Y75H, T77A, Y189H, V214A, G217A and S219A) and at its active site (D32A and D215A) for protein digestion. Mutation of the active site residues Asp32 and Asp215 led to the inactivation of pepsin (both the NA and protein digestion activity), which demonstrated that the active sites of the pepsin protease activity were also important for its nuclease activity. Analysis of the variants revealed that T33A and G217A mutants showed a complete loss of NA digestion activity. In conclusion, residues Asp32, Thr33, Asp215 and Gly217 were related to the pepsin active sites for NA digestion. Moreover, the Y189H and V214A variants showed a loss of digestion activity on double-strand DNA (dsDNA) but only a decrease in digestion activity on single-strand DNA (ssDNA). On the contrary, the G34A variant showed a loss of digestion activity on ssDNA but only a decrease in digestion activity on dsDNA. Our findings are the first to identify the active sites of pepsin nuclease activity and lay the framework for further study of the mechanism of pepsin nuclease activity. PMID:27233129

  6. His-65 in the proton–sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity

    PubMed Central

    Lu, Jade M.-Y.; Bush, Daniel R.

    1998-01-01

    The proton–sucrose symporter that mediates phloem loading is a key component of assimilate partitioning in many higher plants. Previous biochemical investigations showed that a diethyl pyrocarbonate-sensitive histidine residue is at or near the substrate-binding site of the symporter. Among the proton–sucrose symporters cloned to date, only the histidine residue at position 65 of AtSUC1 from Arabidopsis thaliana is conserved across species. To test whether His-65 is involved in the transport reaction, we have used site-directed mutagenesis and functional expression in yeast to determine the significance of this residue in the reaction mechanism. Symporters with mutations at His-65 exhibited a range of activities; for example, the H65C mutant resulted in the complete loss of transport capacity, whereas H65Q was almost as active as wild type. Surprisingly, the H65K and H65R symporters transport sucrose at significantly higher rates (increased Vmax) than the wild-type symporter, suggesting His-65 may be associated with a rate-limiting step in the transport reaction. RNA gel blot and protein blot analyses showed that, with the exception of H65C, the variation in transport activity was not because of alterations in steady-state levels of mRNA or symporter protein. Significantly, those symporters with substitutions of His-65 that remained transport competent were no longer sensitive to inactivation by diethyl pyrocarbonate, demonstrating that this is the inhibitor-sensitive histidine residue. Taken together with our previous results, these data show that His-65 is involved in sucrose binding, and increased rates of transport implicate this region of the protein in the transport reaction. PMID:9671798

  7. cDNA cloning of two isoforms of ornithine carbamoyltransferase from Canavalia lineata leaves and the effect of site-directed mutagenesis of the carbamoyl phosphate binding site.

    PubMed

    Lee, Y; Choi, Y A; Hwang, I D; Kim, S G; Kwon, Y M

    2001-08-01

    The immunoscreening method was used to isolate cDNAs of 1323 bp (ClOCT1) and 1433 bp (ClOCT2) encoding two ornithine carbamoyltransferases (OCT, EC 2.1.3.3) from the cDNA expression library of Canavalia lineata leaves constructed in a lambdaZAP Express vector. ClOCT1 and ClOCT2 encode 359 and 369 amino acids, respectively. The N-terminals of deduced amino acid sequences of the two cDNAs showed typical features of the transit peptide of chloroplast targeting proteins. The ornithine-binding domain (FMHCLP) and catalytic domain (HPXQ) of ClOCT1 and ClOCT2 and the carbamoyl phosphate (CP)-binding site of ClOCT1 (SMRTR) are identical to OCTs of other plant species, pea and Arabidopsis thaliana. However, the CP-binding site sequence of ClOCT2, SLRTH, has not yet been reported. Both ClOCT1 and ClOCT2 cDNAs were expressed in Escherichia coli BL21 (DE3) by using expression vector pET30a. Recombinant ClOCT1 protein showed 14 times higher ornithine-dependent OCT activity than canaline-dependent OCT activity. In contrast, recombinant ClOCT2 protein showed 13 times higher canaline-dependent OCT activity than ornithine-dependent OCT activity. The two amino acids of the CP-binding site of ClOCT2 (SLRTH) were combinatorially changed to those of the CP-binding site of ClOCT1 (SMRTR) by site-directed mutagenesis. When Leu-118 of ClOCT2 was changed to Met, ornithine-dependent activity was increased significantly. It is assumed that the substrate specificity of ClOCT1 or ClOCT2 proteins partially depends on the amino acid sequence of the CP-binding site. PMID:11575720

  8. Mapping the Anopheles gambiae odorant binding protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays.

    PubMed

    Rusconi, B; Maranhao, A C; Fuhrer, J P; Krotee, P; Choi, S H; Grun, F; Thireou, T; Dimitratos, S D; Woods, D F; Marinotti, O; Walter, M F; Eliopoulos, E

    2012-08-01

    The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics. PMID:22564768

  9. Site-directed mutagenesis and feedback-resistant N-acetyl-L-glutamate kinase (NAGK) increase Corynebacterium crenatum L-arginine production.

    PubMed

    Xu, Meijuan; Rao, Zhiming; Dou, Wenfang; Yang, Juan; Jin, Jian; Xu, Zhenghong

    2012-07-01

    N-acetyl-L-glutamate kinase (EC 2.7.2.8) is first committed in the specific L-arginine pathway of Corynebacterium sp. A limited increase of L-arginine production for the argB overexpression in the engineering C. creantum SYPA-CCB strain indicated that L-arginine feedback inhibition plays an influence on the L-arginine production. In this study, we have performed site-directed mutagenesis of the key enzyme (NAGK) and the three mutations (E19R, H26E and H268D) exhibited the increase of I0.5R efficiently. Thereby, the multi-mutated NAGKM3 (including E19R/H26E/H268D) was generated and its I0.5R of L-arginine of the mutant was increased remarkably, whereas the NAGK enzyme activities did not declined. To get a feedback-resistant and robust L-arginine producer, the engineered strains SYPA-CCBM3 were constructed. Introducing the argBM3 gene enabled the NAGK enzyme activity insensitive to the intracellular arginine concentrations resulted in an enhanced arginine biosynthesis flux and decreased formation of by-products. The L-arginine synthesis was largely enhanced due to the overexpression of the argBM3, which is resistant to feedback resistant by L-arginine. Thus L-arginine production could reach 45.6 g/l, about 41.7% higher compared with the initial strain. This is an example of up-modulation of the flux through the L-arginine metabolic pathway by deregulating the key enzyme of the pathway. PMID:21901472

  10. Identification of essential active-site residues in the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis.

    PubMed Central

    Keresztessy, Z; Brown, K; Dunn, M A; Hughes, M A

    2001-01-01

    The coding sequence of the mature cyanogenic beta-glucosidase (beta-glucoside glucohydrolase, EC 3.2.1.21; linamarase) was cloned into the vector pYX243 modified to contain the SUC2 yeast secretion signal sequence and expressed in Saccharomyces cerevisiae. The recombinant enzyme is active, glycosylated and showed similar stability to the plant protein. Michaelis constants for hydrolysis of the natural substrate, linamarin (K(m)=1.06 mM) and the synthetic p-nitrophenyl beta-D-glucopyranoside (PNP-Glc; K(m)=0.36 mM), as well as apparent pK(a) values of the free enzyme and the enzyme-substrate complexes (pK(E)(1)=4.4-4.8, pK(E)(2)=6.7-7.2, pK(ES)(1)=3.9-4.4, pK(ES)(2)=8.3) were very similar to those of the plant enzyme. Site-directed mutagenesis was carried out to study the function of active-site residues based on a homology model generated for the enzyme using the MODELLER program. Changing Glu-413 to Gly destroyed enzyme activity, consistent with it being the catalytic nucleophile. The Gln-339Glu mutation also abolished activity, confirming a function in positioning the catalytic diad. The Ala-201Val mutation shifted the pK(a) of the acid/base catalyst Glu-198 from 7.22 to 7.44, reflecting a change in its hydrophobic environment. A Phe-269Asn change increased K(m) for linamarin hydrolysis 16-fold (16.1 mM) and that for PNP-Glc only 2.5-fold (0.84 mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava beta-glucosidase. PMID:11139381

  11. Crystal structure and site-directed mutagenesis of 3-ketosteroid Δ1-dehydrogenase from Rhodococcus erythropolis SQ1 explain its catalytic mechanism.

    PubMed

    Rohman, Ali; van Oosterwijk, Niels; Thunnissen, Andy-Mark W H; Dijkstra, Bauke W

    2013-12-01

    3-Ketosteroid Δ(1)-dehydrogenases are FAD-dependent enzymes that catalyze the 1,2-desaturation of 3-ketosteroid substrates to initiate degradation of the steroid nucleus. Here we report the 2.0 Å resolution crystal structure of the 56-kDa enzyme from Rhodococcus erythropolis SQ1 (Δ(1)-KSTD1). The enzyme contains two domains: an FAD-binding domain and a catalytic domain, between which the active site is situated as evidenced by the 2.3 Å resolution structure of Δ(1)-KSTD1 in complex with the reaction product 1,4-androstadiene-3,17-dione. The active site contains four key residues: Tyr(119), Tyr(318), Tyr(487), and Gly(491). Modeling of the substrate 4-androstene-3,17-dione at the position of the product revealed its interactions with these residues and the FAD. The C1 and C2 atoms of the substrate are at reaction distance to the N5 atom of the isoalloxazine ring of FAD and the hydroxyl group of Tyr(318), respectively, whereas the C3 carbonyl group is at hydrogen bonding distance from the hydroxyl group of Tyr(487) and the backbone amide of Gly(491). Site-directed mutagenesis of the tyrosines to phenylalanines confirmed their importance for catalysis. The structural features and the kinetic properties of the mutants suggest a catalytic mechanism in which Tyr(487) and Gly(491) work in tandem to promote keto-enol tautomerization and increase the acidity of the C2 hydrogen atoms of the substrate. With assistance of Tyr(119), the general base Tyr(318) abstracts the axial β-hydrogen from C2 as a proton, whereas the FAD accepts the axial α-hydrogen from the C1 atom of the substrate as a hydride ion. PMID:24165124

  12. Evolution of Flavone Synthase I from Parsley Flavanone 3β-Hydroxylase by Site-Directed Mutagenesis1[W][OA

    PubMed Central

    Gebhardt, Yvonne Helen; Witte, Simone; Steuber, Holger; Matern, Ulrich; Martens, Stefan

    2007-01-01

    Flavanone 3β-hydroxylase (FHT) and flavone synthase I (FNS I) are 2-oxoglutarate-dependent dioxygenases with 80% sequence identity, which catalyze distinct reactions in flavonoid biosynthesis. However, FNS I has been reported exclusively from a few Apiaceae species, whereas FHTs are more abundant. Domain-swapping experiments joining the N terminus of parsley (Petroselinum crispum) FHT with the C terminus of parsley FNS I and vice versa revealed that the C-terminal portion is not essential for FNS I activity. Sequence alignments identified 26 amino acid substitutions conserved in FHT versus FNS I genes. Homology modeling, based on the related anthocyanidin synthase structure, assigned seven of these amino acids (FHT/FNS I, M106T, I115T, V116I, I131F, D195E, V200I, L215V, and K216R) to the active site. Accordingly, FHT was modified by site-directed mutagenesis, creating mutants encoding from one to seven substitutions, which were expressed in yeast (Saccharomyces cerevisiae) for FNS I and FHT assays. The exchange I131F in combination with either M106T and D195E or L215V and K216R replacements was sufficient to confer some FNS I side activity. Introduction of all seven FNS I substitutions into the FHT sequence, however, caused a nearly complete change in enzyme activity from FHT to FNS I. Both FHT and FNS I were proposed to initially withdraw the β-face-configured hydrogen from carbon-3 of the naringenin substrate. Our results suggest that the 7-fold substitution affects the orientation of the substrate in the active-site pocket such that this is followed by syn-elimination of hydrogen from carbon-2 (FNS I reaction) rather than the rebound hydroxylation of carbon-3 (FHT reaction). PMID:17535823

  13. Characterization and site-directed mutagenesis of a novel class II 5-enopyruvylshikimate-3-phosphate (EPSP) synthase from the deep-sea bacterium Alcanivorax sp. L27.

    PubMed

    Zhang, Yi; Yi, Licong; Lin, Yongjun; Zhang, Lili; Shao, Zongze; Liu, Ziduo

    2014-09-01

    The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants, which catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). In this study, a novel AroA-encoding gene was identified from the deep sea bacterium Alcanivorax sp. L27 through screening the genomic library and termed as AroAA.sp. A phylogenetic analysis revealed that AroAA.sp (1317 bp and 438 amino acids) is a class II AroA. This enzyme exhibited considerable activity between pH 5.5 and pH 8.0 and notable activity at low temperatures. The KM for PEP and IC50 [glyphosate] values (the concentration of glyphosate that inhibited enzyme activity by 50%) of AroAA.sp were 78 μM and 1.5 mM, respectively. Furthermore, site-directed mutagenesis revealed that the G100A mutant had a 30-fold increase in the IC50 [glyphosate] value; while the L105P mutant showed only 20% catalytic activity compared to wild-type AroAA.sp. The specific activity of the wild-type AroAA.sp, the G100A mutant and the L105P mutant were 7.78 U/mg, 7.26 U/mg and 1.76 U/mg, respectively. This is the first report showing that the G100A mutant of AroA displays considerably improved glyphosate resistance and demonstrates that Leu105 is essential for the enzyme's activity. PMID:25039062

  14. Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism*

    PubMed Central

    Rohman, Ali; van Oosterwijk, Niels; Thunnissen, Andy-Mark W. H.; Dijkstra, Bauke W.

    2013-01-01

    3-Ketosteroid Δ1-dehydrogenases are FAD-dependent enzymes that catalyze the 1,2-desaturation of 3-ketosteroid substrates to initiate degradation of the steroid nucleus. Here we report the 2.0 Å resolution crystal structure of the 56-kDa enzyme from Rhodococcus erythropolis SQ1 (Δ1-KSTD1). The enzyme contains two domains: an FAD-binding domain and a catalytic domain, between which the active site is situated as evidenced by the 2.3 Å resolution structure of Δ1-KSTD1 in complex with the reaction product 1,4-androstadiene-3,17-dione. The active site contains four key residues: Tyr119, Tyr318, Tyr487, and Gly491. Modeling of the substrate 4-androstene-3,17-dione at the position of the product revealed its interactions with these residues and the FAD. The C1 and C2 atoms of the substrate are at reaction distance to the N5 atom of the isoalloxazine ring of FAD and the hydroxyl group of Tyr318, respectively, whereas the C3 carbonyl group is at hydrogen bonding distance from the hydroxyl group of Tyr487 and the backbone amide of Gly491. Site-directed mutagenesis of the tyrosines to phenylalanines confirmed their importance for catalysis. The structural features and the kinetic properties of the mutants suggest a catalytic mechanism in which Tyr487 and Gly491 work in tandem to promote keto-enol tautomerization and increase the acidity of the C2 hydrogen atoms of the substrate. With assistance of Tyr119, the general base Tyr318 abstracts the axial β-hydrogen from C2 as a proton, whereas the FAD accepts the axial α-hydrogen from the C1 atom of the substrate as a hydride ion. PMID:24165124

  15. Site-Directed Mutagenesis from Arg195 to His of a Microalgal Putatively Chloroplastidial Glycerol-3-Phosphate Acyltransferase Causes an Increase in Phospholipid Levels in Yeast

    PubMed Central

    Ouyang, Long-Ling; Li, Hui; Yan, Xiao-Jun; Xu, Ji-Lin; Zhou, Zhi-Gang

    2016-01-01

    To analyze the contribution of glycerol-3-phosphate acyltransferase (GPAT) to the first acylation of glycerol-3-phosphate (G-3-P), the present study focused on a functional analysis of the GPAT gene from Lobosphaera incisa (designated as LiGPAT). A full-length cDNA of LiGPAT consisting of a 1,305-bp ORF, a 1,652-bp 5′-UTR, and a 354-bp 3′-UTR, was cloned. The ORF encoded a 434-amino acid peptide, of which 63 residues at the N-terminus defined a chloroplast transit peptide. Multiple sequence alignment and phylogeny analysis of GPAT homologs provided the convincible bioinformatics evidence that LiGPAT was localized to chloroplasts. Considering the conservation of His among the G-3-P binding sites from chloroplastidial GPATs and the substitution of His by Arg at position 195 in the LiGPAT mature protein (designated mLiGPAT), we established the heterologous expression of either mLiGPAT or its mutant (Arg195His) (sdmLiGPAT) in the GPAT-deficient yeast mutant gat1Δ. Lipid profile analyses of these transgenic yeasts not only validated the acylation function of LiGPAT but also indicated that the site-directed mutagenesis from Arg195 to His led to an increase in the phospholipid level in yeast. Semi-quantitative analysis of mLiGPAT and sdmLiGPAT, together with the structural superimposition of their G-3-P binding sites, indicated that the increased enzymatic activity was caused by the enlarged accessible surface of the phosphate group binding pocket when Arg195 was mutated to His. Thus, the potential of genetic manipulation of GPAT to increase the glycerolipid level in L. incisa and other microalgae would be of great interest. PMID:27014309

  16. Directed modification of the Aspergillus usamii β-mannanase to improve its substrate affinity by in silico design and site-directed mutagenesis.

    PubMed

    Li, Jianfang; Wei, Xihuan; Tang, Cunduo; Wang, Junqing; Zhao, Mei; Pang, Qingfeng; Wu, Minchen

    2014-04-01

    β-Mannanases (EC 3.2.1.78) can catalyze the cleavage of internal β-1,4-D-mannosidic linkages of mannan backbones, and they have found applications in food, feed, pulp and paper, oil, pharmaceutical and textile industries. Suitable amino acid substitution can promote access to the substrate-binding groove and maintain the substrate therein, which probably improves the substrate affinity and, thus, increases catalytic efficiency of the enzyme. In this study, to improve the substrate affinity of AuMan5A, a glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii, had its directed modification conducted by in silico design, and followed by site-directed mutagenesis. The mutant genes, Auman5A (Y111F) and Auman5A (Y115F), were constructed by megaprimer PCR, respectively. Then, Auman5A and its mutant genes were expressed in Pichia pastoris GS115 successfully. The specific activities of purified recombinant β-mannanases (reAuMan5A, reAuMan5A(Y111F) and reAuMan5A(Y115F)) towards locust bean gum were 152.5, 199.6 and 218.9 U mg(-1), respectively. The two mutants were found to be similar to reAuMan5A regarding temperature and pH characteristics. Nevertheless, the K m values of reAuMan5A(Y111F) and reAuMan5A(Y115F), towards guar gum, decreased to 2.95 ± 0.22 and 2.39 ± 0.33 mg ml(-1) from 4.49 ± 0.07 mg ml(-1) of reAuMan5A, which would make reAuMan5A(Y111F) and reAuMan5A(Y115F) promising candidates for industrial processes. Structural analysis showed that the two mutants increased their affinity by decreasing the steric conflicts with those more complicated substrates. The results suggested that subtle conformational modification in the substrate-binding groove could substantially alter the substrate affinity of AuMan5A. This study laid a solid foundation for the directed modification of substrate affinities of β-mannanases and other enzymes. PMID:24493565

  17. Site-Directed Mutagenesis of Human Cytosolic Sulfotransferase (SULT) 2B1b to Phospho-mimetic Ser348Asp Results in an Isoform With Increased Catalytic Activity

    PubMed Central

    Salman, Emily D.; He, Dongning; Runge-Morris, Melissa; Kocarek, Thomas A.; Falany, Charles N.

    2011-01-01

    Human SULT2B1b is distinct from other SULT isoforms due to the presence of unique amino (N)- and carboxy (C)-terminal peptides. Using site-directed mutagenesis, it was determined that phosphorylation of Ser348 was associated with nuclear localization. To investigate the effects of this phosphorylation of Ser348 on activity and cellular localization, an in silico molecular mimic was generated by mutating Ser348 to an Asp. The Asp residue mimics the shape and charge of a phospho-Ser and homology models of SULT2B1b-phospho-S348 and SULT2B1b-S348D suggest a similar significant structural rearrangement in the C-terminal peptide. To evaluate the functional consequences of this post-translational modification and predicted rearrangement, 6His-SULT2B1b-S348D was synthesized, expressed, purified and characterized. The 6His-SULT2B1b-S348D has a specific activity for DHEA sulfation ten-fold higher than recombinant 6His-SULT2B1b (209.6 and 21.8 pmol·min−1·mg−1, respectively). Similar to native SULT2B1b, gel filtration chromatography showed SULT2B1b-S348D was enzymatically active as a homodimer. Stability assays comparing SULT2B1b and SUL2B1b-S348 demonstrated that SULT2B1b is 60% less thermostable than SULT2B1b-348D. The increased stability and sulfation activity allowed for better characterization of the sulfation kinetics for putative substrates as well as the determination of dissociation constants that were difficult to obtain with wild-type (WT) 6His-SULT2B1b. The KDs for DHEA and PAPS binding to 6His-SULT2B1b-S348D were 650 ± 7 nM and 265 ± 4 nM, respectively, whereas KDs for binding of substrates to the WT enzyme could not be determined. Characterization of the molecular mimic SULT2B1b-S348D provides a better understanding for the role of the unique structure of SULT2B1b and its effect on sulfation activity, and has allowed for improved kinetic characterization of the SULT2B1b enzyme. PMID:21855633

  18. Exploration of the ligand binding site of the human 5-HT4 receptor by site-directed mutagenesis and molecular modeling

    PubMed Central

    Mialet, Jeanne; Dahmoune, Yamina; Lezoualc'h, Frank; Berque-Bestel, Isabelle; Eftekhari, Pierre; Hoebeke, Johan; Sicsic, Sames; Langlois, Michel; Fischmeister, Rodolphe

    2000-01-01

    Among the five human 5-HT4 (h5-HT4) receptor isoforms, the h5-HT4(a) receptor was studied with a particular emphasis on the molecular interactions involved in ligand binding. For this purpose, we used site-directed mutagenesis of the transmembrane domain. Twelve mutants were constructed with a special focus on the residue P4.53 of helix IV which substitutes in h5-HT4 receptors the highly conserved S residue among the rhodopsin family receptors. The mutated receptors were transiently expressed in COS-7 cells.Ligand binding or competition studies with two h5-HT4 receptor agonists, serotonin and ML10302 and two h5-HT4 receptor antagonists, [3H]-GR113808 and ML10375 were performed on wild type and mutant receptors. Functional activity of the receptors was evaluated by measuring the ability of serotonin to stimulate adenylyl cyclase.Ligand binding experiments revealed that [3H]-GR113808 did not bind to mutants P4.53A, S5.43A, F6.51A, Y7.43A and to double mutant F6.52V/N6.55L. On the other hand mutations D3.32N, S5.43A and Y7.43A appeared to promote a dramatic decrease of h5-HT4(a) receptor functional activity. From these studies, S5.43 and Y7.43 clearly emerged as common anchoring sites to antagonist [3H]-GR113808 and to serotonin.According to these results, we propose ligand-receptor complex models with serotonin and [3H]-GR113808. For serotonin, three interaction points were selected including ionic interaction with D3.32, a stabilizing interaction of this ion pair by Y7.43 and a hydrogen bond with S5.43. [3H]-GR113808 was also docked, based on the same type of interactions with S5.43 and D3.32: the proposed model suggested a possible role of P4.53 in helix IV structure allowing the involvement of a close hydrophobic residue, W4.50, in a hydrophobic pocket for hydrophobic interactions with the indole ring of [3H]-GR113808. PMID:10821780

  19. Site-directed mutagenesis substituting cysteine for serine in 2-Cys peroxiredoxin (2-Cys Prx A) of Arabidopsis thaliana effectively improves its peroxidase and chaperone functions

    PubMed Central

    Lee, Eun Mi; Lee, Seung Sik; Tripathi, Bhumi Nath; Jung, Hyun Suk; Cao, Guang Ping; Lee, Yuno; Singh, Sudhir; Hong, Sung Hyun; Lee, Keun Woo; Lee, Sang Yeol; Cho, Jae-Young; Chung, Byung Yeoup

    2015-01-01

    Background and Aims The 2-Cys peroxiredoxin (Prx) A protein of Arabidopsis thaliana performs the dual functions of a peroxidase and a molecular chaperone depending on its conformation and the metabolic conditions. However, the precise mechanism responsible for the functional switching of 2-Cys Prx A is poorly known. This study examines various serine-to-cysteine substitutions on α-helix regions of 2-Cys Prx A in Arabidopsis mutants and the effects they have on the dual function of the protein. Methods Various mutants of 2-Cys Prx A were generated by replacing serine (Ser) with cysteine (Cys) at different locations by site-directed mutagenesis. The mutants were then over-expressed in Escherichia coli. The purified protein was further analysed by size exclusion chromatography, polyacrylamide gel electrophoresis, circular dichroism spectroscopy and transmission electron microscopy (TEM) and image analysis. Peroxidase activity, molecular chaperone activity and hydrophobicity of the proteins were also determined. Molecular modelling analysis was performed in order to demonstrate the relationship between mutation positions and switching of 2-Cys Prx A activity. Key Results Replacement of Ser150 with Cys150 led to a marked increase in holdase chaperone and peroxidase activities of 2-Cys Prx A, which was associated with a change in the structure of an important domain of the protein. Molecular modelling demonstrated the relationship between mutation positions and the switching of 2-Cys Prx A activity. Examination of the α2 helix, dimer–dimer interface and C-term loop indicated that the peroxidase function is associated with a fully folded α2 helix and easy formation of a stable reduced decamer, while a more flexible C-term loop makes the chaperone function less likely. Conclusions Substitution of Cys for Ser at amino acid location 150 of the α-helix of 2-Cys Prx A regulates/enhances the dual enzymatic functions of the 2-Cys Prx A protein. If confirmed in planta, this

  20. Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron coordination sites.

    PubMed Central

    Jiang, H; Parales, R E; Lynch, N A; Gibson, D T

    1996-01-01

    The terminal oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISP(TOL)) that requires mononuclear iron for enzyme activity. Alignment of all available predicted amino acid sequences for the large (alpha) subunits of terminal oxygenases showed a conserved cluster of potential mononuclear iron-binding residues. These were between amino acids 210 and 230 in the alpha subunit (TodC1) of ISP(TOL). The conserved amino acids, Glu-214, Asp-219, Tyr-221, His-222, and His-228, were each independently replaced with an alanine residue by site-directed mutagenesis. Tyr-266 in TodC1, which has been suggested as an iron ligand, was treated in an identical manner. To assay toluene dioxygenase activity in the presence of TodC1 and its mutant forms, conditions for the reconstitution of wild-type ISP(TOL) activity from TodC1 and purified TodC2 (beta subunit) were developed and optimized. A mutation at Glu-214, Asp-219, His-222, or His-228 completely abolished toluene dioxygenase activity. TodC1 with an alanine substitution at either Tyr-221 or Tyr-266 retained partial enzyme activity (42 and 12%, respectively). In experiments with [14C]toluene, the two Tyr-->Ala mutations caused a reduction in the amount of Cis-[14C]-toluene dihydrodiol formed, whereas a mutation at Glu-214, Asp-219, His-222, or His-228 eliminated cis-toluene dihydrodiol formation. The expression level of all of the mutated TWO proteins was equivalent to that of wild-type TodC1 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. These results, in conjunction with the predicted amino acid sequences of 22 oxygenase components, suggest that the conserved motif Glu-X3-4,-Asp-X2-His-X4-5-His is critical for catalytic function and the glutamate, aspartate, and histidine residues may act as mononuclear iron ligands at the site of oxygen activation. PMID:8655491

  1. Molybdenum cofactor properties and [Fe-S] cluster coordination in Escherichia coli nitrate reductase A: investigation by site-directed mutagenesis of the conserved his-50 residue in the NarG subunit.

    PubMed

    Magalon, A; Asso, M; Guigliarelli, B; Rothery, R A; Bertrand, P; Giordano, G; Blasco, F

    1998-05-19

    Most of the molybdoenzymes contain, in the amino-terminal region of their catalytic subunits, a conserved Cys group that in some cases binds an [Fe-S] cluster. In dissimilatory nitrate reductases, the first Cys residue of this motif is replaced by a conserved His residue. Site-directed mutagenesis of this residue (His-50) was performed on the NarG subunit from Escherichia coli nitrate reductase A. The results obtained by EPR spectroscopy enable us to exclude the implication of this residue in [Fe-S] binding. Additionally, we showed that the His-50 residue does not coordinate the molybdenum atom, but its substitution by Cys or Ser introduces a perturbation of the hydrogen bonding network around the molybdenum cofactor. From potentiometric studies, it is proposed that the high-pH and the low-pH forms of the Mo(V) are both involved during the redox turnover of the enzyme. Perturbation of the Mo(V) pKV value might be responsible for the low activity reported in the His-50-Cys mutant enzyme. A catalytic model is proposed in which the protonation/deprotonation of the Mo(V) species is an essential step. Thus, one of the two protons involved in the catalytic cycle could be the one coupled to the molybdenum atom in the dissimilatory nitrate reductase of E. coli. PMID:9585550

  2. Effects of Site-Directed Mutagenesis of Escherichia coli Heat-Labile Enterotoxin on ADP-Ribosyltransferase Activity and Interaction with ADP-Ribosylation Factors

    PubMed Central

    A. Stevens, Linda; Moss, Joel; Vaughan, Martha; Pizza, Mariagrazia; Rappuoli, Rino

    1999-01-01

    Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsα, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction. PMID:9864224

  3. Identification of essential residues for binding and activation in the human 5-HT7(a) serotonin receptor by molecular modeling and site-directed mutagenesis.

    PubMed

    Impellizzeri, Agata Antonina Rita; Pappalardo, Matteo; Basile, Livia; Manfra, Ornella; Andressen, Kjetil Wessel; Krobert, Kurt Allen; Messina, Angela; Levy, Finn Olav; Guccione, Salvatore

    2015-01-01

    The human 5-HT7 receptor is expressed in both the central nervous system and peripheral tissues and is a potential drug target in behavioral and psychiatric disorders. We examined molecular determinants of ligand binding and G protein activation by the human 5-HT7(a) receptor. The role of several key residues in the 7th transmembrane domain (TMD) and helix 8 were elucidated combining in silico and experimental mutagenesis. Several single and two double point mutations of the 5-HT7(a) wild type receptor were made (W7.33V, E7.35T, E7.35R, E7.35D, E7.35A, R7.36V, Y7.43A, Y7.43F, Y7.43T, R8.52D, D8.53K; E7.35T-R7.36V, R8.52D-D8.53K), and their effects upon ligand binding were assessed by radioligand binding using a potent agonist (5-CT) and a potent antagonist (SB269970). In addition, the ability of the mutated 5-HT7(a) receptors to activate G protein after 5-HT-stimulation was determined through activation of adenylyl cyclase. In silico investigation on mutated receptors substantiated the predicted importance of TM7 and showed critical roles of residues E7.35, W7.33, R7.36 and Y7.43 in agonist and antagonist binding and conformational changes of receptor structure affecting adenylyl cyclase activation. Experimental data showed that mutants E7.35T and E7.35R were incapable of ligand binding and adenylyl cyclase activation, consistent with a requirement for a negatively charged residue at this position. The mutant R8.52D was unable to activate adenylyl cyclase, despite unaffected ligand binding, consistent with the R8.52 residue playing an important role in the receptor-G protein interface. The mutants Y7.43A and Y7.43T displayed reduced agonist binding and AC agonist potency, not seen in Y7.43F, consistent with a requirement for an aromatic residue at this position. Knowledge of the molecular interactions important in h5-HT7 receptor ligand binding and G protein activation will aid the design of selective h5-HT7 receptor ligands for potential pharmacological use. PMID

  4. Identification of essential residues for binding and activation in the human 5-HT7(a) serotonin receptor by molecular modeling and site-directed mutagenesis

    PubMed Central

    Impellizzeri, Agata Antonina Rita; Pappalardo, Matteo; Basile, Livia; Manfra, Ornella; Andressen, Kjetil Wessel; Krobert, Kurt Allen; Messina, Angela; Levy, Finn Olav; Guccione, Salvatore

    2015-01-01

    The human 5-HT7 receptor is expressed in both the central nervous system and peripheral tissues and is a potential drug target in behavioral and psychiatric disorders. We examined molecular determinants of ligand binding and G protein activation by the human 5-HT7(a) receptor. The role of several key residues in the 7th transmembrane domain (TMD) and helix 8 were elucidated combining in silico and experimental mutagenesis. Several single and two double point mutations of the 5-HT7(a) wild type receptor were made (W7.33V, E7.35T, E7.35R, E7.35D, E7.35A, R7.36V, Y7.43A, Y7.43F, Y7.43T, R8.52D, D8.53K; E7.35T-R7.36V, R8.52D-D8.53K), and their effects upon ligand binding were assessed by radioligand binding using a potent agonist (5-CT) and a potent antagonist (SB269970). In addition, the ability of the mutated 5-HT7(a) receptors to activate G protein after 5-HT-stimulation was determined through activation of adenylyl cyclase. In silico investigation on mutated receptors substantiated the predicted importance of TM7 and showed critical roles of residues E7.35, W7.33, R7.36 and Y7.43 in agonist and antagonist binding and conformational changes of receptor structure affecting adenylyl cyclase activation. Experimental data showed that mutants E7.35T and E7.35R were incapable of ligand binding and adenylyl cyclase activation, consistent with a requirement for a negatively charged residue at this position. The mutant R8.52D was unable to activate adenylyl cyclase, despite unaffected ligand binding, consistent with the R8.52 residue playing an important role in the receptor-G protein interface. The mutants Y7.43A and Y7.43T displayed reduced agonist binding and AC agonist potency, not seen in Y7.43F, consistent with a requirement for an aromatic residue at this position. Knowledge of the molecular interactions important in h5-HT7 receptor ligand binding and G protein activation will aid the design of selective h5-HT7 receptor ligands for potential pharmacological use. PMID

  5. Effects of site-directed mutagenesis of Escherichia coli heat-labile enterotoxin on ADP-ribosyltransferase activity and interaction with ADP-ribosylation factors.

    PubMed

    Stevens, L A; Moss, J; Vaughan, M; Pizza, M; Rappuoli, R

    1999-01-01

    Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsalpha, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction. PMID:9864224

  6. Using site-directed mutagenesis to probe the role of the D2 carotenoid in the secondary electron-transfer pathway of photosystem II.

    PubMed

    Shinopoulos, Katherine E; Yu, Jianfeng; Nixon, Peter J; Brudvig, Gary W

    2014-05-01

    Secondary electron transfer in photosystem II (PSII), which occurs when water oxidation is inhibited, involves redox-active carotenoids (Car), as well as chlorophylls (Chl), and cytochrome b 559 (Cyt b 559), and is believed to play a role in photoprotection. CarD2 may be the initial point of secondary electron transfer because it is the closest cofactor to both P680, the initial oxidant, and to Cyt b 559, the terminal secondary electron donor within PSII. In order to characterize the role of CarD2 and to determine the effects of perturbing CarD2 on both the electron-transfer events and on the identity of the redox-active cofactors, it is necessary to vary the properties of CarD2 selectively without affecting the ten other Car per PSII. To this end, site-directed mutations around the binding pocket of CarD2 (D2-G47W, D2-G47F, and D2-T50F) have been generated in Synechocystis sp. PCC 6803. Characterization by near-IR and EPR spectroscopy provides the first experimental evidence that CarD2 is one of the redox-active carotenoids in PSII. There is a specific perturbation of the Car(∙+) near-IR spectrum in all three mutated PSII samples, allowing the assignment of the spectral signature of Car D2 (∙+) ; Car D2 (∙+) exhibits a near-IR peak at 980 nm and is the predominant secondary donor oxidized in a charge separation at low temperature in ferricyanide-treated wild-type PSII. The yield of secondary donor radicals is substantially decreased in PSII complexes isolated from each mutant. In addition, the kinetics of radical formation are altered in the mutated PSII samples. These results are consistent with oxidation of CarD2 being the initial step in secondary electron transfer. Furthermore, normal light levels during mutant cell growth perturb the shape of the Chl(∙+) near-IR absorption peak and generate a dark-stable radical observable in the EPR spectra, indicating a higher susceptibility to photodamage further linking the secondary electron-transfer pathway to

  7. Testing a structural model for viral DNA packaging motor function by optical tweezers measurements, site directed mutagenesis, and molecular dynamics calculations

    NASA Astrophysics Data System (ADS)

    Keller, Nicholas A.; Migliori, Amy D.; Arya, Gaurav; Rao, Venigalla B.; Smith, Douglas E.

    2013-09-01

    Many double-stranded DNA viruses employ a molecular motor to package DNA into preformed capsid shells. Based on structures of phage T4 motor proteins determined by X-ray crystallography and cryo-electron microscopy, Rao, Rossmann and coworkers recently proposed a structural model for motor function. They proposed that DNA is ratcheted by a large conformational change driven by electrostatic interactions between charged residues at an interface between two globular domains of the motor protein. We have conducted experiments to test this model by studying the effect on packaging under applied load of site-directed changes altering these residues. We observe significant impairment of packaging activity including reductions in packaging rate, percent time packaging, and time active under high load. We show that these measured impairments correlate well with alterations in free energies associated with the conformational change predicted by molecular dynamics simulations.

  8. A QuikChange-like method to realize efficient blunt-ended DNA directional cloning and site-directed mutagenesis simultaneously.

    PubMed

    An, Yingfeng; Lv, Anguo; Wu, Wenfang

    2010-06-25

    Here we present a QuikChange-like method to efficiently realize blunt-ended DNA cloning and conveniently introduce a site-directed mutation to recombinant plasmid at the same time. After blunt-ended DNA ligation and transformation, the plasmid DNA mixture is extracted from pooled transformants and directly used as template for PCR amplification with a pair of complementary mutagenic primers. With this method, sam1 gene was inserted into pUC19 vector by blunt-end ligation, and a unique restriction site Spe I was introduced to the recombinant plasmid at the same time. The randomly selected transformants were analyzed by DNA sequencing, and most of the clones were found to have correct sequences. However, no correct construct was found from randomly selected transformants after traditional blunt-ended DNA ligation and transformation. PMID:20471367

  9. Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity.

    PubMed

    Novo, Carlos; Farnaud, Sebastien; Tata, Renée; Clemente, Alda; Brown, Paul R

    2002-08-01

    The aliphatic amidase from Pseudomonas aeruginosa belongs to the nitrilase superfamily, and Cys(166) is the nucleophile of the catalytic mechanism. A model of amidase was built by comparative modelling using the crystal structure of the worm nitrilase-fragile histidine triad fusion protein (NitFhit; Protein Data Bank accession number 1EMS) as a template. The amidase model predicted a catalytic triad (Cys-Glu-Lys) situated at the bottom of a pocket and identical with the presumptive catalytic triad of NitFhit. Three-dimensional models for other amidases belonging to the nitrilase superfamily also predicted Cys-Glu-Lys catalytic triads. Support for the structure for the P. aeruginosa amidase came from site-direct mutagenesis and from the locations of amino acid residues that altered substrate specificity or binding when mutated. PMID:11955282

  10. Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity.

    PubMed Central

    Novo, Carlos; Farnaud, Sebastien; Tata, Renée; Clemente, Alda; Brown, Paul R

    2002-01-01

    The aliphatic amidase from Pseudomonas aeruginosa belongs to the nitrilase superfamily, and Cys(166) is the nucleophile of the catalytic mechanism. A model of amidase was built by comparative modelling using the crystal structure of the worm nitrilase-fragile histidine triad fusion protein (NitFhit; Protein Data Bank accession number 1EMS) as a template. The amidase model predicted a catalytic triad (Cys-Glu-Lys) situated at the bottom of a pocket and identical with the presumptive catalytic triad of NitFhit. Three-dimensional models for other amidases belonging to the nitrilase superfamily also predicted Cys-Glu-Lys catalytic triads. Support for the structure for the P. aeruginosa amidase came from site-direct mutagenesis and from the locations of amino acid residues that altered substrate specificity or binding when mutated. PMID:11955282

  11. Site-directed mutagenesis of dicarboxylic acids near the active site of Bacillus cereus 5/B/6 beta-lactamase II.

    PubMed Central

    Lim, H M; Iyer, R K; Pène, J J

    1991-01-01

    An amino acid residue functioning as a general base has been proposed to assist in the hydrolysis of beta-lactam antibiotics by the zinc-containing Bacillus cereus beta-lactamase II [Bicknell & Waley (1985) Biochemistry 24, 6876-6887]. Oligonucleotide-directed mutagenesis of cloned Bacillus cereus 5/B/6 beta-lactamase II was used in an 'in vivo' study to investigate the role of carboxy-group-containing amino acids near the active site of the enzyme. Substitution of asparagine for the wild-type aspartic acid residue at position 81 resulted in fully functional enzyme. An aspartic acid residue at position 90 is essential for beta-lactamase II to confer any detectable ampicillin and cephalosporin C resistance to Escherichia coli. Conversion of Asp90 into Asn90 or Glu90 lead to the synthesis of inactive enzyme, suggesting that the spatial position of the beta-carboxy group of Asp90 is critical for enzyme function. Images Fig. 2. Fig. 3. PMID:1904717

  12. Site-directed mutagenesis of the Klebsiella pneumoniae nifL and nifH promoters and in vivo analysis of promoter activity.

    PubMed Central

    Buck, M; Khan, H; Dixon, R

    1985-01-01

    The role of conserved nucleotides in nitrogen-fixation promoter function has been examined using both oligonucleotide and chemical mutagenesis to introduce base changes in the Klebsiella pneumoniae nifL and nifH promoters. Among ten mutations analysed, including six spontaneous mutations, base changes at -12, -13, -14, and -26, located in previously identified conserved sequences, perturbed the activity of the promoters, demonstrating that these sequences are required for transcription. Not all base changes produced similar strong promoter down phenotypes when the nifL and nifH promoters were compared: activation of the nifH promoter by the nifA gene product was less sensitive to base changes in conserved nucleotides than was activation of the equivalently altered nifL promoter by the nifA or ntrC products. We have found that the nifH promoter can be weakly activated by the ntrC product; this activation shows the same down response to base changes seen with ntrC activation of the nifL promoter. We present evidence that the efficient activation of the nifH promoter by nifA (but not ntrC) can be attributed to specific upstream sequences present in the nifH promoter. PMID:3906564

  13. 3D Structure of Sulfolobus solfataricus Carboxypeptidase Developed by Molecular Modeling is Confirmed by Site-Directed Mutagenesis and Small Angle X-Ray Scattering

    PubMed Central

    Occhipinti, Emanuela; Martelli, Pier Luigi; Spinozzi, Francesco; Corsi, Federica; Formantici, Cristina; Molteni, Laura; Amenitsch, Heintz; Mariani, Paolo; Tortora, Paolo; Casadio, Rita

    2003-01-01

    Sulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme with a Mr of 43,000. Taking into account the experimentally determined zinc content of one ion per subunit, we developed two alternative 3D models, starting from the available structures of Thermoactinomyces vulgaris carboxypeptidase (Model A) and Pseudomonas carboxypeptidase G2 (Model B). The former enzyme is monomeric and has one metal ion in the active site, while the latter is dimeric and has two bound zinc ions. The two models were computed by exploiting the structural alignment of the one zinc- with the two zinc-containing active sites of the two templates, and with a threading procedure. Both computed structures resembled the respective template, with only one bound zinc with tetrahedric coordination in the active site. With these models, two different quaternary structures can be modeled: one using Model A with a hexameric symmetry, the other from Model B with a tetrameric symmetry. Mutagenesis experiments directed toward the residues putatively involved in metal chelation in either of the models disproved Model A and supported Model B, in which the metal-binding site comprises His108, Asp109, and His168. We also identified Glu142 as the acidic residue interacting with the water molecule occupying the fourth chelation site. Furthermore, the overall fold and the oligomeric structure of the molecule was validated by small angle x-ray scattering (SAXS). An ab initio original approach was used to reconstruct the shape of the CPSso in solution from the experimental curves. The results clearly support a tetrameric structure. The Monte Carlo method was then used to compare the crystallographic coordinates of the possible quaternary structures for CPSso with the SAXS profiles. The fitting procedure showed that only the model built using the Pseudomonas carboxypeptidase G2 structure as a template fitted the experimental data. PMID:12885660

  14. Development of a Homologous Expression System for and Systematic Site-Directed Mutagenesis Analysis of Thurincin H, a Bacteriocin Produced by Bacillus thuringiensis SF361

    PubMed Central

    Wang, Gaoyan; Manns, David C.; Churey, John J.

    2014-01-01

    Thurincin H is an antimicrobial peptide produced by Bacillus thuringiensis SF361. With a helical back bone, the 31 amino acids of thurincin H form a hairpin structure maintained by four pairs of very unique sulfur-to-α-carbon thioether bonds. The production of thurincin H depends on a putative gene cluster containing 10 open reading frames. The gene cluster includes three tandem structural genes (thnA1, thnA2, and thnA3) encoding three identical 40-amino-acid thurincin H prepeptides and seven other genes putatively responsible for prepeptide processing, regulation, modification, exportation, and self-immunity. A homologous thurincin H expression system was developed by transforming a thurincin H-deficient host with a novel expression vector, pGW133. The host, designated B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3, was constructed by deletion of the three tandem structural genes from the chromosome of the natural thurincin H producer. The thurincin H expression vector pGW133 was constructed by cloning the thurincin H native promoter, thnA1, and a Cry protein terminator into the Escherichia coli-B. thuringiensis shuttle vector pHT315. Thirty-three different pGW133 variants, each containing a different point mutation in the thnA1 gene, were generated and separately transformed into B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3. Those site-directed mutants contained either a single radical or conservative amino acid substitution on the thioether linkage-forming positions or a radical substitution on all other nonalanine amino acids. The bacteriocin activities of B. thuringiensis SF361 ΔthnA1 ΔthnA2 ΔthnA3 carrying different pGW133 variants against three different indicator strains were subsequently compared. PMID:24682301

  15. Expression and site-directed mutagenesis of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus: probing the role of the active site Glu146.

    PubMed

    Martin, S F; Spaller, M R; Hergenrother, P J

    1996-10-01

    A series of site-specific mutants of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) was prepared in which the glutamic acid residue at position 146 was replaced with glutamine, aspartic acid, histidine, and leucine to elucidate what role Glu146 might play in catalysis. An expression system for the native enzyme in Escherichia coli was first developed to provide PLCBc that was fused via an intervening factor Xa protease recognition sequence at its N-terminus to maltose binding protein (MBP). This MBP-PLCBc fusion protein was isolated at levels of 50-70 mg/L of culture; selective trypsin digestion of the MBP-PLCBc fusion protein followed by chromatographic purification yielded recombinant PLCBc at levels of ca. 10 mg/L. Polymerase chain reaction (PCR) mutagenesis on the PLCBc gene (plc) was then used to replace the Glu146 codon with those for glutamine (E146Q), aspartic acid (E146D), histidine (E146H), and leucine (E146L). The catalytic efficiency of the E146Q mutant was 1.6% that of native PLCBc, while the other mutants each possessed activities of 0.2-0.3% of the wild type. The kcat/Km vs pH profiles for both E146Q and native PLCBc have ascending acidic limbs, suggesting that Glu146 does not serve as the general base in the hydrolysis reaction. As measured by circular dichroism, all of the mutant proteins contained less helical structure and underwent denaturation at lower temperatures than the wild type in the order: wild type > E146Q > E146D approximately E146H approximately E146L. Atomic absorption analyses indicated that the mutant proteins also exhibited lower Zn2+ content than the wild type. Thus, the Glu146 residue in PLCBc stabilizes the secondary and tertiary structure of the enzyme and serves as a critical ligand for Zn2, but it does not appear to have any specific catalytic role. PMID:8841144

  16. Site-directed mutagenesis of the greasy slide aromatic residues within the LamB (maltoporin) channel of Escherichia coli: effect on ion and maltopentaose transport.

    PubMed

    Denker, Katrin; Orlik, Frank; Schiffler, Bettina; Benz, Roland

    2005-09-23

    The 3D-structure of the maltooligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) is known from X-ray crystallography. The 3D structure suggests that a number of aromatic residues (Y6, Y41, W74, F229, W358 and W420) within the channel lumen are involved in carbohydrate and ion transport. All aromatic residues were replaced by alanine-scanning mutagenesis. Furthermore, LamB mutants were created in which two, three, four, five and all six aromatic residues were replaced to study their effects on ion and maltopentaose transport through LamB. The purified mutant proteins were reconstituted into lipid bilayer membranes and the single-channel conductance of the mutants was studied in conductance experiments. The results suggest that all aromatic residues provide some steric hindrance for ion transport through LamB. Highest impact is provided by Y6 and Y41 that are localized opposite Y118, which form the central constriction of the LamB channel. Stability constants for binding of maltopentaose to the mutant channels were measured using titration experiments with the carbohydrate. The mutation of one or several aromatic residue(s) led to a substantial decrease of the stability constant of binding. The highest effect was observed when all aromatic residues were replaced by alanine because no binding of maltopentaose could be detected in such a case. However, binding was again possible when Y118 was replaced by tryptophan. The carbohydrate-induced block of the channel function could be used also for the study of current noise through the different mutant LamB-channels. The analysis of the power density spectra of some of the mutants allowed the evaluation of the on-rate and off-rate constants (k1 and k(-1)) of carbohydrate binding to the binding site inside the channels. The results suggest that both on-rate and off-rate constants were affected by the mutations. For most mutants, k1 decreased and k(-1) increased. The possible influence of the

  17. Increased phosphate transport of Arabidopsis thaliana Pht1;1 by site-directed mutagenesis of tyrosine 312 may be attributed to the disruption of homomeric interactions.

    PubMed

    Fontenot, Elena B; Ditusa, Sandra Feuer; Kato, Naohiro; Olivier, Danielle M; Dale, Renee; Lin, Wei-Yi; Chiou, Tzyy-Jen; Macnaughtan, Megan A; Smith, Aaron P

    2015-10-01

    Members of the Pht1 family of plant phosphate (Pi) transporters play vital roles in Pi acquisition from soil and in planta Pi translocation to maintain optimal growth and development. The study of the specificities and biochemical properties of Pht1 transporters will contribute to improving the current understanding of plant phosphorus homeostasis and use-efficiency. In this study, we show through split in vivo interaction methods and in vitro analysis of microsomal root tissues that Arabidopsis thaliana Pht1;1 and Pht1;4 form homomeric and heteromeric complexes. Transient and heterologous expression of the Pht1;1 variants, Pht1;1(Y312D), Pht1;1(Y312A) and Pht1;1(Y312F), was used to analyse the role of a putative Pi binding residue (Tyr 312) in Pht1;1 transporter oligomerization and function. The homomeric interaction among Pht1;1 proteins was disrupted by mutation of Tyr 312 to Asp, but not to Ala or Phe. In addition, the Pht1;1(Y312D) variant conferred enhanced Pi transport when expressed in yeast cells. In contrast, mutation of Tyr 312 to Ala or Phe did not affect Pht1;1 transport kinetics. Our study demonstrates that modifications to the Pht1;1 higher-order structure affects Pi transport, suggesting that oligomerization may serve as a regulatory mechanism for modulating Pi uptake. PMID:25754174

  18. SELEX_DB: a database on in vitro selected oligomers adapted for recognizing natural sites and for analyzing both SNPs and site-directed mutagenesis data.

    PubMed

    Ponomarenko, Julia V; Orlova, Galina V; Frolov, Anatoly S; Gelfand, Mikhail S; Ponomarenko, Mikhail P

    2002-01-01

    SELEX_DB is an online resource containing both the experimental data on in vitro selected DNA/RNA oligomers (aptamers) and the applets for recognition of these oligomers. Since in vitro experimental data are evidently system-dependent, the new release of the SELEX_DB has been supplemented by the database SYSTEM storing the experimental design. In addition, the recognition applet package, SELEX_TOOLS, applying in vitro selected data to annotation of the genome DNA, is accompanied by the cross-validation test database CROSS_TEST discriminating the sites (natural or other) related to in vitro selected sites out of random DNA. By cross-validation testing, we have unexpectedly observed that the recognition accuracy increases with the growth of homology between the training and test sets of protein binding sequences. For natural sites, the recognition accuracy was lower than that for the nearest protein homologs and higher than that for distant homologs and non-homologous proteins binding the common site. The current SELEX_DB release is available at http://wwwmgs.bionet.nsc.ru/mgs/systems/selex/. PMID:11752291

  19. Molecular cloning, homology modeling and site-directed mutagenesis of vanadium-dependent bromoperoxidase (GcVBPO1) from Gracilaria changii (Rhodophyta).

    PubMed

    Baharum, H; Chu, W-C; Teo, S-S; Ng, K-Y; Rahim, R Abdul; Ho, C-L

    2013-08-01

    Vanadium-dependent haloperoxidases belong to a class of vanadium enzymes that may have potential industrial and pharmaceutical applications due to their high stability. In this study, the 5'-flanking genomic sequence and complete reading frame encoding vanadium-dependent bromoperoxidase (GcVBPO1) was cloned from the red seaweed, Fracilaria changii, and the recombinant protein was biochemically characterized. The deduced amino acid sequence of GcVBPO1 is 1818 nucleotides in length, sharing 49% identity with the vanadium-dependent bromoperoxidases from Corralina officinalis and Cor. pilulifera, respectively. The amino acid residues associated with the binding site of vanadate cofactor were found to be conserved. The Km value of recombinant GcVBPO1 for Br(-) was 4.69 mM, while its Vmax was 10.61 μkat mg(-1) at pH 7. Substitution of Arg(379) with His(379) in the recombinant protein caused a lower affinity for Br(-), while substitution of Arg(379) with Phe(379) not only increased its affinity for Br(-) but also enabled the mutant enzyme to oxidize Cl(-). The mutant Arg(379)Phe was also found to have a lower affinity for I(-), as compared to the wild-type GcVBPO1 and mutant Arg(379)His. In addition, the Arg(379)Phe mutant has a slightly higher affinity for H2O2 compared to the wild-type GcVBPO1. Multiple cis-acting regulatory elements associated with light response, hormone signaling, and meristem expression were detected at the 5'-flanking genomic sequence of GcVBPO1. The transcript abundance of GcVBPO1 was relatively higher in seaweed samples treated with 50 parts per thousand (ppt) artificial seawater (ASW) compared to those treated in 10 and 30 ppt ASW, in support of its role in the abiotic stress response of seaweed. PMID:23684235

  20. Site-directed mutagenesis of Escherichia coli acetylglutamate kinase and aspartokinase III probes the catalytic and substrate-binding mechanisms of these amino acid kinase family enzymes and allows three-dimensional modelling of aspartokinase.

    PubMed

    Marco-Marín, Clara; Ramón-Maiques, Santiago; Tavárez, Sandra; Rubio, Vicente

    2003-11-28

    We test, using site-directed mutagenesis, predictions based on the X-ray structure of N-acetyl-L-glutamate kinase (NAGK), the paradigm of the amino acid kinase protein family, about the roles of specific residues on substrate binding and catalysis. The mutations K8R and D162E decreased V([sustrate]= infinity ) 100-fold and 1000-fold, respectively, in agreement with the predictions that K8 catalyzes phosphoryl transfer and D162 organizes the catalytic groups. R66K and N158Q increased selectively K(m)(Asp) three to four orders of magnitude, in agreement with the binding of R66 and N158 to the C(alpha) substituents of NAG. Mutagenesis in parallel of aspartokinase III (AKIII phosphorylates aspartate instead of acetylglutamate), another important amino acid kinase family member of unknown 3-D structure, identified in AKIII two residues, K8 and D202, that appear to play roles similar to those of K8 and D162 of NAGK, and supports the involvement of E119 and R198, similarly to R66 and N158 of NAGK, in the binding of the amino acid substrate, apparently interacting, respectively, with the alpha-NH(3)(+) and alpha-COO(-) of aspartate. These results and an improved alignment of the NAGK and AKIII sequences have guided us into 3-D modelling of the amino acid kinase domain of AKIII using NAGK as template. The model has good stereochemistry and validation parameters. It provides insight into substrate binding and catalysis, agreeing with mutagenesis results with another aspartokinase that were not considered when building the model.AKIII is homodimeric and is inhibited by lysine. Lysine may bind to a regulatory region that is C-terminal to the amino acid kinase domain. We make a C-terminally truncated AKIII (AKIIIt) and show that the C-region is involved in intersubunit interactions, since AKIIIt is found to be monomeric. Further, it is inactive, as demanded if dimer formation is essential for activity. Models for AKIII architecture are proposed that account for these findings

  1. Site-directed mutagenesis reveals a conservation of the copper-binding site and the crucial role of His24 in CopH from Cupriavidus metallidurans CH34.

    PubMed

    Sendra, Véronique; Gambarelli, Serge; Bersch, Beate; Covès, Jacques

    2009-12-01

    CopH is a periplasmic copper-binding protein from Cupriavidus metallidurans CH34 that contains two histidine residues. Both His24 and His26 contribute to the formation of two high-affinity copper-binding sites in wild-type CopH and are likely involved in a 2N2O coordination sphere in the equatorial plane. We have used site-directed mutagenesis, and a series of spectroscopic and calorimetric studies to further characterize the copper-binding sites in CopH. While His24 plays a predominant role in copper affinity, one Cu-binding site was lost when either histidine residue was mutated. However, as shown by NMR and EPR, the mutation of the His residues does not affect the structural organization of the Cu-binding site nor the number of nitrogen ligands involved in copper ligation. In the absence of structural data, we propose a model that conciliates most of the spectroscopic data recorded during this study. PMID:19857899

  2. Expression of Bacillus protease (Protease BYA) from Bacillus sp. Y in Bacillus subtilis and enhancement of its specific activity by site-directed mutagenesis-improvement in productivity of detergent enzyme-.

    PubMed

    Tobe, Seiichi; Shimogaki, Hisao; Ohdera, Motoyasu; Asai, Yoshio; Oba, Kenkichi; Iwama, Masanori; Irie, Masachika

    2006-01-01

    An attempt was made to express protease BYA produced by an alkalophilic Bacillus sp. Y in Bacillus subtilis by gene engineering methods. The gene encoding protease BYA was cloned from Bacillus sp. Y, and expression vector pTA71 was constructed from the amylase promoter of Bacillus licheniformis, DNA fragments encoding the open reading frame of protease BYA, and pUB110. Protease BYA was secreted at an activity level of 5100 APU/ml in the common industrial culture medium of Bacillus subtilis transformed with pTA71. We then attempted to increase the specific activity of protease BYA by site-directed mutagenesis. Amino acid residue Ala29 next to catalytic Asp30 was replaced by one of three uncharged amino acid residues (Val29, Leu29, Ile29), and each mutant enzyme was expressed and isolated from the culture medium. Val29 mutant enzyme was secreted at an activity level of greater than 7000 APU/ml in culture medium, and its specific activity was 1.5-fold higher than that of the wild-type enzyme. Other mutant enzymes had specific activity similar to that of the original one and were less stabile than the wild-type enzyme. It can be thought that the substitution at amino acid residue 29 affects the level of activity and stability of protease BYA. PMID:16394504

  3. Methanol:coenzyme M methyltransferase from Methanosarcina barkeri--identification of the active-site histidine in the corrinoid-harboring subunit MtaC by site-directed mutagenesis.

    PubMed

    Sauer, K; Thauer, R K

    1998-05-01

    The enzyme system catalyzing the formation of methyl-coenzyme M from methanol and coenzyme M in Methanosarcina barkeri is composed of the three different polypeptides MtaA, MtaB and MtaC of which MtaC harbors a corrinoid prosthetic group. The heterologous expression of mtaA and mtaB in Escherichia coli has been described previously. We report here on the overproduction of the apoprotein of MtaC in E. coli, on its reconstitution to the active holoprotein with either cob(II)alamin or methyl-cob(III)alamin, and on the properties of the reconstituted corrinoid protein. Reconstituted MtaC was found to contain 1 mol bound cobamide/mol. EPR spectroscopic evidence is presented for a His residue as an axial ligand to Co2+ of the bound corrinoid. This active-site His was identified by site-directed mutagenesis as His136 in the MtaC sequence that contains four His residues. The reconstituted MtaC, in the cob(I)amide oxidation state, was methylated with methanol in the presence of MtaB and demethylated with coenzyme M in the presence of MtaA. In the presence of both MtaB and MtaA, methyl-coenzyme M was formed from methanol and coenzyme M at specific rates comparable to those determined for the enzyme system purified from M. barkeri. M. barkeri contains an isoenzyme of MtaA designated MtbA. The isoenzyme reacted with MtaC with only 2.5% of the activity of MtaA. PMID:9654068

  4. Expression of recombinant human serum amyloid A in mammalian cells and demonstration of the region necessary for high-density lipoprotein binding and amyloid fibril formation by site-directed mutagenesis.

    PubMed Central

    Patel, H; Bramall, J; Waters, H; De Beer, M C; Woo, P

    1996-01-01

    Site-directed mutagenesis of the acute-phase human serum amyloid A (SAA1 alpha) protein was used to evaluate the importance of the N-terminal amino acid residues, namely RSFFSFLGEAF The full-length cDNA clone of SAA1 alpha (pA1.mod.) was used to create two mutations, namely Gly-8 to Asp-8 and an 11 amino acid truncation between Arg-1 and Phe-11 respectively. Wild-type and mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells under the control of the human cytomegalovirus promoter, which resulted in the secretion of the processed proteins into the culture media. Wild-type recombinant human SAA (rSAA) protein was shown to have pI values of 6.0 and 6.4, similar to the human SAA isoform SAA1 alpha and SAA1 alpha desArg found in acute-phase plasma. N-terminal sequencing of 56 residues confirmed its identity with human SAA1 alpha. The total yield of wild-type rSAA measured by ELISA was between 3.5 and 30 mg/l. The two mutations resulted in reduced expression levels of the mutant SAA proteins (3-10 mg/l). Further measurements of rSAA concentration in lipid fractions of culture medium collected at a density of 1.21 g/ml (high-density liporotein; HDL) and 1.063-1.18 g/ml (very-low-density lipoprotein/low-density lipoprotein; VLDL/LDL) showed that 76% of the wild-type protein was found in the HDL fraction and the remaining 24% in the infranatant non-lipid fraction. In contrast the relative concentration of mutant rSAA in HDL and infranatant fractions was reversed. This is consistent with the previously proposed involvement of the 11 amino acid peptide in anchoring. SAA protein on to HDL3 [Turnell, Sarra, Glover, Baum, Caspi, Baltz and Pepys (1986) Mol. Biol. Med. 3, 387-407]. Wild-type rSAA protein was shown to from amyloid fibrils in vitro under acidic conditions as shown by electron microscopy, and stained positive with Congo Red and exhibited apple-green birefringence when viewed under polarized light. Under the same conditions mutSAA(G8D) and mutSAA delta 1

  5. Study of the roles of Arg-104 and Arg-225 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis.

    PubMed Central

    Rider, M H; Crepin, K M; De Cloedt, M; Bertrand, L; Vertommen, D; Hue, L

    1995-01-01

    The roles of Arg-104 and Arg-225 located in the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) have been studied by site-directed mutagenesis. In recombinant rat liver PFK-2/FBPase-2, mutation of Arg-225 to Ser increased the Km of PFK-2 for fructose-6-phosphate (Fru-6-P) 7-fold at pH 6 and decreased PFK-2 activity at suboptimal substrate concentrations between pH 6 and 9.5. The mutation had no effect on the Vmax of PFK-2 or on the Km of PFK-2 for MgATP. The mutation also increased the Vmax. of FBPase-2 4-fold without changing the Km for Fru-2,6-P2 or IC50 of Fru-6-P. These findings are in agreement with a previous study [Rider and Hue (1992) Eur. J. Biochem. 207, 967-972] on the protection by Fru-6-P of the labelling of Arg-225 by phenylglyoxal, and suggest that Arg-225 participates in Fru-6-P binding. In recombinant rat muscle PFK-2/FBPase-2, mutation of Arg-104 to Ser increased the Km for Fru-6-P 60-fold, increased the IC50 of citrate, increased the Vmax. 1.5-3-fold at pH 8.5 and altered the pH profile of PFK-2 activity. It did not affect the Km of PFK-2 for MgATP. The mutation also decreased the Vmax. of FBPase-2 3-fold, increased the Km for Fru-2,6-P2 70-fold and increased the IC50 of Fru-6-P at least 300-fold. Although the dimeric structure was maintained in the mutant, its PFK-2 activity was more sensitive towards inactivation by guanidinium chloride than the wild-type enzyme activity. The findings indicate that Arg-104 is involved in Fru-6-P binding in the PFK-2 domain and that it might also bind citrate. Structural changes resulting from the mutation might be responsible for the changes in kinetic properties of FBPase-2. PMID:7619077

  6. Structure-function relationships in the Na,K-ATPase. cap alpha. subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

    SciTech Connect

    Price, E.M.; Lingrel, J.B.

    1988-11-01

    Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the ..cap alpha..1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat ..cap alpha..1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase ..cap alpha.. subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep ..cap alpha..1 subunit was changed to that of the rat. When expressed in HeLa cells, this mutated sheep ..cap alpha..1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep ..cap alpha..1 cDNA containing only two amino acid substitutions. The resistant cells, whether transfected with the rat ..cap alpha..1 cDNA, the rat/sheep chimera, or the mutant sheep ..cap alpha..1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, /sup 86/Rb/sup +/ uptake, and Na,K-ATPase activity. These results demonstrate that the presence of arginine and aspartic acid on the amino end and carboxyl end, respectively, of the H1-H2 extracellular domain of the Na,K-ATPase ..cap alpha.. subunit together is responsible for the ouabain-resistant character of the rat enzyme and the corresponding residues in the sheep ..cap alpha..1 subunit (glutamine and asparagine) are somehow involved in ouabain binding.

  7. Probing the Effect of the Non-Active-Site Mutation Y229W in New Delhi Metallo-β-lactamase-1 by Site-Directed Mutagenesis, Kinetic Studies, and Molecular Dynamics Simulations

    PubMed Central

    Shi, Yun; Hu, Feng; Lao, Xingzhen; Gao, Xiangdong; Zheng, Heng; Yao, Wenbing

    2013-01-01

    New Delhi metallo-β-lactmase-1 (NDM-1) has attracted extensive attention for its high catalytic activities of hydrolyzing almost all β-lactam antibiotics. NDM-1 shows relatively higher similarity to subclass B1 metallo-β-lactmases (MβLs), but its residue at position 229 is identical to that of B2/B3 MβLs, which is a Tyr instead of a B1-MβL-conserved Trp. To elucidate the possible role of Y229 in the bioactivity of NDM-1, we performed mutagenesis study and molecular dynamics (MD) simulations. Although residue Y229 is spatially distant from the active site and not contacting directly with the substrate or zinc ions, the Y229W mutant was found to have higher kcat and Km values than those of wild-type NDM-1, resulting in 1∼7 fold increases in kcat/Km values against tested antibiotics. In addition, our MD simulations illustrated the enhanced flexibility of Loop 2 upon Y229W mutation, which could increase the kinetics of both substrate entrance (kon) and product egress (koff). The enhanced flexibility of Loop 2 might allow the enzyme to adjust the geometry of its active site to accommodate substrates with different structures, broadening its substrate spectrum. This study indicated the possible role of the residue at position 229 in the evolution of NDM-1. PMID:24339993

  8. Mutagenesis protocols in Saccharomyces cerevisiae by in vivo overlap extension.

    PubMed

    Alcalde, Miguel

    2010-01-01

    A high recombination frequency and its ease of manipulation has made Saccharomyces cerevisiae a unique model eukaryotic organism to study homologous recombination. Indeed, the well-developed recombination machinery in S. cerevisiae facilitates the construction of mutant libraries for directed evolution experiments. In this context, in vivo overlap extension (IVOE) is a particularly attractive protocol that takes advantage of the eukaryotic apparatus to carry out combinatorial saturation mutagenesis, site-directed recombination or site-directed mutagenesis, avoiding ligation steps and additional PCR reactions that are common to standard in vitro protocols. PMID:20676972

  9. Specific mutagenesis of a chlorophyll-binding protein. Progress report.

    SciTech Connect

    Eaton-Rye, Dr., Julian; Shen, Gaozhong

    1990-01-01

    During the first phase of the project regarding specific mutagenesis of the chlorophyll-binding protein CP47 in photosystem II (PS II) most of the time has been devoted to (1) establishment of an optimal procedure for the reintroduction of psbB (the gene encoding CP47) carrying a site-directed mutation into the experimental organism, the cyanobacterium Synechocystis sp. PCC 6803, (2) preparations for site-directed mutagenesis, and (3) creation and analysis of chimaeric spinach/cyanobacterial CP47 mutants of Synechocystis. In the coming year, psbB constructs with site-directed mutations in potential chlorophyll-binding regions of CP47 will be introduced into the Synechocystis genome, and site-directed mutants will be characterized according to procedures described in the original project description. In addition, analysis of chimaeric CP47 mutants will be continued.

  10. The maturation mechanism of γ-glutamyl transpeptidases: Insights from the crystal structure of a precursor mimic of the enzyme from Bacillus licheniformis and from site-directed mutagenesis studies.

    PubMed

    Pica, Andrea; Chi, Meng-Chun; Chen, Yi-Yu; d'Ischia, Marco; Lin, Long-Liu; Merlino, Antonello

    2016-02-01

    γ-Glutamyl transpeptidases (γ-GTs) are members of N-terminal nucleophile hydrolase superfamily. They are synthetized as single-chain precursors, which are then cleaved to form mature enzymes. Basic aspects of autocatalytic processing of these pro-enzymes are still unknown. Here we describe the X-ray structure of the precursor mimic of Bacillus licheniformis γ-GT (BlGT), obtained by mutating catalytically important threonine to alanine (T399A-BlGT), and report results of autoprocessing of mutants of His401, Thr415, Thr417, Glu419 and Arg571. Data suggest that Thr417 is in a competent position to activate the catalytic threonine (Thr399) for nucleophilic attack of the scissile peptide bond and that Thr415 plays a major role in assisting the process. On the basis of these new structural results, a possible mechanism of autoprocessing is proposed. This mechanism, which guesses the existence of a six-membered transition state involving one carbonyl and two hydroxyl groups, is in agreement with all the available experimental data collected on γ-GTs from different species and with our new Ala-scanning mutagenesis data. PMID:26536828

  11. Site-directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of Rab7b paralogue in the model eukaryote Paramecium octaurelia.

    PubMed

    Wyroba, E; Kwaśniak, P; Miller, K; Kobyłecki, K; Osińska, M

    2016-01-01

    Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue - distinct from that of Rab7a directly involved in phagocytosis - was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala200 resulted in diminished incorporation of [P32] by 37.4% and of 32 [C14-]UDP-glucose by 24%, respectively, into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells contrary to non- mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.63+ characteristic for the glycan group attached to Thr200 in Rab7b using nano LC-MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P32]and  [C14-]UDP- glucose, the suggested composition of the adduct attached to Thr200 might be (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Rab7b in Paramecium is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a. PMID:27349314

  12. Site-Directed Mutagenesis, in Vivo Electroporation and Mass Spectrometry in Search for Determinants of the Subcellular Targeting of Rab7b Paralogue in the Model Eukaryote Paramecium Octaurelia

    PubMed Central

    Wyroba, E.; Kwaśniak, P.; Miller, K.; Kobyłecki, K.; Osińska, M.

    2016-01-01

    Protein products of paralogous genes resulting from whole genome duplication may acquire new functions. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue (distinct from that of Rab7a directly involved in phagocytosis) was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala diminished the incorporation of [P32] by 37% and of [C14-]UDP-glucose by 24% into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells in contrast to non-mutagenized recombinant Rab7b correctly incorporated in the cytostome area. Using nano LC-MS/MS to compare the peptide map of Rab7b with that after deglycosylation with a mixture of five enzymes of different specificity we identified a peptide ion at m/z=677.63+ representing a glycan group attached to Thr200. Based on its mass and quantitative assays with [P32] and [C14]UDP-glucose, the suggested composition of the adduct attached to Thr200 is (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Paramecium octaurelia Rab7b is crucial for the proper localization/function of this protein. Moreover, the two Rab7 paralogues differ also in another PTM: substantially more phosphorylated amino acid residues are in Rab7b than in Rab7a. PMID:27349314

  13. Acid–base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu237→Gln mutant enzymes

    PubMed Central

    Schwarz, Alexandra; Brecker, Lothar; Nidetzky, Bernd

    2007-01-01

    The role of acid–base catalysis in the two-step enzymatic mechanism of α-retaining glucosyl transfer by Leuconostoc mesenteroides sucrose phosphorylase has been examined through site-directed replacement of the putative catalytic Glu237 and detailed comparison of purified wild-type and Glu237→Gln mutant enzymes using steady-state kinetics. Reactions with substrates requiring Brønsted catalytic assistance for glucosylation or deglucosylation were selectively slowed at the respective step, about 105-fold, in E237Q. Azide, acetate and formate but not halides restored catalytic activity up to 300-fold in E237Q under conditions in which the deglucosylation step was rate-determining, and promoted production of the corresponding α-glucosides. In situ proton NMR studies of the chemical rescue of E237Q by acetate and formate revealed that enzymatically formed α-glucose 1-esters decomposed spontaneously via acyl group migration and hydrolysis. Using pH profiles of kcat/Km, the pH dependences of kinetically isolated glucosylation and deglucosylation steps were analysed for wild-type and E237Q. Glucosylation of the wild-type proceeded optimally above and below apparent pKa values of about 5.6 and 7.2 respectively whereas deglucosylation was dependent on the apparent single ionization of a group of pKa≈5.8 that must be deprotonated for reaction. Glucosylation of E237Q was slowed below apparent pKa≈6.0 but had lost the high pH dependence of the wild-type. Deglucosylation of E237Q was pH-independent. The results allow unequivocal assignment of Glu237 as the catalytic acid–base of sucrose phosphorylase. They support a mechanism in which the pKa of Glu237 cycles between ≈7.2 in free enzyme and ≈5.8 in glucosyl enzyme intermediate, ensuring optimal participation of the glutamate residue side chain at each step in catalysis. Enzyme deglucosylation to an anionic nucleophile took place with Glu237 protonated or unprotonated. The results delineate how conserved active

  14. Structural and Mechanistic Analysis of Trichodiene Synthase Using Site-Directed Mutagenesis: Probing the Catalytic Function of Tryosine-295 and the Asparagine-225/Serine-229/Glutamate-233-Mg2+ B Motif

    SciTech Connect

    Vedula,L.; Jiang, J.; Zakharian, T.; Cane, D.; Christianson, D.

    2008-01-01

    Trichodiene synthase from Fusarium sporotrichioides contains two metal ion-binding motifs required for the cyclization of farnesyl diphosphate: the 'aspartate-rich' motif D100DXX(D/E) that coordinates to Mg{sup 2+}{sub A} and Mg{sup 2+}{sub C} source, and the 'NSE/DTE' motif N225DXXSXXXE that chelates Mg{sup 2+}{sub b} (boldface indicates metal ion ligands). Here, we report steady-state kinetic parameters, product array analyses, and X-ray crystal structures of trichodiene synthase mutants in which the fungal NSE motif is progressively converted into a plant-like DDXXTXXXE motif, resulting in a degradation in both steady-state kinetic parameters and product specificity. Each catalytically active mutant generates a different distribution of sesquiterpene products, and three newly detected sesquiterpenes are identified. In addition, the kinetic and structural properties of the Y295F mutant of trichodiene synthase were found to be similar to those of the wild-type enzyme, thereby ruling out a proposed role for Y295 in catalysis.

  15. Effects of ligand binding on the conformation and internal dynamics in specific regions of porcine pancreatic phospholipase A2 with tryptophan as a probe: a study combining time-resolved fluorescence spectroscopy and site-directed mutagenesis (same as p. 100)

    NASA Astrophysics Data System (ADS)

    Kuipers, Oscar; Vincent, Michel; Brochon, Jean-Claude; Verheij, Bert; de Haas, Gerard; Gallay, Jacques

    1990-05-01

    Exploration of the effect of ligand-protein interactions on conformational substates and internal dynamics in different regions of phospholipase A2 from porcine pancreas (PLA2), was performed by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) in the wild type protein was replaced by a phenylalanine residue, whereafter Trp was substituted either for leucine-31 ,located in the calcium binding loop, or for phenylalanine-94, located at the "back side" of the enzyme, in a-helix E (Dijkstra et al., J. Mol. Biol., 147, 97-123, 1981). Analyses by the Maximum Entropy Method (MIEM) of the total fluorescence intensity decays, provide in each case a distribution of separate lifetime classes, which can be interpreted as reflecting the existence of discrete conformational substates in slow exchange with respect to the time-scale of the decay kinetics. The fluorescence decay of the W94 mutant is dominated by an extremely short excited state lifetime of ~60 ps, probably arising from the presence of two proximate disulfide bridges. Time-resolved fluorescence anisotropy studies show that the Trp residue near the NH2 terminus (Trp-3) undergoes a more limited rotational motion than the Trp-3 1 located in the calcium binding loop. The widest angular rotation is observed at position 94, in a-helix E. Calcium binding displays the strongest influence on the lifetime distribution of Trp-31: a major local conformation corresponding to a lifetime class with a barycenter value of ~5.5 ns and contributing to ~50% of the decay is selected. The conformations giving rise to the short lifetimes ((tau)1 and (tau)2 lifetime classes) become less important. The contribution of the third lifetime class (c3) stays at a constant value of 30%. In the presence of calcium, the amplitude of motion is wider than without the ion. There is virtually no effect of calcium binding on the lifetime distribution of the Trp residue at the 3 or the

  16. Effects of ligand binding on the conformation and internal dynamics in specific regions of porcine pancreatic phospholipase A2 with tryptophan as a probe: a study combinging time-resolved fluorescence spectroscopy and site-directed mutagenesis (same as p. 628)

    NASA Astrophysics Data System (ADS)

    Kuipers, Oscar; Vincent, Michel; Brochon, Jean-Claude; Verheij, Bert; de Haas, Gerard; Gallay, Jacques

    1990-05-01

    Exploration of the effect of ligand-protein interactions on conformational substates and internal dynamics in different regions of phospholipase A2 from porcine pancreas (PLA2), was performed by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) in the wild type protein was replaced by a phenylalanine residue, whereafter Tip was substituted either for leucine-3 1 ,located in the calcium binding ioop, or for phenylalanine-94, located at the "back side" of the enzyme, in a-helix E (Dijkstra et al., J. Mol. Biol., 147, 97-123, 1981). Analyses by the Maximum Entropy Method (MEM) of the total fluorescence intensity decays, provide in each case a distribution of separate lifetime classes, which can be interpreted as reflecting the existence of discrete conformational substates in slow exchange with respect to the time-scale of the decay kinetics. The fluorescence decay of the W94 mutant is. dominated by an extremely short excited state lifetime of ~60 ps, probably arising from the presence of two proximate disulfide bridges. Time-resolved fluorescence anisotropy studies show that the Trp residue near the NH2 terminus (Trp-3) undergoes a more limited rotational motion than the Trp-3 1 located in the calcium binding loop. The widest angular rotation is observed at position 94, in a-helix E. Calcium binding displays the strongest influence on the lifetime distribution of Trp-3 1: a major local conformation corresponding to a lifetime class with a barycenter value of -5.5 ns and contributing to ~50% of the decay is selected. The conformations giving rise to the short lifetimes (τ1 and τ2 lifetime classes) become less important. The contribution of the third lifetime class (c3) stays at a constant value of 30%. In the presence of calcium, the amplitude of motion is wider than without the ion. There is virtually no effect of calcium binding on the lifetime distribution of the Trp residue at the 3 or the 94

  17. Evaluation of aflatoxin B/sub 1/ mutagenesis: addition of glutathione and glutathione-S-transferase to the Salmonella mutagenicity assay

    SciTech Connect

    Jorgensen, K.V.; Clayton, J.W.; Price, R.L.

    1987-01-01

    The effects of glutathione (GSH) and the combination of GSH and glutathione-S-transferase (GST) on aflatoxin B/sub 1/ (AFB/sub 1/) mutagenesis in the Salmonella mutagenicity assay using Salmonella typhimurium strains TA98 and TA100 were tested. Ten concentrations of AFB/sub 1/ (0-1.0 ..mu..g/plate) were added to a liver microsomal homogenate (S9 mix) or to S9 mix containing GSH or S9 mix containing the combination of GSH + GST. One third of the samples were plated directly. Two-thirds were incubated for 30 min at 37/sup 0/C prior to plating, and of those, half included bacteria. The results show that the addition of GSH and GSH + GST affected AFB/sub 1/ mutagenesis by forming the AFB/sub 1/-GSH conjugate and decreasing the availability of AFB/sub 1/-8,9-epoxide. The effect of GST on GSH activity varied with the strain because of the different amounts of S9 mix used. The formation of the AFB/sub 1/-GSH conjugate was verified by using reverse-phase high-performance liquid chromatography for quantitation of AFB/sub 1/ and detection of AFB/sub 1/-GSH.

  18. Faux Mutagenesis: Teaching Troubleshooting through Controlled Failure

    ERIC Educational Resources Information Center

    Hartberg, Yasha

    2006-01-01

    By shifting pedagogical goals from obtaining successful mutations to teaching students critical troubleshooting skills, it has been possible to introduce site-directed mutagenesis into an undergraduate teaching laboratory. Described in this study is an inexpensive laboratory exercise in which students follow a slightly modified version of…

  19. Optimization of Combinatorial Mutagenesis

    NASA Astrophysics Data System (ADS)

    Parker, Andrew S.; Griswold, Karl E.; Bailey-Kellogg, Chris

    Protein engineering by combinatorial site-directed mutagenesis evaluates a portion of the sequence space near a target protein, seeking variants with improved properties (stability, activity, immunogenicity, etc.). In order to improve the hit-rate of beneficial variants in such mutagenesis libraries, we develop methods to select optimal positions and corresponding sets of the mutations that will be used, in all combinations, in constructing a library for experimental evaluation. Our approach, OCoM (Optimization of Combinatorial Mutagenesis), encompasses both degenerate oligonucleotides and specified point mutations, and can be directed accordingly by requirements of experimental cost and library size. It evaluates the quality of the resulting library by one- and two-body sequence potentials, averaged over the variants. To ensure that it is not simply recapitulating extant sequences, it balances the quality of a library with an explicit evaluation of the novelty of its members. We show that, despite dealing with a combinatorial set of variants, in our approach the resulting library optimization problem is actually isomorphic to single-variant optimization. By the same token, this means that the two-body sequence potential results in an NP-hard optimization problem. We present an efficient dynamic programming algorithm for the one-body case and a practically-efficient integer programming approach for the general two-body case. We demonstrate the effectiveness of our approach in designing libraries for three different case study proteins targeted by previous combinatorial libraries - a green fluorescent protein, a cytochrome P450, and a beta lactamase. We found that OCoM worked quite efficiently in practice, requiring only 1 hour even for the massive design problem of selecting 18 mutations to generate 107 variants of a 443-residue P450. We demonstrate the general ability of OCoM in enabling the protein engineer to explore and evaluate trade-offs between quality and

  20. Optimization of combinatorial mutagenesis.

    PubMed

    Parker, Andrew S; Griswold, Karl E; Bailey-Kellogg, Chris

    2011-11-01

    Protein engineering by combinatorial site-directed mutagenesis evaluates a portion of the sequence space near a target protein, seeking variants with improved properties (e.g., stability, activity, immunogenicity). In order to improve the hit-rate of beneficial variants in such mutagenesis libraries, we develop methods to select optimal positions and corresponding sets of the mutations that will be used, in all combinations, in constructing a library for experimental evaluation. Our approach, OCoM (Optimization of Combinatorial Mutagenesis), encompasses both degenerate oligonucleotides and specified point mutations, and can be directed accordingly by requirements of experimental cost and library size. It evaluates the quality of the resulting library by one- and two-body sequence potentials, averaged over the variants. To ensure that it is not simply recapitulating extant sequences, it balances the quality of a library with an explicit evaluation of the novelty of its members. We show that, despite dealing with a combinatorial set of variants, in our approach the resulting library optimization problem is actually isomorphic to single-variant optimization. By the same token, this means that the two-body sequence potential results in an NP-hard optimization problem. We present an efficient dynamic programming algorithm for the one-body case and a practically-efficient integer programming approach for the general two-body case. We demonstrate the effectiveness of our approach in designing libraries for three different case study proteins targeted by previous combinatorial libraries--a green fluorescent protein, a cytochrome P450, and a beta lactamase. We found that OCoM worked quite efficiently in practice, requiring only 1 hour even for the massive design problem of selecting 18 mutations to generate 10⁷ variants of a 443-residue P450. We demonstrate the general ability of OCoM in enabling the protein engineer to explore and evaluate trade-offs between quality and

  1. Site-directed isotope labelling and FTIR spectroscopy of bacteriorhodopsin.

    PubMed

    Sonar, S; Lee, C P; Coleman, M; Patel, N; Liu, X; Marti, T; Khorana, H G; RajBhandary, U L; Rothschild, K J

    1994-08-01

    Insight into integral membrane proteins function is presently limited by the difficulty of producing three-dimensional crystals. In addition, X-ray structures of proteins normally do not provide information about the protonation state and structural changes of individual residues. We report here the first use of site-directed isotope labelling and Fourier transform infrared (FTIR) difference spectroscopy to detect structural changes at the level of single residues in an integral membrane protein. Two site-directed isotope labeled (SDIL) tyrosine analogues of bacteriorhodopsin were produced which exhibit normal activity. FTIR spectroscopy shows that out of 11 tyrosines, only Tyr 185 is structurally active during the early photocycle and may be part of a proton wire. PMID:7664078

  2. Theoretical site-directed mutagenesis: Asp168Ala mutant of lactate dehydrogenase

    PubMed Central

    Ferrer, Silvia; Tuñón, Iñaki; Moliner, Vicent; Williams, Ian H.

    2008-01-01

    Molecular simulations based on the use of hybrid quantum mechanics/molecular mechanics methods are able to provide detailed information about the complex enzymatic reactions and the consequences of specific mutations on the activity of the enzyme. In this work, the reduction of pyruvate to lactate catalysed by wild-type and Asp168Ala mutant lactate dehydrogenase (LDH) has been studied by means of simulations using a very flexible molecular model consisting of the full tetramer of the enzyme, together with the cofactor NADH, the substrate and solvent water molecules. Our results indicate that the Asp168Ala mutation provokes a shift in the pKa value of Glu199 that becomes unprotonated at neutral pH in the mutant enzyme. This change compensates the loss of the negative charge of Asp168, rendering a still active enzyme. Thus, our methodology gives a calculated barrier height for the Asp168Ala mutant 3 kcal mol−1 higher than that for wild-type LDH, which is in very good agreement with the experiment. The computed potential energy surfaces reveal the reaction pathways and transition structures for the wild-type and mutant enzymes. Hydride transfer is less advanced and the proton transfer is more advanced in the Asp168Ala mutant than in the wild type. This approach provides a very powerful tool for the analysis of the roles of key active-site residues. PMID:18682365

  3. Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II.

    PubMed

    Verma, Shikha; Mehta, Ranjit Kumar; Maiti, Prasanta; Röhm, Klaus-Heinrich; Sonawane, Avinash

    2014-07-01

    Bacterial asparaginases (EC 3.5.1.1) have attracted considerable attention because enzymes of this group are used in the therapy of certain forms of leukemia. Class II asparaginase from Escherichia coli (EcA), a homotetramer with a mass of 138 kDa, is especially effective in cancer therapy. However, the therapeutic potential of EcA is impaired by the limited stability of the enzyme in vivo and by the induction of antibodies in the patients. In an attempt to modify the properties of EcA, several variants with amino acid replacements at subunit interfaces were constructed and characterized. Chemical and thermal denaturation analysis monitored by activity, fluorescence, circular dichroism, and differential scanning calorimetry showed that certain variants with exchanges that weaken dimer-dimer interactions exhibited complex denaturation profiles with active dimeric and/or inactive monomeric intermediates appearing at low denaturant concentrations. By contrast, other EcA variants showed considerably enhanced activity and stability as compared to the wild-type enzyme. Thus, even small changes at a subunit interface may markedly affect EcA stability without impairing its catalytic properties. Variants of this type may have a potential for use in the asparaginase therapy of leukemia. PMID:24721562

  4. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site.

    PubMed

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called 'catalytic residues' are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. PMID:25902402

  5. A site-directed mutagenesis analysis of tNOX functional domains

    NASA Technical Reports Server (NTRS)

    Chueh, Pin-Ju; Morre, Dorothy M.; Morre, D. James

    2002-01-01

    Constitutive NADH oxidase proteins of the mammalian cell surface exhibit two different activities, oxidation of hydroquinones (or NADH) and protein disulfide-thiol interchange which alternate to yield oscillatory patterns with period lengths of 24 min. A drug-responsive tNOX (tumor-associated NADH oxidase) has a period length of about 22 min. The tNOX cDNA has been cloned and expressed. These two proteins are representative of cycling oxidase proteins of the plant and animal cell surface. In this report, we describe a series of eight amino acid replacements in tNOX which, when expressed in Escherichia coli, were analyzed for enzymatic activity, drug response and period length. Replacement sites selected include six cysteines that lie within the processed plasma membrane (34 kDa) form of the protein, and amino acids located in putative drug and adenine nucleotide (NADH) binding domains. The latter, plus two of the cysteine replacements, resulted in a loss of enzymatic activity. The recombinant tNOX with the modified drug binding site retained activity but the activity was no longer drug-responsive. The four remaining cysteine replacements were of interest in that both activity and drug response were retained but the period length for both NADH oxidation and protein disulfide-thiol interchange was increased from 22 min to 36 or 42 min. The findings confirm the correctness of the drug and adenine nucleotide binding motifs within the tNOX protein and imply a potential critical role of cysteine residues in determining the period length.

  6. Distinct ETA receptor binding mode of macitentan as determined by site directed mutagenesis.

    PubMed

    Gatfield, John; Mueller Grandjean, Celia; Bur, Daniel; Bolli, Martin H; Nayler, Oliver

    2014-01-01

    The competitive endothelin receptor antagonists (ERA) bosentan and ambrisentan, which have long been approved for the treatment of pulmonary arterial hypertension, are characterized by very short (1 min) occupancy half-lives at the ET(A) receptor. The novel ERA macitentan, displays a 20-fold increased receptor occupancy half-life, causing insurmountable antagonism of ET-1-induced signaling in pulmonary arterial smooth muscle cells. We show here that the slow ET(A) receptor dissociation rate of macitentan was shared with a set of structural analogs, whereas compounds structurally related to bosentan displayed fast dissociation kinetics. NMR analysis showed that macitentan adopts a compact structure in aqueous solution and molecular modeling suggests that this conformation tightly fits into a well-defined ET(A) receptor binding pocket. In contrast the structurally different and negatively charged bosentan-type molecules only partially filled this pocket and expanded into an extended endothelin binding site. To further investigate these different ET(A) receptor-antagonist interaction modes, we performed functional studies using ET(A) receptor variants harboring amino acid point mutations in the presumed ERA interaction site. Three ET(A) receptor residues significantly and differentially affected ERA activity: Mutation R326Q did not affect the antagonist activity of macitentan, however the potencies of bosentan and ambrisentan were significantly reduced; mutation L322A rendered macitentan less potent, whereas bosentan and ambrisentan were unaffected; mutation I355A significantly reduced bosentan potency, but not ambrisentan and macitentan potencies. This suggests that--in contrast to bosentan and ambrisentan--macitentan-ET(A) receptor binding is not dependent on strong charge-charge interactions, but depends predominantly on hydrophobic interactions. This different binding mode could be the reason for macitentan's sustained target occupancy and insurmountable antagonism. PMID:25226600

  7. Site-directed mutagenesis and PBAN activation of the Helicoverpa zea PBAN-receptor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insect neuropeptides are produced in the central or peripheral nerve tissues, and released to regulate various physiological and behavioral actions during development and reproduction. Pheromone biosynthesis-activating neuropeptide (PBAN)/Pyrokinin is a major neuropeptide family characterized with a...

  8. Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cellobiose 2-epimerase from the thermophile Caldicellulosiruptor saccharolyticus (CsCE) catalyzes the isomerization of lactose into lactulose, a non-digestible disaccharide widely used in food and pharmaceutical industries. Semi-rational approaches were applied to enhance the thermostability of CsCE...

  9. Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin

    PubMed Central

    Gasymov, Oktay K.; Abduragimov, Adil R.; Glasgow, Ben J.

    2008-01-01

    The absorption spectra of N-acetyl-L-tryptophanamide in various solvents were resolved into the sums of the 1La and 1Lb components. The relative intensities of the 0-0 transitions of the 1Lb bands correlate linearly with the solvent polarity values (ETN). A novel strategy, which utilizes a set of the experimental 1Lb bands, was employed to resolve the near-UV CD spectra of tryptophanyl residues. Resolved spectral parameters from the single-tryptophan mutants of tear lipocalin (TL), F99W and Y87W, corroborate the fluorescence as well as structural data of TL. Analysis of the 1Lb bands of the Trp CD spectra in proteins is a valuable tool to obtain the local features. The “DMSO-like” 1Lb band of Trp CD spectra may be used as a “fingerprint” to identify the tryptophanyl side chains in situations where the benzene rings of Trp have van der Waals interactions with the side chains of its nearest neighbor. In addition, the signs and intensities of the components hold information about the side-chain conformations and dynamics in proteins. Combined with Trp mutagenesis, this method we call site-directed circular dichroism is broadly applicable to various proteins to obtain the position-specific data. PMID:18047823

  10. Site-directed circular dichroism of proteins: 1Lb bands of Trp resolve position-specific features in tear lipocalin.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2008-03-15

    The absorption spectra of N-acetyl-L-tryptophanamide in various solvents were resolved into the sums of the (1)L(a) and (1)L(b) components. The relative intensities of the 0-0 transitions of the (1)L(b) bands correlate linearly with the solvent polarity values (E(T)(N)). A novel strategy that uses a set of the experimental (1)L(b) bands was employed to resolve the near-UV circular dichroism (CD) spectra of tryptophanyl residues. Resolved spectral parameters from the single-tryptophan mutants of tear lipocalin (TL), F99W and Y87W, corroborate the fluorescence and structural data of TL. Analysis of the (1)L(b) bands of the Trp CD spectra in proteins is a valuable tool to obtain the local features. The dimethyl sulfoxide (DMSO)-like (1)L(b) band of Trp CD spectra may be used as a "fingerprint" to identify the tryptophanyl side chains in situations where the benzene rings of Trp have van der Waals interactions with the side chains of its nearest neighbor. In addition, the signs and intensities of the components hold information about the side chain conformations and dynamics in proteins. Combined with Trp mutagenesis, this method, which we call site-directed circular dichroism, is broadly applicable to various proteins to obtain the position-specific data. PMID:18047823

  11. The Parasol Protocol for computational mutagenesis.

    PubMed

    Aronica, P G A; Verma, C; Popovic, B; Leatherbarrow, R J; Gould, I R

    2016-07-01

    To aid in the discovery and development of peptides and proteins as therapeutic agents, a virtual screen can be used to predict trends and direct workflow. We have developed the Parasol Protocol, a dynamic method implemented using the AMBER MD package, for computational site-directed mutagenesis. This tool can mutate between any pair of amino acids in a computationally expedient, automated manner. To demonstrate the potential of this methodology, we have employed the protocol to investigate a test case involving stapled peptides, and have demonstrated good agreement with experiment. PMID:27255759

  12. Preliminary Work in Obtaining Site-Directed Mutants of Hen Egg White Lysozyme

    NASA Technical Reports Server (NTRS)

    Holmes, Leonard D.

    1996-01-01

    < ------ > dimer < ------- > tetramer < ------ > octamer < ------ > higher order. It is believed that multimer aggregation of lysozyme occurs by interaction at specific binding sites on the surface of the protein crystals. If the presence of discrete binding sites and the aggregation hypothesis is true, then it follows that the alteration of the binding site(s) should have significant effect on the measurements obtained during growth experiments. Site-directed mutagenesis allows the specific alteration of proteins by replacement, deletion or addition of specific amino acid residues. This report outlines the approach for this strategy and the progress made thus far toward that end.

  13. Computer Simulation of Mutagenesis.

    ERIC Educational Resources Information Center

    North, J. C.; Dent, M. T.

    1978-01-01

    A FORTRAN program is described which simulates point-substitution mutations in the DNA strands of typical organisms. Its objective is to help students to understand the significance and structure of the genetic code, and the mechanisms and effect of mutagenesis. (Author/BB)

  14. 2004 Mutagenesis Gordon Conference

    SciTech Connect

    Dr. Sue Jinks-Robertson

    2005-09-16

    Mutations are genetic alterations that drive biological evolution and cause many, if not all, human diseases. Mutation originates via two distinct mechanisms: ''vertical'' variation is de novo change of one or few bases, whereas ''horizontal'' variation occurs by genetic recombination, which creates new mosaics of pre-existing sequences. The Mutagenesis Conference has traditionally focused on the generation of mutagenic intermediates during normal DNA synthesis or in response to environmental insults, as well as the diverse repair mechanisms that prevent the fixation of such intermediates as permanent mutations. While the 2004 Conference will continue to focus on the molecular mechanisms of mutagenesis, there will be increased emphasis on the biological consequences of mutations, both in terms of evolutionary processes and in terms of human disease. The meeting will open with two historical accounts of mutation research that recapitulate the intellectual framework of this field and thereby place the current research paradigms into perspective. The two introductory keynote lectures will be followed by sessions on: (1) mutagenic systems, (2) hypermutable sequences, (3) mechanisms of mutation, (4) mutation avoidance systems, (5) mutation in human hereditary and infectious diseases, (6) mutation rates in evolution and genotype-phenotype relationships, (7) ecology, mutagenesis and the modeling of evolution and (8) genetic diversity of the human population and models for human mutagenesis. The Conference will end with a synthesis of the meeting as the keynote closing lecture.

  15. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  16. Solution structure by site directed tryptophan fluorescence in tear lipocalin.

    PubMed

    Gasymov, O K; Abduragimov, A R; Yusifov, T N; Glasgow, B J

    1997-10-01

    The solution structure of the G strand of human tear lipocalin was deduced by site directed tryptophan fluorescence (SDTF). The fluorescent amino acid, tryptophan, was sequentially substituted for each native amino acid in the sequence of the G strand. The fluorescent properties resolved alternating periodicity as predicted for beta sheet structure, twists in the beta sheet, strand orientation in the lipocalin cavity, and the relative depth of residues in the cavity. A distribution of microstates with various orientations of dipoles in the side chain environments of the G strand revealed mobility on the nanosecond time scale. SDTF is broadly applicable to most proteins and will complement x-ray crystallography, site directed spin labeling by electron paramagnetic resonance (EPR), and nuclear magnetic resonance (NMR) in the determination of solution structure. PMID:9345294

  17. Mapping the lipoylation site of Arabidopsis thaliana plastidial dihydrolipoamide S-acetyltransferase using mass spectrometry and site-directed mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The catalytic enhancement achieved by the pyruvate dehydrogenase complex (PDC) results from a combination of substrate channeling plus active-site coupling. The mechanism for active-site coupling involves lipoic acid prosthetic groups covalently attached to Lys residues in the primary ...

  18. Comparison of covalent with reversible inhibitor binding sites of the gastric H,K-ATPase by site-directed mutagenesis.

    PubMed

    Lambrecht, N; Munson, K; Vagin, O; Sachs, G

    2000-02-11

    The gastric H,K-ATPase is covalently inhibited by substituted pyridyl-methylsulfinyl-benzimidazoles, such as omeprazole, that convert to thiophilic probes of luminally accessible cysteines in the acid space. The K(+) competitive inhibitor, SCH28080, prevented inhibition of acid transport by omeprazole. In stably expressing HEK293 cells, the benzimidazole-reactive cysteines, Cys-321 (transmembrane helix (TM) 3), Cys-813 and Cys-822 (TM5/6), and Cys-892 (TM7/8) were mutated to the amino acids found in the SCH28080-resistant Na,K-ATPase and kinetic parameters of H,K-ATPase activity analyzed. Mutations of Cys-822 and Cys-892 had insignificant effects on the K(i(app)), K(m(app)) or V(max), but mutations of Cys-813 to threonine and Cys-321 to alanine decreased the affinity for SCH28080. Mutation of Cys-321 to alanine produced mixed kinetics of inhibition, still with higher affinity for the cation-free form of phosphoenzyme. Since the phenylmethoxy ring of the imidazo-pyridine inhibitors binds to TM1/2, as shown by earlier photoaffinity studies, and the mutations in TM6 (Cys-813 --> Thr) as well as the end of TM3 (Cys-321 --> Ala) decrease the affinity for SCH28080, the TM1/2, TM3, and TM6 helices lie within approximately 16 A of each other based on the size of the active, extended conformation of SCH28080. PMID:10660561

  19. Domain folding and flexibility of Escherichia coli FtsZ determined by tryptophan site-directed mutagenesis

    PubMed Central

    Díaz-Espinoza, Rodrigo; Garcés, Andrea P.; Arbildua, José J.; Montecinos, Felipe; Brunet, Juan E.; Lagos, Rosalba; Monasterio, Octavio

    2007-01-01

    FtsZ has two domains, the amino GTPase domain with a Rossmann fold, and the carboxyl domain that resembles the chorismate mutase fold. Bioinformatics analyses suggest that the interdomain interaction is stronger than the interaction of the protofilament longitudinal interfaces. Crystal B factor analysis of FtsZ and detected conformational changes suggest a connection between these domains. The unfolding/folding characteristics of each domain of FtsZ were tested by introducing tryptophans into the flexible region of the amino (F135W) and the carboxyl (F275W and I294W) domains. As a control, the mutation F40W was introduced in a more rigid part of the amino domain. These mutants showed a native-like structure with denaturation and renaturation curves similar to wild type. However, the I294W mutant showed a strong loss of functionality, both in vivo and in vitro when compared to the other mutants. The functionality was recovered with the double mutant I294W/F275A, which showed full in vivo complementation with a slight increment of in vitro GTPase activity with respect to the single mutant. The formation of a stabilizing aromatic interaction involving a stacking between the tryptophan introduced at position 294 and phenylalanine 275 could account for these results. Folding/unfolding of these mutants induced by guanidinium chloride was compatible with a mechanism in which both domains within the protein show the same stability during FtsZ denaturation and renaturation, probably because of strong interface interactions. PMID:17656575

  20. Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression.

    PubMed

    Xu, Jian-zhong; Zhang, Wei-guo

    2016-02-01

    With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators. PMID:26834010

  1. Molecular cloning, modeling, and site-directed mutagenesis of type III polyketide synthase from Sargassum binderi (Phaeophyta).

    PubMed

    Baharum, Hariyanti; Morita, Hiroyuki; Tomitsuka, Akifumi; Lee, Fong-Chin; Ng, Kim-Yong; Rahim, Raha Abdul; Abe, Ikuro; Ho, Chai-Ling

    2011-10-01

    Type III polyketide synthases (PKSs) produce an array of metabolites with diverse functions. In this study, we have cloned the complete reading frame encoding type III PKS (SbPKS) from a brown seaweed, Sargassum binderi, and characterized the activity of its recombinant protein biochemically. The deduced amino acid sequence of SbPKS is 414 residues in length, sharing a higher sequence similarity with bacterial PKSs (38% identity) than with plant PKSs. The Cys-His-Asn catalytic triad of PKS is conserved in SbPKS with differences in some of the residues lining the active and CoA binding sites. The wild-type SbPKS displayed broad starter substrate specificity to aliphatic long-chain acyl-CoAs (C(6)-C(14)) to produce tri- and tetraketide pyrones. Mutations at H(331) and N(364) caused complete loss of its activity, thus suggesting that these two residues are the catalytic residues for SbPKS as in other type III PKSs. Furthermore, H227G, H227G/L366V substitutions resulted in increased tetraketide-forming activity, while wild-type SbPKS produces triketide α-pyrone as a major product. On the other hand, mutant H227G/L366V/F93A/V95A demonstrated a dramatic decrease of tetraketide pyrone formation. These observations suggest that His(227) and Leu(366) play an important role for the polyketide elongation reaction in SbPKS. The conformational changes in protein structure especially the cavity of the active site may have more significant effect to the activity of SbPKS compared with changes in individual residues. PMID:21181422

  2. Site-directed mutagenesis of an energy transducing protein: Bacteriorhodopsin. Final report, July 15, 1992--July 14, 1996

    SciTech Connect

    Needleman, R.

    1998-05-01

    The objective was to understand at the molecular level how bacteriorhodopsin (BR) transports protons. The work involves the synthesis of mutant BRs, their expression in the natural host, H. halobium, and an investigation of their photocycles. This final report has led to the development of a greatly improved expression system and to an increased understanding of the mechanism of proton transport. At the beginning of the award period a central concern was establishing the details of the photocycle. This phase was essentially complete by mid 1994. The author then investigated the energy coupling mechanism which allows uni-directional proton transfer and found that a major determinant was the coupling of the proton release to changes in the pKa of D85.

  3. Hypericum perforatum hydroxyalkylpyrone synthase involved in sporopollenin biosynthesis--phylogeny, site-directed mutagenesis, and expression in nonanther tissues.

    PubMed

    Jepson, Christina; Karppinen, Katja; Daku, Rhys M; Sterenberg, Brian T; Suh, Dae-Yeon

    2014-09-01

    Anther-specific chalcone synthase-like enzyme (ASCL), an ancient plant type III polyketide synthase, is involved in the biosynthesis of sporopollenin, the stable biopolymer found in the exine layer of the wall of a spore or pollen grain. The gene encoding polyketide synthase 1 from Hypericum perforatum (HpPKS1) was previously shown to be expressed mainly in young flower buds, but also in leaves and other tissues at lower levels. Angiosperm ASCLs, identified by sequence and phylogenetic analyses, are divided into two sister clades, the Ala-clade and the Val-clade, and HpPKS1 belongs to the Ala-clade. Recombinant HpPKS1 produced triketide and, to a lesser extent, tetraketide alkylpyrones from medium-chain (C6) to very long-chain (C24) fatty acyl-CoA substrates. Like other ASCLs, HpPKS1 also preferred hydroxyl fatty acyl-CoA esters over the analogous unsubstituted fatty acyl-CoA esters. To study the structural basis of the substrate preference, mutants of Ala200 and Ala215 at the putative active site and Arg202 and Asp211 at the modeled acyl-binding tunnel were constructed. The A200T/A215Q mutant accepted decanoyl-CoA, a poor substrate for the wild-type enzyme, possibly because of active site constriction by bulkier substitutions. The substrate preference of the A215V and A200T/A215Q mutants shifted toward nonhydroxylated, medium-chain to long-chain fatty acyl-CoA substrates. The R202L/D211V double mutant was selective for acyl-CoA with chain lengths of C16-C18, and showed a diminished preference for the hydroxylated acyl-CoA substrates. Transient upregulation by abscisic acid and downregulation by jasmonic acid and wounding suggested that HpPKS1, and possibly other Ala-clade ASCLs, may be involved in the biosynthesis of minor cell wall components in nonanther tissues. PMID:25040801

  4. Defining a Conformational Consensus Motif in Cotransin-Sensitive Signal Sequences: A Proteomic and Site-Directed Mutagenesis Study

    PubMed Central

    Klein, Wolfgang; Westendorf, Carolin; Schmidt, Antje; Conill-Cortés, Mercè; Rutz, Claudia; Blohs, Marcus; Beyermann, Michael; Protze, Jonas; Krause, Gerd; Krause, Eberhard; Schülein, Ralf

    2015-01-01

    The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity. PMID:25806945

  5. Site-directed mutagenesis of two aromatic residues lining the active site pocket of the yeast Ltp1.

    PubMed

    Paoli, Paolo; Modesti, Alessandra; Magherini, Francesca; Gamberi, Tania; Caselli, Anna; Manao, Giampaolo; Raugei, Giovanni; Camici, Guido; Ramponi, Giampietro

    2007-05-01

    We mutated Trp(134) and Tyr(135) of the yeast LMW-PTP to explore their catalytic roles, demonstrating that the mutations of Trp(134) to Tyr or Ala, and Tyr(135) to Ala, all interfere with the formation of the phosphorylenzyme intermediate, a phenomenon that can be seen by the decrease in the kinetic constant of the chemical step (k(3)). Furthermore, we noted that the Trp(134) to Ala mutation causes a dramatic drop in k(cat)/K(m) and a slight enhancement of the dissociation constant K(s). The conservative mutant W134Y shows a k(cat)/K(m) very close to that of wild type, probably compensating the two-fold decrease of k(3) with an increase in substrate affinity. The Y135A mutation enhances the substrate affinity, but reduces the enzyme phosphorylation rate. The replacement of Trp(134) with alanine interferes with the partition between phosphorylenzyme hydrolysis and phosphotransfer from the phosphorylenzyme to glycerol and abolish the enzyme activation by adenine. Finally, we found that mutation of Trp(134) to Ala causes a dramatic change in the pH-rate profile that becomes similar to that of the D132A mutant, suggesting that an aromatic residue in position 134 is necessary to assist the proper positioning of the proton donor in the transition state of the chemical step. PMID:17296269

  6. A catalytic role for threonine-12 of E. coli asparaginase II as established by site-directed mutagenesis.

    PubMed

    Harms, E; Wehner, A; Aung, H P; Röhm, K H

    1991-07-01

    A threonine-12 to alanine mutant of E. coli asparaginase II (EC 3.5.1.1) has less than 0.01% of the activity of wild-type enzyme. Both tertiary and quaternary structure of the enzyme are essentially unaffected by the mutation; thus the activity loss seems to be the result of a direct impairment of catalytic function. As aspartate is still bound by the mutant enzyme, Thr-12 appears not be involved in substrate binding. PMID:1906013

  7. Engineering lower inhibitor affinities in beta-D-xylosidase of Selenomonas ruminantium by site-directed mutagenesis of Trp145

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme reported for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D-xylose. One property that could use improvement is its relatively high affinities for D-glucose and D-xylose (Ki~10 mM), wh...

  8. Structure-function studies of the muscle nicotinic acetylcholine receptor by site-directed mutagenesis in the pore region

    NASA Astrophysics Data System (ADS)

    Zhang, Haiyun

    In nicotinic acetylcholine receptors (nAChRs), as in glycine, GABA A, serotonin 5-HT3, and GluCl glutamate receptors, a leucine residue at the approximate midpoint (the 9' position) of the M2 transmembrane domain is conserved across all known subunits. We expressed the embryonic mouse muscle nAChRs with varying numbers (m* s) of subunits (2 αs, 1 β, 1 γ, and 1 δ) mutated at this position in Xenopus oocytes and discovered that mutations to serine (Leu9'Ser) result in a tenfold higher receptor sensitivity to acetylcholine (ACh) for each subunit mutated. Moreover, increases of side-chain polarity increase the sensitivity to ACh when other natural and unnatural residues are incorporated into this position. The data also indicated an especially strong interaction between the γ and δ subunits in the pore region, suggesting a specific arrangement of subunits within the pentamer. Detailed single-channel kinetic studies reveal that Leu9'Ser AChRs have (1) longer voltage- relaxation time constants, (2) longer ACh-induced openings and bursts, and (3) more frequent spontaneous openings. These effects increase with m* s. Synthesized postsynaptic currents were produced with a piezoelectric micromanipulator that delivered brief ACh pulses to multi-channel patches. Their decay time constants were, as expected, similar to the channel burst duration. Thus, both longer and more frequent openings contribute to the >=104-fold increase in the receptor sensitivity to ACh from the wild-type receptor to the receptor with m*s=4; and the highly conserved 9' leucine is crucial for the brief synaptic events that are normally observed. We also explored the effects of ligand-binding domain mutations: γD174N and δD180N (aspartic acid (D) to asparagine (N)). Macroscopic dose-response relations revealed that these mutations decrease the receptor's sensitivity to ACh. The combined effect with Leu9'Ser, however, differs from that predicted from a linear or independent sum of effects from individual mutations. The single-channel recordings of the α2Leu9'SerγD174N and α2Leu9'Ser-γD174NδD180N receptors showed that they have channel open times between those of the wild-type and α2Leu9'Ser receptors, but their channel opening rates are slower. Therefore these ligand- binding domain mutations change the channel gating properties and are possibly involved in signal transduction between the ligand binding and channel gating.

  9. Aminopeptidase B, a glucagon-processing enzyme: site directed mutagenesis of the Zn2+-binding motif and molecular modelling

    PubMed Central

    Pham, Viet-Laï; Cadel, Marie-Sandrine; Gouzy-Darmon, Cécile; Hanquez, Chantal; Beinfeld, Margery C; Nicolas, Pierre; Etchebest, Catherine; Foulon, Thierry

    2007-01-01

    Background Aminopeptidase B (Ap-B; EC 3.4.11.6) catalyzes the cleavage of basic residues at the N-terminus of peptides and processes glucagon into miniglucagon. The enzyme exhibits, in vitro, a residual ability to hydrolyze leukotriene A4 into the pro-inflammatory lipid mediator leukotriene B4. The potential bi-functional nature of Ap-B is supported by close structural relationships with LTA4 hydrolase (LTA4H ; EC 3.3.2.6). A structure-function analysis is necessary for the detailed understanding of the enzymatic mechanisms of Ap-B and to design inhibitors, which could be used to determine the complete in vivo functions of the enzyme. Results The rat Ap-B cDNA was expressed in E. coli and the purified recombinant enzyme was characterized. 18 mutants of the H325EXXHX18E348 Zn2+-binding motif were constructed and expressed. All mutations were found to abolish the aminopeptidase activity. A multiple alignment of 500 sequences of the M1 family of aminopeptidases was performed to identify 3 sub-families of exopeptidases and to build a structural model of Ap-B using the x-ray structure of LTA4H as a template. Although the 3D structures of the two enzymes resemble each other, they differ in certain details. The role that a loop, delimiting the active center of Ap-B, plays in discriminating basic substrates, as well as the function of consensus motifs, such as RNP1 and Armadillo domain are discussed. Examination of electrostatic potentials and hydrophobic patches revealed important differences between Ap-B and LTA4H and suggests that Ap-B is involved in protein-protein interactions. Conclusion Alignment of the primary structures of the M1 family members clearly demonstrates the existence of different sub-families and highlights crucial residues in the enzymatic activity of the whole family. E. coli recombinant enzyme and Ap-B structural model constitute powerful tools for investigating the importance and possible roles of these conserved residues in Ap-B, LTA4H and M1 aminopeptidase catalytic sites and to gain new insight into their physiological functions. Analysis of Ap-B structural model indicates that several interactions between Ap-B and proteins can occur and suggests that endopeptidases might form a complex with Ap-B during hormone processing. PMID:17974014

  10. Effects of site-directed mutagenesis of mglA on motility and swarming of Myxococcus xanthus

    PubMed Central

    2010-01-01

    Background The mglA gene from the bacterium Myxococcus xanthus encodes a 22kDa protein related to the Ras superfamily of monomeric GTPases. MglA is required for the normal function of A-motility (adventurous), S-motility (social), fruiting body morphogenesis, and sporulation. MglA and its homologs differ from all eukaryotic and other prokaryotic GTPases because they have a threonine (Thr78) in place of the highly conserved aspartate residue of the consensus PM3 (phosphate-magnesium binding) region. To identify residues critical for MglA function or potential protein interactions, and explore the function of Thr78, the phenotypes of 18 mglA mutants were characterized. Results Nine mutants, with mutations predicted to alter residues that bind the guanine base or coordinate magnesium, did not produce detectable MglA. As expected, these mutants were mot- dev- because MglA is essential for these processes. Of the remaining nine mutants, seven showed a wild-type distribution pattern for MglA but fell into two categories with regard to function. Five of the seven mutants exhibited mild phenotypes, but two mutants, T78D and P80A, abolished motility and development. The localization pattern of MglA was abolished in two mutants that were mot- spo- and dev-. These two mutants were predicted to alter surface residues at Asp52 and Thr54, which suggests that these residues are critical for proper localization and may define a protein interaction site. Improving the consensus match with Ras at Thr78 abolished function of MglA. Only the conservative serine substitution was tolerated at this position. Merodiploid constructs revealed that a subset of alleles, including mglAD52A, were dominant and also illustrated that changing the balance of MglA and its co-transcribed partner, MglB, affects A-motility. Conclusion Our results suggest that GTP binding is critical for stability of MglA because MglA does not accumulate in mutants that cannot bind GTP. The threonine in PM3 of MglA proteins represents a novel modification of the highly conserved GTPase consensus at this position. The requirement for a hydroxyl group at this position may indicate that MglA is subject to modification under certain conditions. Proper localization of MglA is critical for both motility and development and likely involves protein interactions mediated by residues Asp52 and Thr54. PMID:21083931

  11. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

    PubMed

    Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst. PMID:27402448

  12. Catalytic surface radical in dye-decolorizing peroxidase: a computational, spectroscopic and site-directed mutagenesis study

    PubMed Central

    Linde, Dolores; Pogni, Rebecca; Cañellas, Marina; Lucas, Fátima; Guallar, Victor; Baratto, Maria Camilla; Sinicropi, Adalgisa; Sáez-Jiménez, Verónica; Coscolín, Cristina; Romero, Antonio; Medrano, Francisco Javier; Ruiz-Dueñas, Francisco J.; Martínez, Angel T.

    2014-01-01

    Dye-decolorizing peroxidase (DyP) of Auricularia auricula-judae has been expressed in Escherichia coli as a representative of a new DyP family, and subjected to mutagenic, spectroscopic, crystallographic and computational studies. The crystal structure of DyP shows a buried haem cofactor, and surface tryptophan and tyrosine residues potentially involved in long-range electron transfer from bulky dyes. Simulations using PELE (Protein Energy Landscape Exploration) software provided several binding-energy optima for the anthraquinone-type RB19 (Reactive Blue 19) near the above aromatic residues and the haem access-channel. Subsequent QM/MM (quantum mechanics/molecular mechanics) calculations showed a higher tendency of Trp-377 than other exposed haem-neighbouring residues to harbour a catalytic protein radical, and identified the electron-transfer pathway. The existence of such a radical in H2O2-activated DyP was shown by low-temperature EPR, being identified as a mixed tryptophanyl/tyrosyl radical in multifrequency experiments. The signal was dominated by the Trp-377 neutral radical contribution, which disappeared in the W377S variant, and included a tyrosyl contribution assigned to Tyr-337 after analysing the W377S spectra. Kinetics of substrate oxidation by DyP suggests the existence of high- and low-turnover sites. The high-turnover site for oxidation of RB19 (kcat> 200 s−1) and other DyP substrates was assigned to Trp-377 since it was absent from the W377S variant. The low-turnover site/s (RB19 kcat ~20 s−1) could correspond to the haem access-channel, since activity was decreased when the haem channel was occluded by the G169L mutation. If a tyrosine residue is also involved, it will be different from Tyr-337 since all activities are largely unaffected in the Y337S variant. PMID:25495127

  13. Enhancement of thermal stability of chondroitinase ABC I by site-directed mutagenesis: an insight from Ramachandran plot.

    PubMed

    Nazari-Robati, Mahdieh; Khajeh, Khosro; Aminian, Mahdi; Mollania, Nasrin; Golestani, Abolfazl

    2013-02-01

    The application of chondroitinase ABC I (cABC I) in damaged nervous tissue is believed to prune glycosaminoglycan chains of proteoglycans, thereby facilitates axon regeneration. However, the utilization of cABC I as therapeutics is notably restricted due to its thermal instability. In the present study, we have explored the possibility of thermostabilization of cABC I through release of its conformational strain using Ramachandran plot information. In this regard, Gln140 with non-optimal φ and ψ values were replaced with Gly, Ala and Asn. The results indicated that Q140G and Q140A mutants were able to improve both activity and thermal stability of the enzyme while Q140N variant reduced the enzyme activity and destabilized it. Moreover, the two former variants displayed a remarkable resistance to trypsin degradation. Structural analysis of all mutants showed an increase in intrinsic fluorescence intensity and secondary structure content of Q140G and Q140A compared to the wild type which indicated more compact structure upon mutation. This investigation demonstrated that relief of conformational tension can be considered as a possible approach to increase the stability of the protein. PMID:23159774

  14. Catalytic Mechanism of Heparinase II Investigated by Site-directed Mutagenesis and the Crystal Structure with Its Substrate

    SciTech Connect

    Shaya, D.; Zhao, W; Garron, M; Xiao, Z; Cui, Q; Zhang, Z; Sulea, T; Linhardt, R; Cygler, M

    2010-01-01

    Heparinase II (HepII) is an 85-kDa dimeric enzyme that depolymerizes both heparin and heparan sulfate glycosaminoglycans through a {beta}-elimination mechanism. Recently, we determined the crystal structure of HepII from Pedobacter heparinus (previously known as Flavobacterium heparinum) in complex with a heparin disaccharide product, and identified the location of its active site. Here we present the structure of HepII complexed with a heparan sulfate disaccharide product, proving that the same binding/active site is responsible for the degradation of both uronic acid epimers containing substrates. The key enzymatic step involves removal of a proton from the C5 carbon (a chiral center) of the uronic acid, posing a topological challenge to abstract the proton from either side of the ring in a single active site. We have identified three potential active site residues equidistant from C5 and located on both sides of the uronate product and determined their role in catalysis using a set of defined tetrasaccharide substrates. HepII H202A/Y257A mutant lost activity for both substrates and we determined its crystal structure complexed with a heparan sulfate-derived tetrasaccharide. Based on kinetic characterization of various mutants and the structure of the enzyme-substrate complex we propose residues participating in catalysis and their specific roles.

  15. Exploring the Mechanisms of the Reductase Activity of Neuroglobin by Site-Directed Mutagenesis of the Heme Distal Pocket

    PubMed Central

    2016-01-01

    Neuroglobin (Ngb) is a six-coordinate globin that can catalyze the reduction of nitrite to nitric oxide. Although this reaction is common to heme proteins, the molecular interactions in the heme pocket that regulate this reaction are largely unknown. We have shown that the H64L Ngb mutation increases the rate of nitrite reduction by 2000-fold compared to that of wild-type Ngb [Tiso, M., et al. (2011) J. Biol. Chem. 286, 18277–18289]. Here we explore the effect of distal heme pocket mutations on nitrite reduction. For this purpose, we have generated mutations of Ngb residues Phe28(B10), His64(E7), and Val68(E11). Our results indicate a dichotomy in the reactivity of deoxy five- and six-coordinate globins toward nitrite. In hemoglobin and myoglobin, there is a correlation between faster rates and more negative potentials. However, in Ngb, reaction rates are apparently related to the distal pocket volume, and redox potential shows a poor relationship with the rate constants. This suggests a relationship between the nitrite reduction rate and heme accessibility in Ngb, particularly marked for His64(E7) mutants. In five-coordinate globins, His(E7) facilitates nitrite reduction, likely through proton donation. Conversely, in Ngb, the reduction mechanism does not rely on the delivery of a proton from the histidine side chain, as His64 mutants show the fastest reduction rates. In fact, the rate observed for H64A Ngb (1120 M–1 s–1) is to the best of our knowledge the fastest reported for a heme nitrite reductase. These differences may be related to a differential stabilization of the iron–nitrite complexes in five- and six-coordinate globins. PMID:25554946

  16. Engineering lower inhibitor affinities in beta-D-xylosidase by site-directed mutagenesis of Trp 145

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beta- D-xylosidase catalyzes hydrolysis of xylooligosaccharides to D-xylose monosaccharides. beta-Xylosidase from Selenomonas ruminantium, SXA, is the most active catalyst known for the reaction; however, its activity is inhibited by D-xylose and D-glucose (Ki values of ~10-2 M). Higher Ki’s could...

  17. Exploring the Interaction of SV2A with Racetams Using Homology Modelling, Molecular Dynamics and Site-Directed Mutagenesis

    PubMed Central

    Lee, Joanna; Daniels, Veronique; Sands, Zara A.; Lebon, Florence; Shi, Jiye; Biggin, Philip C.

    2015-01-01

    The putative Major Facilitator Superfamily (MFS) transporter, SV2A, is the target for levetiracetam (LEV), which is a successful anti-epileptic drug. Furthermore, SV2A knock out mice display a severe seizure phenotype and die after a few weeks. Despite this, the mode of action of LEV is not known at the molecular level. It would be extremely desirable to understand this more fully in order to aid the design of improved anti-epileptic compounds. Since there is no structure for SV2A, homology modelling can provide insight into the ligand-binding site. However, it is not a trivial process to build such models, since SV2A has low sequence identity to those MFS transporters whose structures are known. A further level of complexity is added by the fact that it is not known which conformational state of the receptor LEV binds to, as multiple conformational states have been inferred by tomography and ligand binding assays or indeed, if binding is exclusive to a single state. Here, we explore models of both the inward and outward facing conformational states of SV2A (according to the alternating access mechanism for MFS transporters). We use a sequence conservation analysis to help guide the homology modelling process and generate the models, which we assess further with Molecular Dynamics (MD). By comparing the MD results in conjunction with docking and simulation of a LEV-analogue used in radioligand binding assays, we were able to suggest further residues that line the binding pocket. These were confirmed experimentally. In particular, mutation of D670 leads to a complete loss of binding. The results shed light on the way LEV analogues may interact with SV2A and may help with the on-going design of improved anti-epileptic compounds. PMID:25692762

  18. Oligonucleotide-directed site-specific mutagenesis in Drosophila melanogaster.

    PubMed Central

    Banga, S S; Boyd, J B

    1992-01-01

    An efficient technique has been developed for performing in vivo site-directed mutagenesis in Drosophila melanogaster. This procedure involves directed repair of P-element-induced DNA lesions after injection of a modified DNA sequence into early embryos. An oligonucleotide of 50 base pairs, whose sequence spans the P-element insertion site, mediates base replacement in the endogenous gene. Restriction mapping, DNA sequencing, and polymerase chain reaction analysis demonstrate that base substitutions present in an injected oligonucleotide are incorporated into genomic sequences flanking a P insertion site in the white gene. This analysis suggests that progeny bearing directed mutations are recovered with a frequency of about 0.5 x 10(-3). Because Drosophila remains a premier organism for the analysis of eukaryotic gene regulation, this system should find strong application in that analysis as well as in the analysis of DNA recombination, conversion, repair, and mutagenesis. Images PMID:1311850

  19. Mutagenesis of the borage Delta(6) fatty acid desaturase.

    PubMed

    Sayanova, O; Beaudoin, F; Libisch, B; Shewry, P; Napier, J

    2000-12-01

    The consensus sequence of the third histidine box of a range of Delta(5), Delta(6), Delta(8) and sphingolipid desaturases differs from that of the membrane-bound non-fusion Delta(12) and Delta(15) desaturases in the presence of glutamine instead of histidine. We have used site-directed mutagenesis to determine the importance of glutamine and other residues of the third histidine box and created a chimaeric enzyme to determine the ability of the Cyt b(5) fusion domain from the plant sphingolipid desaturase to substitute for the endogenous domain of the Delta(6) desaturase. PMID:11171152

  20. Site-specific mutagenesis of the D1 subunit of photosystem II in wild-type Chlamydomonas.

    PubMed Central

    Przibilla, E; Heiss, S; Johanningmeier, U; Trebst, A

    1991-01-01

    The structure and functional mode of photosystem II reaction center protein D1 can be studied by analyzing the effects of amino acid substitutions within the binding niche for QB, the second stable electron acceptor of photosystem II, on herbicide binding. Here we report on site-directed mutagenesis of the psbA gene coding for the D1 protein in the unicellular alga Chlamydomonas reinhardtii. The chloroplasts of wild-type cells were transformed using the particle gun. The plasmids introduced carried an in vitro mutated fragment of the psbA gene. We obtained a double mutant with replacements of amino acids 264 and 266 and a triple mutant having an additional substitution in position 259. The sensitivities of both mutants toward several types of herbicides are given and compared with those of a mutant having only a substitution at position 264. PMID:1840907

  1. Heat shock and herpes virus: enhanced reactivation without untargeted mutagenesis

    SciTech Connect

    Lytle, C.D.; Carney, P.G.

    1988-01-01

    Enhanced reactivation of Ultraviolet-irradiated virus has been reported to occur in heat-shocked host cells. Since enhanced virus reactivation is often accompanied by untargeted mutagenesis, we investigated whether such mutagenesis would occur for herpes simplex virus (HSV) in CV-1 monkey kidney cells subjected to heat shock. In addition to expressing enhanced reactivation, the treated cells were transiently more susceptible to infection by unirradiated HSV. No mutagenesis of unirradiated HSV was found whether infection occurred at the time of increased susceptibility to infection or during expression of enhanced viral reactivation.

  2. Structure-Based and Random Mutagenesis Approaches Increase the Organophosphate-Degrading Activity of a Phosphotriesterase Homologue from Deinococcus radiodurans

    SciTech Connect

    Hawwa, Renda; Larsen, Sonia D.; Ratia, Kiira; Mesecar, Andrew D.

    2010-11-09

    An enzyme from the amidohydrolase family from Deinococcus radiodurans (Dr-OPH) with homology to phosphotriesterase has been shown to exhibit activity against both organophosphate (OP) and lactone compounds. We have characterized the physical properties of Dr-OPH and have found it to be a highly thermostable enzyme, remaining active after 3 h of incubation at 60 C and withstanding incubation at temperatures up to 70 C. In addition, it can withstand concentrations of at least 200 mg/mL. These properties make Dr-OPH a promising candidate for development in commercial applications. However, compared to the most widely studied OP-degrading enzyme, that from Pseudomonas diminuta, Dr-OPH has low hydrolytic activity against certain OP substrates. Therefore, we sought to improve the OP-degrading activity of Dr-OPH, specifically toward the pesticides ethyl and methyl paraoxon, using structure-based and random approaches. Site-directed mutagenesis, random mutagenesis, and site-saturation mutagenesis were utilized to increase the OP-degrading activity of Dr-OPH. Out of a screen of more than 30,000 potential mutants, a total of 26 mutant enzymes were purified and characterized kinetically. Crystal structures of w.t. Dr-OPH, of Dr-OPH in complex with a product analog, and of 7 mutant enzymes were determined to resolutions between 1.7 and 2.4 {angstrom}. Information from these structures directed the design and production of 4 additional mutants for analysis. In total, our mutagenesis efforts improved the catalytic activity of Dr-OPH toward ethyl and methyl paraoxon by 126- and 322-fold and raised the specificity for these two substrates by 557- and 183-fold, respectively. Our work highlights the importance of an iterative approach to mutagenesis, proving that large rate enhancements are achieved when mutations are made in already active mutants. In addition, the relationship between the kinetic parameters and the introduced mutations has allowed us to hypothesize on those

  3. Intranasal immunogenicity and adjuvanticity of site-directed mutant derivatives of cholera toxin.

    PubMed

    Douce, G; Fontana, M; Pizza, M; Rappuoli, R; Dougan, G

    1997-07-01

    Genetically modified derivatives of cholera toxin (CT), harboring a single amino acid substitution in and around the NAD binding cleft of the A subunit, were isolated following site-directed mutagenesis of the ctxA gene. Two mutants of CT, designated CTS106 (with a proline-to-serine change at position 106) and CTK63 (with a serine-to-lysine change at position 63), were found to have substantially reduced ADP-ribosyltransferase activity and toxicity; CTK63 was completely nontoxic in all assays, whereas CTS106 was 10(4) times less toxic than wild-type CT. The mucosal adjuvanticity and immunogenicity of derivatives of CT were assessed by intranasal immunization of mice, with either ovalbumin or fragment C of tetanus toxin as a bystander antigen. Mice immunized with wild-type CT produced both local (immunoglobulin A in mucosal washes) and systemic immune responses to both CT and bystander antigens. CTS106 showed good local and systemic responses to bystander proteins and to itself. Interestingly, mice immunized with the nontoxic derivative of CT, CTK63, generated weak immune responses to the bystander antigens which were similar to those achieved when CT B subunit was used as an adjuvant. In parallel experiments, an equivalent nontoxic mutant of the Escherichia coli heat-labile enterotoxin, LTK63 (with a serine-to-lysine change at position 63), was tested (9). In contrast to CTK63, LTK63 was found to be more immunogenic and a better intranasal adjuvant than recombinant heat-labile enterotoxin B subunit or CTK63. This information, together with data on immunoglobulin subclass responses, suggests that although highly homologous, CT and heat-labile enterotoxin should not be considered biologically identical in terms of their ability to act as intranasal adjuvants. PMID:9199455

  4. A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice.

    PubMed

    Nishizawa-Yokoi, Ayako; Cermak, Tomas; Hoshino, Tomoki; Sugimoto, Kazuhiko; Saika, Hiroaki; Mori, Akiko; Osakabe, Keishi; Hamada, Masao; Katayose, Yuichi; Starker, Colby; Voytas, Daniel F; Toki, Seiichi

    2016-02-01

    We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing. PMID:26668331

  5. A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice1[OPEN

    PubMed Central

    Cermak, Tomas; Sugimoto, Kazuhiko; Saika, Hiroaki; Mori, Akiko; Osakabe, Keishi; Hamada, Masao; Katayose, Yuichi; Voytas, Daniel F.

    2016-01-01

    We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type. In addition, the ratio of large deletions (greater than 10 bp) and deletions repaired by microhomology-mediated end joining (MMEJ) to total deletion mutations in lig4 null mutant calli was higher than that in the lig4 heterozygous mutant or wild type. Furthermore, almost all insertions (2 bp or greater) were shown to be processed via copy and paste of one or more regions around the TALENs cleavage site and rejoined via MMEJ regardless of genetic background. Taken together, our findings indicate that the dysfunction of cNHEJ leads to a shift in the repair pathway from cNHEJ to altNHEJ or synthesis-dependent strand annealing. PMID:26668331

  6. A mutagenesis-free approach to assignment of (19)F NMR resonances in biosynthetically labeled proteins.

    PubMed

    Kitevski-LeBlanc, Julianne L; Al-Abdul-Wahid, M Sameer; Prosser, R Scott

    2009-02-18

    Solution NMR studies of protein structure and dynamics using fluorinated amino acid probes are a valuable addition to the repertoire of existing (13)C, (15)N, and (1)H experiments. Despite the numerous advantages of the (19)F nucleus in NMR, protein studies are complicated by the dependence of resonance assignments on site-directed mutagenesis methods which are laborious and often problematic. Here we report an NMR-based route to the assignment of fluorine resonances in (13)C,(15)N-3-fluoro-l-tyrosine labeled calmodulin. The assignment begins with the correlation of the fluorine nucleus to the delta proton in the novel (13)C,(15)N-enriched probe which is achieved using a CT-HCCF-COSY experiment. Connection to the backbone is made through two additional solution NMR experiments, namely the (H(beta))C(beta)(C(gamma)C(delta))H(delta) and HNCACB. Assignments are completed using either previously published backbone chemical shift data or obtained experimentally provided uniform (13)C,(15)N labeling procedures are employed during protein expression. Additional benefits of the (13)C,(15)N-3-fluoro-l-tyrosine probe include the reduction of spectral overlap through ((13)C(19)F) CT-HSQCs, as well as the ability to monitor side chain dynamics using (19)F T(1), T(2), and the (13)C-(19)F NOE. PMID:19173647

  7. Key Sites for P2X Receptor Function and Multimerization: Overview of Mutagenesis Studies on a Structural Basis

    PubMed Central

    Hausmann, Ralf; Kless, Achim; Schmalzing, Günther

    2015-01-01

    P2X receptors constitute a seven-member family (P2X1-7) of extracellular ATP-gated cation channels of widespread expression. Because P2X receptors have been implicated in neurological, inflammatory and cardiovascular diseases, they constitute promising drug targets. Since the first P2X cDNA sequences became available in 1994, numerous site-directed mutagenesis studies have been conducted to disclose key sites of P2X receptor function and oligomerization. The publication of the 3-Å crystal structures of the zebrafish P2X4 (zfP2X4) receptor in the homotrimeric apo-closed and ATP-bound open states in 2009 and 2012, respectively, has ushered a new era by allowing for the interpretation of the wealth of molecular data in terms of specific three-dimensional models and by paving the way for designing more-decisive experiments. Thanks to these structures, the last five years have provided invaluable insight into our understanding of the structure and function of the P2X receptor class of ligandgated ion channels. In this review, we provide an overview of mutagenesis studies of the pre- and post-crystal structure eras that identified amino acid residues of key importance for ligand binding, channel gating, ion flow, formation of the pore and the channel gate, and desensitization. In addition, the sites that are involved in the trimerization of P2X receptors are reviewed based on mutagenesis studies and interface contacts that were predicted by the zfP2X4 crystal structures. PMID:25439586

  8. Environmental stress induces trinucleotide repeat mutagenesis in human cells.

    PubMed

    Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A; Yotnda, Patricia; Wilson, John H

    2015-03-24

    The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)-the cause of multiple human diseases-have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential. PMID:25775519

  9. Environmental stress induces trinucleotide repeat mutagenesis in human cells

    PubMed Central

    Chatterjee, Nimrat; Lin, Yunfu; Santillan, Beatriz A.; Yotnda, Patricia; Wilson, John H.

    2015-01-01

    The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)—the cause of multiple human diseases—have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential. PMID:25775519

  10. Conversed mutagenesis of an inactive peptide to ASIC3 inhibitor for active sites determination.

    PubMed

    Osmakov, Dmitry I; Koshelev, Sergey G; Andreev, Yaroslav A; Dyachenko, Igor A; Bondarenko, Dmitry A; Murashev, Arkadii N; Grishin, Eugene V; Kozlov, Sergey A

    2016-06-15

    Peptide Ugr9-1 from the venom of sea anemone Urticina grebelnyi selectively inhibits the ASIC3 channel and significantly reverses inflammatory and acid-induced pain in vivo. A close homolog peptide Ugr 9-2 does not have these features. To find the pharmacophore residues and explore structure-activity relationships of Ugr 9-1, we performed site-directed mutagenesis of Ugr 9-2 and replaced several positions by the corresponding residues from Ugr 9-1. Mutant peptides Ugr 9-2 T9F and Ugr 9-2 Y12H were able to inhibit currents of the ASIC3 channels 2.2 times and 1.3 times weaker than Ugr 9-1, respectively. Detailed analysis of the spatial models of Ugr 9-1, Ugr 9-2 and both mutant peptides revealed the presence of the basic-aromatic clusters on opposite sides of the molecule, each of which is responsible for the activity. Additionally, Ugr9-1 mutant with truncated N- and C-termini retained similar with the Ugr9-1 action in vitro and was equally potent in vivo model of thermal hypersensitivity. All together, these results are important for studying the structure-activity relationships of ligand-receptor interaction and for the future development of peptide drugs from animal toxins. PMID:26686983

  11. Forward and reverse mutagenesis in C. elegans

    PubMed Central

    Kutscher, Lena M.; Shaham, Shai

    2014-01-01

    Mutagenesis drives natural selection. In the lab, mutations allow gene function to be deciphered. C. elegans is highly amendable to functional genetics because of its short generation time, ease of use, and wealth of available gene-alteration techniques. Here we provide an overview of historical and contemporary methods for mutagenesis in C. elegans, and discuss principles and strategies for forward (genome-wide mutagenesis) and reverse (target-selected and gene-specific mutagenesis) genetic studies in this animal. PMID:24449699

  12. Optogenetic mutagenesis in Caenorhabditis elegans

    PubMed Central

    Noma, Kentaro; Jin, Yishi

    2015-01-01

    Reactive oxygen species (ROS) can modify and damage DNA. Here we report an optogenetic mutagenesis approach that is free of toxic chemicals and easy to perform by taking advantage of a genetically encoded ROS generator. This method relies on the potency of ROS generation by His-mSOG, the mini singlet oxygen generator, miniSOG, fused to a histone. Caenorhabditis elegans expressing His-mSOG in the germline behave and reproduce normally, without photoinduction. Following exposure to blue light, the His-mSOG animals produce progeny with a wide range of heritable phenotypes. We show that optogenetic mutagenesis by His-mSOG induces a broad spectrum of mutations including single-nucleotide variants (SNVs), chromosomal deletions, as well as integration of extrachromosomal transgenes, which complements those derived from traditional chemical or radiation mutagenesis. The optogenetic mutagenesis expands the toolbox for forward genetic screening and also provides direct evidence that nuclear ROS can induce heritable and specific genetic mutations. PMID:26632265

  13. Mixed Exciton–Charge-Transfer States in Photosystem II: Stark Spectroscopy on Site-Directed Mutants

    PubMed Central

    Romero, Elisabet; Diner, Bruce A.; Nixon, Peter J.; Coleman, Wiliam J.; Dekker, Jan P.; van Grondelle, Rienk

    2012-01-01

    We investigated the electronic structure of the photosystem II reaction center (PSII RC) in relation to the light-induced charge separation process using Stark spectroscopy on a series of site-directed PSII RC mutants from the cyanobacterium Synechocystis sp. PCC 6803. The site-directed mutations modify the protein environment of the cofactors involved in charge separation (PD1, PD2, ChlD1, and PheD1). The results demonstrate that at least two different exciton states are mixed with charge-transfer (CT) states, yielding exciton states with CT character: (PD2δ+PD1δ−ChlD1)∗673nm and (ChlD1δ+PheD1δ−)∗681nm (where the subscript indicates the wavelength of the electronic transition). Moreover, the CT state PD2+PD1− acquires excited-state character due to its mixing with an exciton state, producing (PD2+PD1−)δ∗684nm. We conclude that the states that initiate charge separation are mixed exciton-CT states, and that the degree of mixing between exciton and CT states determines the efficiency of charge separation. In addition, the results reveal that the pigment-protein interactions fine-tune the energy of the exciton and CT states, and hence the mixing between these states. This mixing ultimately controls the selection and efficiency of a specific charge separation pathway, and highlights the capacity of the protein environment to control the functionality of the PSII RC complex. PMID:22853895

  14. A computer program to display codon changes caused by mutagenesis.

    PubMed

    Sirotkin, K

    1988-04-01

    A FORTRAN program for displaying the correspondence between codon changes and different possible base changes is presented. Changes of both single bases and dimers are considered. The user can specify the mutagenesis spectrum. Additionally, the user can choose whether or not to consider single or double events in a codon and whether or not to consider the possibility that the change of two bases (a dimer) can overlap a codon boundary. Furthermore, a variety of ways may be chosen to display and summarize the codon changes that can result from the specified mutagenesis. A user-supplied sequence or the genetic code table can be analyzed. PMID:3167596

  15. Yeast cytochrome c peroxidase: mutagenesis and expression in Escherichia coli show tryptophan-51 is not the radical site in compound I

    SciTech Connect

    Fishel, L.A.; Villafranca, J.E.; Mauro, J.M.; Kraut, J.

    1987-01-27

    Using oligonucleotide-directed site-specific mutagenesis, they have constructed a system for the mutation and expression of yeast cytochrome c peroxidase (CCP, EC 1.11.1.5) in Escherichia coli and applied it to test the hypothesis that Trp-51 is the locus of the free radical observed in compound I of CCP. The system was created by substituting a CCP gene modified by site-directed mutagenesis, CCP(MI), for the fol gene in a vector previously used for mutagenesis and overexpression of dihydrofolate reductase. E. coli transformed with the resulting plasmid produced the CCP(MI) enzyme in large quantities, more than 15 mg/L of cell culture, of which 10% is holo- and 90% is apo-CCP(MI). The apoenzyme was easily converted to holoenzyme by the addition of bovine hemin. Purified CCP(MI) has the same catalytic activity and spectra as bakers' yeast CCP. A mutation has been made in CCP(MI), Trp-51 to Phe. The Phe-51 mutant protein CCP(MI,F51) is fully active, and the electron paramagnetic resonance (EPR) spectrum, at 89 K, of its oxidized intermediate, compound I, displays a strong sharp resonance at g = 2.004, which is very similar to the signal observed for compound I of both bakers' yeast CCP and CCP(MI). However, UV-visible and EPR spectroscopy revealed that the half-life of CCP(MI,F51) compound I at 23 /sup 0/C is only 1.4% of that observed for the compound I forms of CCP(MI) or bakers' yeast CCP. Thus, Trp-51 is not necessary for the formation of the free radical observed in compound I but appears to exert a significant influence on its stability.

  16. Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

    PubMed

    Gagnon, James A; Valen, Eivind; Thyme, Summer B; Huang, Peng; Akhmetova, Laila; Ahkmetova, Laila; Pauli, Andrea; Montague, Tessa G; Zimmerman, Steven; Richter, Constance; Schier, Alexander F

    2014-01-01

    The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes. PMID:24873830

  17. A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data*

    PubMed Central

    Ahuja, Shivani; Jahr, Nicole; Im, Sang-Choul; Vivekanandan, Subramanian; Popovych, Nataliya; Le Clair, Stéphanie V.; Huang, Rui; Soong, Ronald; Xu, Jiadi; Yamamoto, Kazutoshi; Nanga, Ravi P.; Bridges, Angela; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2013-01-01

    Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes. PMID:23709268

  18. Site-directed isotope labeling and FTIR spectroscopy: assignment of tyrosine bands in the bR-->M difference spectrum of bacteriorhodopsin.

    PubMed

    Liu, X M; Sonar, S; Lee, C P; Coleman, M; RajBhandary, U L; Rothschild, K J

    1995-01-01

    Fourier transform infrared difference spectroscopy has been used extensively to probe structural changes in bacteriorthodopsin and other retinal proteins. However, the absence of a general method to assign bands to individual chemical groups in a protein has limited the application of this technique. While site-directed mutagenesis has been successful in special cases for such assignments, in general, this approach induces perturbations in the structure and function of the protein, thereby preventing unambiguous band assignments. A new approach has recently been reported (Sonar et al., Nature Struct. Biol. 1 (1994) 512-517) which involves cell-free expression of bacteriorhodopsin and site-directed isotope labeling (SDIL). We have now used this method to re-examine bands assigned in the bR-->M difference spectrum to tyrosine residues. Our results show that out of 11 tyrosines in bR, only Tyr 185 is structurally active. This work further demonstrates the power of SDIL and FTIR to probe conformational changes at the level of individual amino acid residues in proteins. PMID:7662870

  19. A low-toxic site-directed mutant of Clostridium perfringens ε-toxin as a potential candidate vaccine against enterotoxemia.

    PubMed

    Li, Qing; Xin, Wenwen; Gao, Shan; Kang, Lin; Wang, Jinglin

    2013-11-01

    Clostridium perfringens epsilon toxin (ETX), one of the most potent toxins known, is a potential biological weapon; therefore, the development of an effective vaccine is important for preventing intoxication or disease by ETX. In this study, genetically detoxified epsilon toxin mutants were developed as candidate vaccines. We used site-directed mutagenesis to mutate the essential amino acid residues (His106, Ser111 and Phe199). Six site-directed mutants of ETX (mETX (H106P) , mETX (S111H) , mETX (S111Y) , mETX (F199H) , mETX (F199E) , mETX (S111YF199E) ) were generated and then expressed in Escherichia coli. Both mETX (F199E) and mETX (H106P) with low or non-cytotoxicity that retained their immunogenicity were selected to immunize mice 3 times, and the mouse survival data were recorded after challenging with recombinant wild-type ETX. mETX (F199E) induces the same protection as mETX (H106P) , which was reported previously as a promising toxin mutant for vaccine, and both of them could protect immunized mice against a 100× LD₅₀ dose of active wild-type recombinant ETX. This work showed that mETX (F199E) is another promising candidate vaccine against enterotoxemia and other diseases caused by ETX. PMID:23835363

  20. A low-toxic site-directed mutant of Clostridium perfringens ε-toxin as a potential candidate vaccine against enterotoxemia

    PubMed Central

    Li, Qing; Xin, Wenwen; Gao, Shan; Kang, Lin; Wang, Jinglin

    2013-01-01

    Clostridium perfringens epsilon toxin (ETX), one of the most potent toxins known, is a potential biological weapon; therefore, the development of an effective vaccine is important for preventing intoxication or disease by ETX. In this study, genetically detoxified epsilon toxin mutants were developed as candidate vaccines. We used site-directed mutagenesis to mutate the essential amino acid residues (His106, Ser111 and Phe199). Six site-directed mutants of ETX (mETXH106P, mETXS111H, mETXS111Y, mETXF199H, mETXF199E, mETXS111YF199E) were generated and then expressed in Escherichia coli. Both mETXF199E and mETXH106P with low or non-cytotoxicity that retained their immunogenicity were selected to immunize mice 3 times, and the mouse survival data were recorded after challenging with recombinant wild-type ETX. mETXF199E induces the same protection as mETXH106P, which was reported previously as a promising toxin mutant for vaccine, and both of them could protect immunized mice against a 100× LD50 dose of active wild-type recombinant ETX. This work showed that mETXF199E is another promising candidate vaccine against enterotoxemia and other diseases caused by ETX. PMID:23835363

  1. Disaggregation of Alzheimer β -Amyloid by Site-Directed mAb

    NASA Astrophysics Data System (ADS)

    Solomon, Beka; Koppel, Rela; Frankel, Dan; Hanan-Aharon, Eilat

    1997-04-01

    In Alzheimer disease, β -amyloid peptide accumulates in the brain as insoluble amyloid plaques. Amyloid filaments, similar to those found in amyloid plaques, can be assembled in vitro from chemically synthesized β -peptides. In this study, we report that antibodies raised against the N-terminal region (1-28) of the β -amyloid peptide bind to the in vitro-formed β -amyloid assemblies, leading to disaggregation of the fibrils and partial restoration of the peptide's solubility. The concomitant addition of fibrillar β -amyloid with these antibodies to PC 12 cells leads to the inhibition of the neurotoxic effects of β -amyloid. Some of the mAbs raised against soluble β -peptide (1-28) have been found to prevent in vitro fibrillar aggregation of β -amyloid peptide. These experimental data suggest that site-directed mAbs interfere with the aggregation of β -amyloid and trigger reversal to its nontoxic, normal components. The above findings give hints on how to convert in vivo senile plaques into nontoxic, diffuse components and may have therapeutic interest for those studying Alzheimer disease and other human diseases related to amyloidogenic properties of physiological peptides and proteins.

  2. Structural insights from random mutagenesis of Campylobacter jejuni oligosaccharyltransferase PglB

    PubMed Central

    2012-01-01

    Background Protein glycosylation is of fundamental importance in many biological systems. The discovery of N-glycosylation in bacteria and the functional expression of the N-oligosaccharyltransferase PglB of Campylobacter jejuni in Escherichia coli enabled the production of engineered glycoproteins and the study of the underlying molecular mechanisms. A particularly promising application for protein glycosylation in recombinant bacteria is the production of potent conjugate vaccines where polysaccharide antigens of pathogenic bacteria are covalently bound to immunogenic carrier proteins. Results In this study capsular polysaccharides of the clinically relevant pathogen Staphylococcus aureus serotype 5 (CP5) were expressed in Escherichia coli and linked in vivo to a detoxified version of Pseudomonas aeruginosa exotoxin (EPA). We investigated which amino acids of the periplasmic domain of PglB are crucial for the glycosylation reaction using a newly established 96-well screening system enabling the relative quantification of glycoproteins by enzyme-linked immunosorbent assay. A random mutant library was generated by error-prone PCR and screened for inactivating amino acid substitutions. In addition to 15 inactive variants with amino acid changes within the previously known, strictly conserved WWDYG motif of N-oligosaccharyltransferases, 8 inactivating mutations mapped to a flexible loop in close vicinity of the amide nitrogen atom of the acceptor asparagine as revealed in the crystal structure of the homologous enzyme C. lari PglB. The importance of the conserved loop residue H479 for glycosylation was confirmed by site directed mutagenesis, while a change to alanine of the adjacent, non-conserved L480 had no effect. In addition, we investigated functional requirements in the so-called MIV motif of bacterial N-oligosaccharyltransferases. Amino acid residues I571 and V575, which had been postulated to interact with the acceptor peptide, were subjected to cassette

  3. TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice.

    PubMed

    Li, Ting; Liu, Bo; Chen, Chih Ying; Yang, Bing

    2016-05-20

    Over the last decades, much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology. The breakthrough has been made in recent years with the advent of sequence-specific endonucleases, especially zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) guided nucleases (e.g., Cas9). In higher eukaryotic organisms, site-directed mutagenesis usually can be achieved through non-homologous end-joining (NHEJ) repair to the DNA double-strand breaks (DSBs) caused by the exogenously applied nucleases. However, site-specific gene replacement or genuine genome editing through homologous recombination (HR) repair to DSBs remains a challenge. As a proof of concept gene replacement through TALEN-based HR in rice (Oryza sativa), we successfully produced double point mutations in rice acetolactate synthase gene (OsALS) and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations. After ballistic delivery into rice calli of TALEN construct and donor DNA, nine HR events with different genotypes of OsALS were obtained in T0 generation at the efficiency of 1.4%-6.3% from three experiments. The HR-mediated gene edits were heritable to the progeny of T1 generation. The edited T1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance. The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms. PMID:27180265

  4. Beta-D-xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-directed Mutagenesis, Alternate Substrates, and Inhibitor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium (SXA) is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D-xylose. Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic ...

  5. Site-directed mutagenesis indicates an important role of cysteines 76 and 181 in the catalysis of hydantoin racemase from Sinorhizobium meliloti

    PubMed Central

    Martínez-Rodríguez, Sergio; Andújar-Sánchez, Montserrat; Neira, Jose L.; Clemente-Jiménez, Josefa M.; Jara-Pérez, Vicente; Rodríguez-Vico, Felipe; Las Heras-Vázquez, Francisco J.

    2006-01-01

    Hydantoin racemase enzyme plays a crucial role in the reaction cascade known as “hydantoinase process.” In conjunction with a stereoselective hydantoinase and a stereospecific carbamoylase, it allows the total conversion from D,L-5-monosubstituted hydantoins, with a low rate of racemization, to optically pure D- or L-amino acids. Residues Cys76 and Cys181 belonging to hydantoin racemase from Sinorhizobium meliloti (SmeHyuA) have been proved to be involved in catalysis. Here, we report biophysical data of SmeHyuA Cys76 and Cys181 to alanine mutants, which point toward a two-base mechanism for the racemization of 5-monosubstituted hydantoins. The secondary and the tertiary structure of the mutants were not significantly affected, as shown by circular dichroism. Calorimetric and fluorescence experiments have shown that Cys76 is responsible for recognition and proton retrieval of D-isomers, while Cys181 is responsible for L-isomer recognition and racemization. This recognition process is further supported by measurements of protein stability followed by chemical denaturation in the presence of the corresponding compound. PMID:17132860

  6. Engineering of Recombinant Poplar Deoxy-D-Xylulose-5-Phosphate Synthase (PtDXS) by Site-Directed Mutagenesis Improves Its Activity

    PubMed Central

    Banerjee, Aparajita; Preiser, Alyssa L.

    2016-01-01

    Deoxyxylulose 5-phosphate synthase (DXS), a thiamine diphosphate (ThDP) dependent enzyme, plays a regulatory role in the methylerythritol 4-phosphate (MEP) pathway. Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the end products of this pathway, inhibit DXS by competing with ThDP. Feedback inhibition of DXS by IDP and DMADP constitutes a significant metabolic regulation of this pathway. The aim of this work was to experimentally test the effect of key residues of recombinant poplar DXS (PtDXS) in binding both ThDP and IDP. This work also described the engineering of PtDXS to improve the enzymatic activity by reducing its inhibition by IDP and DMADP. We have designed and tested modifications of PtDXS in an attempt to reduce inhibition by IDP. This could possibly be valuable by removing a feedback that limits the usefulness of the MEP pathway in biotechnological applications. Both ThDP and IDP use similar interactions for binding at the active site of the enzyme, however, ThDP being a larger molecule has more anchoring sites at the active site of the enzyme as compared to the inhibitors. A predicted enzyme structure was examined to find ligand-enzyme interactions, which are relatively more important for inhibitor-enzyme binding than ThDP-enzyme binding, followed by their modifications so that the binding of the inhibitors can be selectively affected compared to ThDP. Two alanine residues important for binding ThDP and the inhibitors were mutated to glycine. In two of the cases, both the IDP inhibition and the overall activity were increased. In another case, both the IDP inhibition and the overall activity were reduced. This provides proof of concept that it is possible to reduce the feedback from IDP on DXS activity. PMID:27548482

  7. X-ray structures of recombinant yeast cytochrome c peroxidase and three heme-cleft mutants prepared by site-directed mutagenesis

    SciTech Connect

    Wang, J.; Mauro, J.M.; Edwards, S.L.; Oatley, S.J.; Fishel, L.A.; Ashford, V.A.; Xuong, Nguyenhuu; Kraut, J. )

    1990-08-07

    The 2.2-{angstrom} x-ray structure for CCP(MI), a plasmid-encoded form of Saccharomyces cerevisiae cytochrome c peroxidase (CCP) expressed in Escherichia coli has been solved, together with the structures of three specifically designed single-site heme-cleft mutants. The structure of CCP(MI) was solved by using molecular replacement methods, since its crystals grow differently from the crystals of CCP isolated from bakers' yeast used previously for structural solution. Small distal-side differences between CCP(MI) and bakers' yeast CCP are observed, presumably due to a strain-specific Thr-53 {yields} Ile substitution in CCP(MI). The observation of a vacant sixth coordination site in this structure differs from the results of solution resonance Raman studies, which predict hexacoordinated high-spin iron. The coordination behavior of this W51F mutant is apparently altered in the presence of a precipitating agent, 30% 2-methyl-2,4-pentanediol. A proximal Trp-191 {yields} Phe mutant that has substantially diminished enzyme activity and altered magnetic properties accommodates the substitution by allowing the side chain of Phe-191, together with the segment of backbone to which it is attached, to move toward the heme. This relatively large local perturbation is accompanied by numerous small adjustments resulting in a slight overall compression of the enzyme's proximal domain; however, the iron coordination sphere is essentially unchanged. This structures rules out a major alteration in protein conformation as a reason for the dramatically decreased activity of the W191F mutant. From the alteration of local structure that occurs in this mutant, coupled with the results of preliminary functional studies, the authors infer that Asp-235 exerts influence on the heme iron so as to keep its sixth coordination site vacant, and hence reactive with peroxide substrate, over a wide pH range.

  8. Insights into the key interactions between human protein phosphatase 5 and cantharidin using molecular dynamics and site-directed mutagenesis bioassays.

    PubMed

    Liu, Ji-Yuan; Chen, Xi-En; Zhang, Ya-Lin

    2015-01-01

    Serine/threonine protein phosphatase 5 (PP5) is a promising novel target for anticancer therapies. This work aims to uncover the key interactions at the atomic level between PP5 and three inhibitors (cantharidin, norcantharidin and endothall). We found that, unlike previous report, Arg 100 contributes less to PP5-inhibitor binding, and the residues His 69, Asn 128, His 129, Arg 225, His 252 and Arg 250 are of importance to PP5-inhibitor binding. The hydrophobic interactions established between the residues Val 254, Phe 271 and Tyr 276, especially Glu 253, are very important to enhance the inhibitive interaction. We suggested that, to increase the inhibitory activity, the interactions of inhibitor with three negatively charged unfavorable interaction residues, Asp 99, Glu 130 and Asp 213, should be avoided. However, the interactions of inhibitor with favorable interaction residue Arg 250 could enhance the inhibitory activity. The Manganese ion 2 (MN2) unfavorably contribute to the total interaction free energies. The coordination between MN2 and chemical group of inhibitor should be eliminated. This work provides insight into how cantharidin and its analogs bind to PP5c at the atomic level and will facilitate modification of cantharidin-like chemicals to rationally develop more specific and less cytotoxic anti-cancer drugs. PMID:26190207

  9. Site-directed mutagenesis of Lys-174, Asp-179 and Asp-191 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

    PubMed Central

    Bertrand, L; Deprez, J; Vertommen, D; Di Pietro, A; Hue, L; Rider, M H

    1997-01-01

    In a structural model of the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase based on the analogy with adenylate kinase, Lys-174, Asp-179 and Asp-191 residues are located in the putative active site. Asp-179 and Asp-191 are conserved in all known 6-phosphofructo-2-kinase sequences. In contrast, Lys-174 is conserved except in a yeast isoenzyme, fbp26, where it is replaced by glycine. Yeast fbp26 possesses fructose-2,6-bisphosphatase activity, but is devoid of 6-phosphofructo-2-kinase activity. Mutation of Asp-179 and Asp-191 of the rat liver isoenzyme to alanine increased the Km of 6-phosphofructo-2-kinase for fructose 6-phosphate 2000- and 1000-fold respectively, whereas mutation of Lys-174 to glycine decreased the Vmax of 6-phosphofructo-2-kinase more than 4000-fold. In contrast, none of the mutations affected the kinetic parameters of fructose-2,6-bisphosphatase. CD and fluorescence measurements indicated that the mutations had no effect on the structure and stability of the recombinant proteins. The results show that Asp-179 and Asp-191 participate in fructose 6-phosphate binding, whereas Lys-174 is important for catalysis. Therefore the natural mutation of Lys-174 to glycine in the fbp26 yeast isoenzyme could explain the lack of 6-phosphofructo-2-kinase activity. These results support a novel 6-phosphofructo-2-kinase structure model based on adenylate kinase. PMID:9032446

  10. Molecular Insights into the Coding Region Determinant-binding Protein-RNA Interaction through Site-directed Mutagenesis in the Heterogeneous Nuclear Ribonucleoprotein-K-homology Domains*

    PubMed Central

    Barnes, Mark; van Rensburg, Gerrit; Li, Wai-Ming; Mehmood, Kashif; Mackedenski, Sebastian; Chan, Ching-Man; King, Dustin T.; Miller, Andrew L.; Lee, Chow H.

    2015-01-01

    The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function. PMID:25389298

  11. Beta-D-xylosidase from Selenomonas ruminantium: role of glutamate 186 in catalysis revealed by site-directed mutagenesis, alternate substrates, and inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beta-D-xylosidase from Selenomonas ruminantium is the best catalyst known for promoting hydrolysis of 1,4-beta-D-xylooligosaccharides, and it has potential utility in industrial saccharification processes. Kinetic parameters, kcat and kcat/Km, are more than 10-fold larger than those reported for th...

  12. Probing the mechanistic role of the long α-helix in subunit L of respiratory Complex I from Escherichia coli by site-directed mutagenesis

    PubMed Central

    Belevich, Galina; Knuuti, Juho; Verkhovsky, Michael I; Wikström, Mårten; Verkhovskaya, Marina

    2011-01-01

    The C-terminus of the NuoL subunit of Complex I includes a long amphipathic α-helix positioned parallel to the membrane, which has been considered to function as a piston in the proton pumping machinery. Here, we have introduced three types of mutations into the nuoL gene to test the piston-like function. First, NuoL was truncated at its C- and N-termini, which resulted in low production of a fragile Complex I with negligible activity. Second, we mutated three partially conserved residues of the amphipathic α-helix: Asp and Lys residues and a Pro were substituted for acidic, basic or neutral residues. All these variants exhibited almost a wild-type phenotype. Third, several substitutions and insertions were made to reduce rigidity of the amphipathic α-helix, and/or to change its geometry. Most insertions/substitutions resulted in a normal growth phenotype, albeit often with reduced stability of Complex I. In contrast, insertion of six to seven amino acids at a site of the long α-helix between NuoL and M resulted in substantial loss of proton pumping efficiency. The implications of these results for the proton pumping mechanism of Complex I are discussed. PMID:22060017

  13. Insights into the key interactions between human protein phosphatase 5 and cantharidin using molecular dynamics and site-directed mutagenesis bioassays

    PubMed Central

    Liu, Ji-Yuan; Chen, Xi-En; Zhang, Ya-Lin

    2015-01-01

    Serine/threonine protein phosphatase 5 (PP5) is a promising novel target for anticancer therapies. This work aims to uncover the key interactions at the atomic level between PP5 and three inhibitors (cantharidin, norcantharidin and endothall). We found that, unlike previous report, Arg 100 contributes less to PP5-inhibitor binding, and the residues His 69, Asn 128, His 129, Arg 225, His 252 and Arg 250 are of importance to PP5-inhibitor binding. The hydrophobic interactions established between the residues Val 254, Phe 271 and Tyr 276, especially Glu 253, are very important to enhance the inhibitive interaction. We suggested that, to increase the inhibitory activity, the interactions of inhibitor with three negatively charged unfavorable interaction residues, Asp 99, Glu 130 and Asp 213, should be avoided. However, the interactions of inhibitor with favorable interaction residue Arg 250 could enhance the inhibitory activity. The Manganese ion 2 (MN2) unfavorably contribute to the total interaction free energies. The coordination between MN2 and chemical group of inhibitor should be eliminated. This work provides insight into how cantharidin and its analogs bind to PP5c at the atomic level and will facilitate modification of cantharidin-like chemicals to rationally develop more specific and less cytotoxic anti-cancer drugs. PMID:26190207

  14. Effect of Site-directed Mutagenesis of Methylglyoxal- Modifiable Arginine Residues on the Structure and Chaperone Function of Human αA-crystallin

    PubMed Central

    Biswas, Ashis; Miller, Antonia; Oya-Ito, Tomoko; Santhoshkumar, Puttur; Bhat, Manjunatha; Nagaraj, Ram H.

    2008-01-01

    We reported previously that chemical modification of human αA-crystallin by a metabolic dicarbonyl compound, methylglyoxal (MGO), enhances its chaperone-like function, a phenomenon which we attributed to formation of argpyrimidine at arginine residues (R) 21, 49 and 103. This structural change removes the positive charge on the arginine residues. To explore this mechanism further, we replaced these three R residues with a neutral alanine (A) residue one at time or in combination and examined the impact on the structure and chaperone function. Measurement of intrinsic tryptophan fluorescence and near-UV CD spectra revealed alteration of the microenvironment of aromatic amino acid residue in mutant proteins. When compared to wild type (wt) αA-crystallin, the chaperone function of R21A and R103A mutants increased 20% and 18% as measured by the insulin aggregation assay, and increased it as much as 39% and 28% when measured by the citrate synthase (CS) aggregation assay. While the R49A mutant lost most of its chaperone function, R21A/R103A and R21A/R49A/R103A mutants had slightly better function (6–14% and 10–14%) than the wt protein in these assays. R21A and R103A mutants had higher surface hydrophobicity than wt αA-crystallin, but the R49A mutant had lower hydrophobicity. R21A and R103A mutants, but not the R49A mutant, were more efficient than wt protein in refolding guanidine hydrochloride-treated malate dehydrogenase to its native state. Our findings indicate that the positive charges on R21, R49 and R103 are important determinants of the chaperone function of αA-crystallin and suggest that chemical modification of arginine residues may play a role in protein aggregation during lens aging and cataract formation. PMID:16584192

  15. Beta-D-xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-Directed Mutagenesis, Alternate Substrates, and Active-site Inhibitor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium (SXA) is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D xylose. Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic ...

  16. Improvement of expression level of keratinase Sfp2 from Streptomyces fradiae by site-directed mutagenesis of its N-terminal pro-sequence.

    PubMed

    Li, Junxia; Chen, Dongdong; Yu, Zhanqiao; Zhao, Longmei; Zhang, Rijun

    2013-05-01

    The keratinase Sfp2, produced by Streptomyces fradiae var. k11, is a serine alkaline protease first synthesized as pre-pro-mature precursor, of which the N-terminal propeptide must be autocatalytically cleaved on the C-terminal of P1 amino acid to produce mature enzyme. Single amino acid substitutions were introduced at positions -1 and -2 to improve the expression level of mature Sfp2. The specific activity of L(-1)F mutant (48935 U/mg) was nine times that of wild-type Sfp2, whereas the mutants L(-1)D, L(-1)G, L(-1)H, K(-2)E, and K(-2)L had 2-52 % of the specific activity of wild-type. The yield of mature Sfp2 of L(-1)F mutant was estimated to be 800 μg/mg total protein and 112 mg/l culture supernatant, nine and twice that of wild-type, respectively. The L(-1)F mutant exhibited similar enzymatic properties to wild-type. PMID:23355035

  17. Site-directed mutagenesis of the human DNA repair enzyme HAP1: identification of residues important for AP endonuclease and RNase H activity.

    PubMed

    Barzilay, G; Walker, L J; Robson, C N; Hickson, I D

    1995-05-11

    HAP1 protein, the major apurinic/apyrimidinic (AP) endonuclease in human cells, is a member of a homologous family of multifunctional DNA repair enzymes including the Escherichia coli exonuclease III and Drosophila Rrp1 proteins. The most extensively characterised member of this family, exonuclease III, exhibits both DNA- and RNA-specific nuclease activities. Here, we show that the RNase H activity characteristic of exonuclease III has been conserved in the human homologue, although the products resulting from RNA cleavage are dissimilar. To identify residues important for enzymatic activity, five mutant HAP1 proteins containing single amino acid substitutions were purified and analysed in vitro. The substitutions were made at sites of conserved amino acids and targeted either acidic or histidine residues because of their known participation in the active sites of hydrolytic nucleases. One of the mutant proteins (replacement of Asp-219 by alanine) showed a markedly reduced enzymatic activity, consistent with a greatly diminished capacity to bind DNA and RNA. In contrast, replacement of Asp-90, Asp-308 or Glu-96 by alanine led to a reduction in enzymatic activity without significantly compromising nucleic acid binding. Replacement of His-255 by alanine led to only a very small reduction in enzymatic activity. Our data are consistent with the presence of a single catalytic active site for the DNA- and RNA-specific nuclease activities of the HAP1 protein. PMID:7784208

  18. Identification of the residues responsible for the alkaline inhibition of Cu,Zn superoxide dismutase: a site-directed mutagenesis approach.

    PubMed Central

    Polticelli, F.; Battistoni, A.; O'Neill, P.; Rotilio, G.; Desideri, A.

    1996-01-01

    The catalytic rate of wild type, two single (Lys 120-->Leu, Lys 134-->Thr), and one double (Lys 120-->Leu-Lys 134-->Thr) mutants of Xenopus laevis B Cu,Zn superoxide dismutase has been studied by pulse radiolysis as a function of pH. The pH dependence curve of the wild-type enzyme can be deconvoluted by two deprotonation equilibria, at pH 9.3 (pK1) and at pH 11.3 (pK2). Catalytic rate measurements on single and double mutants indicate that pK1 is mainly due to the deprotonation of Lys 120 and Lys 134, with only a minor contribution from other surface basic residues, whereas pK2 is due to titration of the invariant Arg 141, likely coupled to deprotonation of the copper-bound water molecule. Accordingly, Brownian dynamics simulations carried out as a function of pH reproduce well the pH dependence of the catalytic rate, when the experimentally determined pKs are assigned to Lys 120, Lys 134, and Arg 141. PMID:8745402

  19. Enhancement of the alcoholytic activity of alpha-amylase AmyA from Thermotoga maritima MSB8 (DSM 3109) by site-directed mutagenesis.

    PubMed

    Damián-Almazo, Juanita Yazmin; Moreno, Alina; López-Munguía, Agustin; Soberón, Xavier; González-Muñoz, Fernando; Saab-Rincón, Gloria

    2008-08-01

    AmyA, an alpha-amylase from the hyperthermophilic bacterium Thermotoga maritima, is able to hydrolyze internal alpha-1,4-glycosidic bonds in various alpha-glucans at 85 degrees C as the optimal temperature. Like other glycoside hydrolases, AmyA also catalyzes transglycosylation reactions, particularly when oligosaccharides are used as substrates. It was found that when methanol or butanol was used as the nucleophile instead of water, AmyA was able to catalyze alcoholysis reactions. This capability has been evaluated in the past for some alpha-amylases, with the finding that only the saccharifying fungal amylases from Aspergillus niger and from Aspergillus oryzae present measurable alcoholysis activity (R. I. Santamaria, G. Del Rio, G. Saab, M. E. Rodriguez, X. Soberon, and A. Lopez, FEBS Lett. 452:346-350, 1999). In the present work, we found that AmyA generates larger quantities of alkyl glycosides than any amylase reported so far. In order to increase the alcoholytic activity observed in AmyA, several residues were identified and mutated based on previous analogous positions in amylases, defining the polarity and geometry of the active site. Replacement of residue His222 by glutamine generated an increase in the alkyl glucoside yield as a consequence of a higher alcoholysis/hydrolysis ratio. The same change in specificity was observed for the mutants H222E and H222D, but instability of these mutants toward alcohols decreased the yield of alkyl glucoside. PMID:18552192

  20. Spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene.

    PubMed Central

    Dolja, V V; Herndon, K L; Pirone, T P; Carrington, J C

    1993-01-01

    The RNA genome of tobacco etch potyvirus (TEV) was engineered to express bacterial beta-glucuronidase (GUS) fused to the virus helper component proteinase (HC-Pro). It was shown previously that prolonged periods (approximately 1 month) of TEV-GUS propagation in plants resulted in the appearance of spontaneous deletion variants. Nine deletion mutants were identified by nucleotide sequence analysis of 40 cDNA clones obtained after polymerase chain reaction amplification. The mutants were missing between 1,741 and 2,074 nucleotides from TEV-GUS, including the sequences coding for most of GUS and the N-terminal region of HC-Pro. This region of HC-Pro contains determinants involved in helper component activity during aphid transmission, as well as a highly conserved series of cysteine residues. The deletion variants were shown to replicate and move systemically without the aid of a helper virus. Infectious viruses harboring the two largest HC-Pro deletions (termed TEV-2del and TEV-7del) were reconstructed by subcloning the corresponding mutated regions into full-length DNA copies of the TEV genome. Characterization of these and additional variants derived by site-directed mutagenesis demonstrated that deletion of sequences coding for the HC-Pro N-terminal domain had a negative effect on accumulation of viral RNA and coat protein. The TEV-2del variant possessed an aphid-nontransmissible phenotype that could be rescued partially by prefeeding of aphids on active HC-Pro from another potyvirus. These data suggest that the N-terminal domain of HC-Pro or its coding sequence enhances virus replication or genome expression but does not provide an activity essential for these processes. The function of this domain, as well as a proposed deletion mechanism involving nonhomologous recombination, is discussed. Images PMID:8371351

  1. Perturbation of bacteriochlorophyll molecules in Fenna-Matthews-Olson protein complexes through mutagenesis of cysteine residues.

    PubMed

    Saer, Rafael; Orf, Gregory S; Lu, Xun; Zhang, Hao; Cuneo, Matthew J; Myles, Dean A A; Blankenship, Robert E

    2016-09-01

    The Fenna-Matthews-Olson (FMO) pigment-protein complex in green sulfur bacteria transfers excitation energy from the chlorosome antenna complex to the reaction center. In understanding energy transfer in the FMO protein, the individual contributions of the bacteriochlorophyll pigments to the FMO complex's absorption spectrum could provide detailed information with which molecular and energetic models can be constructed. The absorption properties of the pigments, however, are such that their spectra overlap significantly. To overcome this, we used site-directed mutagenesis to construct a series of mutant FMO complexes in the model green sulfur bacterium Chlorobaculum tepidum (formerly Chlorobium tepidum). Two cysteines at positions 49 and 353 in the C. tepidum FMO complex, which reside near hydrogen bonds between BChls 2 and 3, and their amino acid binding partner serine 73 and tyrosine 15, respectively, were changed to alanine residues. The resulting C49A, C353A, and C49A C353A double mutants were analyzed with a combination of optical absorption and circular dichroism (CD) spectroscopies. Our results revealed changes in the absorption properties of several underlying spectral components in the FMO complex, as well as the redox behavior of the complex in response to the reductant sodium dithionite. A high-resolution X-ray structure of the C49A C353A double mutant reveals that these spectral changes appear to be independent of any major structural rearrangements in the FMO mutants. Our findings provide important tests for theoretical calculations of the C. tepidum FMO absorption spectrum, and additionally highlight a possible role for cysteine residues in the redox activity of the pigment-protein complex. PMID:27114180

  2. Mutagenesis for improvement of activity and thermostability of amylomaltase from Corynebacterium glutamicum.

    PubMed

    Nimpiboon, Pitchanan; Kaulpiboon, Jarunee; Krusong, Kuakarun; Nakamura, Shigeyoshi; Kidokoro, Shun-Ichi; Pongsawasdi, Piamsook

    2016-05-01

    This work aims to improve thermostability of amylomaltase from a mesophilic Corynebacterium glutamicum (CgAM) by random and site-directed mutagenesis. From error prone PCR, a mutated CgAM with higher thermostability at 50°C compared to the wild-type was selected and sequenced. The result showed that the mutant contains a single mutation of A406V. Site-directed mutagenesis was then performed to construct A406V and A406L. Both mutated CgAMs showed higher intermolecular transglucosylation activity with an upward shift in the optimum temperature and a slight increase in the optimum pH for disproportionation and cyclization reactions. Thermostability of both mutated CgAMs at 35-40°C was significantly increased with a higher peak temperature from DSC spectra when compared to the wild-type. A406V had a greater effect on activity and thermostability than A406L. The catalytic efficiency values kcat/Km of A406V- and A406L-CgAMs were 2.9 and 1.4 times higher than that of the wild-type, respectively, mainly due to a significant increase in kcat. LR-CD product analysis demonstrated that A406V gave higher product yield, especially at longer incubation time and higher temperature, in comparison to the wild-type enzyme. PMID:26875536

  3. MECHANISM OF CHEMICAL MUTAGENESIS IV.

    PubMed Central

    Lorkiewicz, Z.; Szybalski, Waclaw

    1961-01-01

    Lorkiewicz, Z. (University of Wisconsin, Madison), and Waclaw Szybalski. Mechanism of chemical mutagenesis. IV. Reaction between triethylene melamine and nucleic acid components. J. Bacteriol. 82: 195–201. 1961.—Triethylene melamine interacts primarily with phosphorylated intracellular deoxyribonucleic acid (DNA) precursors and not with DNA. It was found by direct chemical and chromatographic analysis that only pyrimidine precursors of nucleic acids are attacked by triethylene melamine. In the course of the triethylene melamine-deoxycytidine reaction the mutagenicity of the reaction mixture is lost, but the mutagenicity of the triethylene melamine-thymidine reaction products significantly increases above that of the reaction substrates. Several steps are postulated to explain the mechanism of the triethylene melamine-initiated mutagenic reaction: (i) Reaction I, semireversible uptake of triethylene melamine; (ii) reaction II, chemical interaction between triethylene melamine and intracellular thymidine mono- or triphosphate with the production of a functional analogue of the latter; (iii) incorporation of this fraudulent analogue into the newly formed DNA strand; (iv) occurrence of self-perpetuating errors in the sequence of natural bases during subsequent rounds of replication of the analogue-containing DNA strand. It is postulated that the mechanism of mutagenic responses to different types of mutagens can fit either a simplified (mutagenic base analogues) or extended version (radiation) of this schema. PMID:16561917

  4. CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening.

    PubMed

    Ito, Yasuhiro; Nishizawa-Yokoi, Ayako; Endo, Masaki; Mikami, Masafumi; Toki, Seiichi

    2015-11-01

    Site-directed mutagenesis using genetic approaches can provide a wealth of resources for crop breeding as well as for biological research. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 endonuclease (CRISPR/Cas9) system is a novel strategy used to induce mutations in a specific genome region; the system functions in a variety of organisms, including plants. Here, we report application of the CRISPR/Cas9 system to efficient mutagenesis of the tomato genome. In this study, we targeted the tomato RIN gene, which encodes a MADS-box transcription factor regulating fruit ripening. Three regions within the gene were targeted and mutations consisting either of a single base insertion or deletion of more than three bases were found at the Cas9 cleavage sites in T0 regenerated plants. The RIN-protein-defective mutants produced incomplete-ripening fruits in which red color pigmentation was significantly lower than that of wild type, while heterologous mutants expressing the remaining wild-type gene reached full-ripening red color, confirming the important role of RIN in ripening. Several mutations that were generated at three independent target sites were inherited in the T1 progeny, confirming the applicability of this mutagenesis system in tomato. PMID:26408904

  5. Site-directed spin labeling of proteins for distance measurements in vitro and in cells.

    PubMed

    Roser, P; Schmidt, M J; Drescher, M; Summerer, D

    2016-06-15

    Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy allows studying the structure, dynamics, and interactions of proteins via distance measurements in the nanometer range. We here give an overview of available spin labels, the strategies for their introduction into proteins, and the associated potentials for protein structural studies in vitro and in the context of living cells. PMID:27181459

  6. Targeted mutagenesis of Arabidopsis thaliana using engineered TAL effector nucleases.

    PubMed

    Christian, Michelle; Qi, Yiping; Zhang, Yong; Voytas, Daniel F

    2013-10-01

    Custom TAL effector nucleases (TALENs) are increasingly used as reagents to manipulate genomes in vivo. Here, we used TALENs to modify the genome of the model plant, Arabidopsis thaliana. We engineered seven TALENs targeting five Arabidopsis genes, namely ADH1, TT4, MAPKKK1, DSK2B, and NATA2. In pooled seedlings expressing the TALENs, we observed somatic mutagenesis frequencies ranging from 2-15% at the intended targets for all seven TALENs. Somatic mutagenesis frequencies as high as 41-73% were observed in individual transgenic plant lines expressing the TALENs. Additionally, a TALEN pair targeting a tandemly duplicated gene induced a 4.4-kb deletion in somatic cells. For the most active TALEN pairs, namely those targeting ADH1 and NATA2, we found that TALEN-induced mutations were transmitted to the next generation at frequencies of 1.5-12%. Our work demonstrates that TALENs are useful reagents for achieving targeted mutagenesis in this important plant model. PMID:23979944

  7. Structure-based site-directed photo-crosslinking analyses of multimeric cell-adhesive interactions of voltage-gated sodium channel β subunits

    PubMed Central

    Shimizu, Hideaki; Miyazaki, Haruko; Ohsawa, Noboru; Shoji, Shisako; Ishizuka-Katsura, Yoshiko; Tosaki, Asako; Oyama, Fumitaka; Terada, Takaho; Sakamoto, Kensaku; Shirouzu, Mikako; Sekine, Shun-ichi; Nukina, Nobuyuki; Yokoyama, Shigeyuki

    2016-01-01

    The β1, β2, and β4 subunits of voltage-gated sodium channels reportedly function as cell adhesion molecules. The present crystallographic analysis of the β4 extracellular domain revealed an antiparallel arrangement of the β4 molecules in the crystal lattice. The interface between the two antiparallel β4 molecules is asymmetric, and results in a multimeric assembly. Structure-based mutagenesis and site-directed photo-crosslinking analyses of the β4-mediated cell-cell adhesion revealed that the interface between the antiparallel β4 molecules corresponds to that in the trans homophilic interaction for the multimeric assembly of β4 in cell-cell adhesion. This trans interaction mode is also employed in the β1-mediated cell-cell adhesion. Moreover, the β1 gene mutations associated with generalized epilepsy with febrile seizures plus (GEFS+) impaired the β1-mediated cell-cell adhesion, which should underlie the GEFS+ pathogenesis. Thus, the structural basis for the β-subunit-mediated cell-cell adhesion has been established. PMID:27216889

  8. Incorporation of a lambda phage recombination system and EGFP detection to simplify mutagenesis of Herpes simplex virus bacterial artificial chromosomes

    PubMed Central

    Schmeisser, Falko; Weir, Jerry P

    2007-01-01

    Background Targeted mutagenesis of the herpesvirus genomes has been facilitated by the use of bacterial artificial chromosome (BAC) technology. Such modified genomes have potential uses in understanding viral pathogenesis, gene identification and characterization, and the development of new viral vectors and vaccines. We have previously described the construction of a herpes simplex virus 2 (HSV-2) BAC and the use of an allele replacement strategy to construct HSV-2 recombinants. While the BAC mutagenesis procedure is a powerful method to generate HSV-2 recombinants, particularly in the absence of selective marker in eukaryotic culture, the mutagenesis procedure is still difficult and cumbersome. Results Here we describe the incorporation of a phage lambda recombination system into an allele replacement vector. This strategy enables any DNA fragment containing the phage attL recombination sites to be efficiently inserted into the attR sites of the allele replacement vector using phage lambda clonase. We also describe how the incorporation of EGFP into the allele replacement vector can facilitate the selection of the desired cross-over recombinant BACs when the allele replacement reaction is a viral gene deletion. Finally, we incorporate the lambda phage recombination sites directly into an HSV-2 BAC vector for direct recombination of gene cassettes using the phage lambda clonase-driven recombination reaction. Conclusion Together, these improvements to the techniques of HSV BAC mutagenesis will facilitate the construction of recombinant herpes simplex viruses and viral vectors. PMID:17501993

  9. Economical analysis of saturation mutagenesis experiments.

    PubMed

    Acevedo-Rocha, Carlos G; Reetz, Manfred T; Nov, Yuval

    2015-01-01

    Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced. PMID:26190439

  10. Economical analysis of saturation mutagenesis experiments

    PubMed Central

    Acevedo-Rocha, Carlos G.; Reetz, Manfred T.; Nov, Yuval

    2015-01-01

    Saturation mutagenesis is a powerful technique for engineering proteins, metabolic pathways and genomes. In spite of its numerous applications, creating high-quality saturation mutagenesis libraries remains a challenge, as various experimental parameters influence in a complex manner the resulting diversity. We explore from the economical perspective various aspects of saturation mutagenesis library preparation: We introduce a cheaper and faster control for assessing library quality based on liquid media; analyze the role of primer purity and supplier in libraries with and without redundancy; compare library quality, yield, randomization efficiency, and annealing bias using traditional and emergent randomization schemes based on mixtures of mutagenic primers; and establish a methodology for choosing the most cost-effective randomization scheme given the screening costs and other experimental parameters. We show that by carefully considering these parameters, laboratory expenses can be significantly reduced. PMID:26190439

  11. Pulsed EPR Distance Measurements in Soluble Proteins by Site-directed Spin-labeling (SDSL)

    PubMed Central

    de Vera, Ian Mitchelle S.; Blackburn, Mandy E.; Galiano, Luis; Fanucci, Gail E.

    2015-01-01

    The resurgence of pulsed electron paramagnetic resonance (EPR) in structural biology centers on recent improvements in distance measurements using the double electron-electron resonance (DEER) technique. This unit focuses on EPR-based distance measurements by site-directed spin-labeling (SDSL) of engineered cysteine residues in soluble proteins, with HIV-1 protease used as a model. To elucidate conformational changes in proteins, experimental protocols were optimized and existing data analysis programs were employed to derive distance distribution profiles. Experimental considerations, sample preparation and error analysis for artifact suppression are also outlined here. PMID:24510645

  12. Mutagenesis in Newts: Protocol for Iberian Ribbed Newts.

    PubMed

    Hayashi, Toshinori; Takeuchi, Takashi

    2016-01-01

    Newts have the remarkable capability of organ/tissue regeneration, and have been used as a unique experimental model for regenerative biology. The Iberian ribbed newt (Pleurodeles waltl) is suitable as a model animal. We have established methods for artificial insemination and efficient transgenesis using P. waltl newts. In addition to the transgenic technique, development of TALENs enables targeting mutagenesis in the newts. We have reported that TALENs efficiently disrupted targeted genes in newt embryos. In this chapter, we introduce a protocol for TALEN-mediated gene targeting in Iberian ribbed newts. PMID:26443218

  13. Crystallization and preliminary crystallographic studies of human septin 1 with site-directed mutations

    SciTech Connect

    Hu, Hao; Yu, Wen-bo; Li, Shu-xing; Ding, Xiang-ming; Yu, Long; Bi, Ru-Chang

    2006-02-01

    The homogeneity of septin 1 has been improved by site-directed mutation of serine residues and only a small alteration in the secondary structure is observed to arise from the mutations. Crystals of the septin 1 mutant were grown and diffraction data were collected to 2.5 Å resolution. Septin 1 is a member of an evolutionarily conserved family of GTP-binding and filament-forming proteins named septins, which function in diverse processes including cytokinasis, vesicle trafficking, apoptosis, remodelling of the cytoskeleton, infection, neurodegeneration and neoplasia. Human septin 1 has been expressed and purified, but suffers from severe aggregation. Studies have shown that septin 1 with site-directed mutations of five serine residues (Ser19, Ser206, Ser307, Ser312 and Ser315) has a much lower degree of aggregation and better structural homogeneity and that the mutations cause only slight perturbations in the secondary structure of septin 1. This septin 1 mutant was crystallized and diffraction data were collected to 2.5 Å resolution. The space group is P422, with unit-cell parameters a = b = 106.028, c = 137.852 Å.

  14. Detection of the TCDD Binding-Fingerprint within the Ah Receptor Ligand Binding Domain by Structurally Driven Mutagenesis and Functional Analysis†

    PubMed Central

    Pandini, Alessandro; Soshilov, Anatoly A.; Song, Yujuan; Zhao, Jing; Bonati, Laura; Denison, Michael S.

    2010-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent, basic helix–loop–helix Per-Arnt-Sim (PAS)-containing transcription factor that can bind and be activated by structurally diverse chemicals, including the toxic environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Our previous three-dimensional homology model of the mouse AhR (mAhR) PAS B ligand binding domain allowed identification of the binding site and its experimental validation. We have extended this analysis by conducting comparative structural modeling studies of the ligand binding domains of six additional high-affinity mammalian AhRs. These results, coupled with site-directed mutagenesis and AhR functional analysis, have allowed detection of the “TCDD binding-fingerprint” of conserved residues within the ligand binding cavity necessary for high-affinity TCDD binding and TCDD-dependent AhR transformation DNA binding. The essential role of selected residues was further evaluated using molecular docking simulations of TCDD with both wild-type and mutant mAhRs. Taken together, our results dramatically improve our understanding of the molecular determinants of TCDD binding and provide a basis for future studies directed toward rationalizing the observed species differences in AhR sensitivity to TCDD and understanding the mechanistic basis for the dramatic diversity in AhR ligand structure. PMID:19456125

  15. CHALLENGES FOR THE FUTURE IN ENVIRONMENTAL MUTAGENESIS

    EPA Science Inventory

    CHALLENGES FOR THE FUTURE IN ENVIRONMENTAL MUTAGENESIS
    Michael D. Waters
    US Environmental Protection Agency, MD-51A, Research Triangle Park, NC 27711 USA

    Our rapidly growing understanding of the structure of the human genome is forming the basis for numerous new...

  16. MAMMALIAN CELL MUTAGENESIS, BANBURY CONFERENCE (JOURNAL VERSION)

    EPA Science Inventory

    A conference on mammalian cell mutagenesis was held at the Banbury Center, Cold Spring Harbor, NY, USA, March 22-25, 1987. The objective of the conference was to provide a forum for discussions concerning the genetic, biochemical, and molecular basis of induced mutations in stand...

  17. Mutagenesis and heterologous expression in yeast of a plant Delta6-fatty acid desaturase.

    PubMed

    Sayanova, O; Beaudoin, F; Libisch, B; Castel, A; Shewry, P R; Napier, J A

    2001-07-01

    Membrane-bound microsomal fatty acid desaturases are known to have three conserved histidine boxes, comprising a total of up to eight histidine residues. Recently, a number of deviations from this consensus have been reported, with the substitution of a glutamine for the first histidine residue of the third histidine box being present in the so called 'front end' desaturases. These enzymes are also characterized by the presence of a cytochrome b5 domain at the protein N-terminus. Site-directed mutagenesis has been used to probe the functional importance of a number of amino acid residues which comprise the third histidine box of a 'front end' desaturase, the borage Delta6-fatty acid desaturase. This showed that the variant glutamine in the third histidine box is essential for enzyme activity and that histidine is not able to substitute for this residue. PMID:11457919

  18. Cloning of human epidermal growth factor as a bacterial secretory protein, its properties and mutagenesis

    SciTech Connect

    Engler, D.A.; Matsunami, R.K.; Campion, S.R.; Foote, R.S.; Mural, R.J.; Larimer, F.W.; Stevens, A.; Niyogi, S.K.

    1987-05-01

    A chimeric gene, containing the DNA coding for the human epidermal growth factor (EGF) and that for the signal peptide of E. coli alkaline phosphatase, was constructed by the annealing and subsequent ligation of appropriate DNA oligonucleotides synthesized in an automated DNA synthesizer. The gene was then cloned into a bacterial plasmid under the transcriptional control of the E. coli trp-lac (tac) promoter, and then transformed into E. coli. Following induction with isopropylthiogalactoside, the secretion of EGF into the E. coli periplasmic space and some into the growth medium was confirmed by its specific binding to the EGF receptor and stimulation of the EGF receptor tyrosine kinase activity. The size and physicochemical properties of the purified protein mimicked those of authentic human EGF. Studies of structure/function relationships by specific alterations of targeted amino acid residues in the EGF molecule have been initiated by utilizing site-directed mutagenesis.

  19. X-Ray Structure and Mutagenesis Studies of the N-Isopropylammelide Isopropylaminohydrolase, AtzC

    PubMed Central

    Newman, Janet; Briggs, Lyndall J.; Scott, Colin; Peat, Thomas S.

    2015-01-01

    The N-isopropylammelide isopropylaminohydrolase from Pseudomonas sp. strain ADP, AtzC, provides the third hydrolytic step in the mineralization of s-triazine herbicides, such as atrazine. We obtained the X-ray crystal structure of AtzC at 1.84 Å with a weak inhibitor bound in the active site and then used a combination of in silico docking and site-directed mutagenesis to understand the interactions between AtzC and its substrate, isopropylammelide. The substitution of an active site histidine residue (His249) for an alanine abolished the enzyme’s catalytic activity. We propose a plausible catalytic mechanism, consistent with the biochemical and crystallographic data obtained that is similar to that found in carbonic anhydrase and other members of subtype III of the amidohydrolase family PMID:26390431

  20. Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin.

    PubMed Central

    Gasymov, O. K.; Abduragimov, A. R.; Yusifov, T. N.; Glasgow, B. J.

    2000-01-01

    The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is

  1. Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin.

    PubMed

    Gasymov, O K; Abduragimov, A R; Yusifov, T N; Glasgow, B J

    2000-02-01

    The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues interact with the ligand. Stern-Volmer plots are inconsistent with a ligand that is

  2. Engineering the donor selectivity of D-fructose-6-phosphate aldolase for biocatalytic asymmetric cross-aldol additions of glycolaldehyde.

    PubMed

    Szekrenyi, Anna; Soler, Anna; Garrabou, Xavier; Guérard-Hélaine, Christine; Parella, Teodor; Joglar, Jesús; Lemaire, Marielle; Bujons, Jordi; Clapés, Pere

    2014-09-22

    D-Fructose-6-phosphate aldolase (FSA) is a unique catalyst for asymmetric cross-aldol additions of glycolaldehyde. A combination of a structure-guided approach of saturation mutagenesis, site-directed mutagenesis, and computational modeling was applied to construct a set of FSA variants that improved the catalytic efficiency towards glycolaldehyde dimerization up to 1800-fold. A combination of mutations in positions L107, A129, and A165 provided a toolbox of FSA variants that expand the synthetic possibilities towards the preparation of aldose-like carbohydrate compounds. The new FSA variants were applied as highly efficient catalysts for cross-aldol additions of glycolaldehyde to N-carbobenzyloxyaminoaldehydes to furnish between 80-98 % aldol adduct under optimized reaction conditions. Donor competition experiments showed high selectivity for glycolaldehyde relative to dihydroxyacetone or hydroxyacetone. These results demonstrate the exceptional malleability of the active site in FSA, which can be remodeled to accept a wide spectrum of donor and acceptor substrates with high efficiency and selectivity. PMID:25146467

  3. A model system for investigating lineshape/structure correlations in RNA site-directed spin labeling☆

    PubMed Central

    Qin, Peter Z.; Iseri, Jennifer; Oki, Arisa

    2008-01-01

    In RNA site-directed spin labeling (SDSL) studies, structural and dynamic information at the individual RNA nucleotide level is derived from the observed electron paramagnetic resonance spectrum of a covalently attached nitroxide. A systematic approach for RNA SDSL is to establish a library that categorizes observed spectral lineshapes based on known RNA structures, thus enabling lineshape-based structure identification at any RNA site. To establish the first RNA SDSL library, selective secondary structure elements have been systematically engineered into a model RNA. Nitroxide lineshapes reporting features specific to each element were obtained utilizing a new avidin-tethering scheme for suppressing spectral effects due to uniform RNA tumbling. The data demonstrated two key features required for a SDSL library with a predicting power: (i) spectral divergence—distinctive lineshape for different elements; and (ii) spectral convergence—similar lineshape for the same element in different contexts. This sets the foundation for further RNA SDSL library development. PMID:16530169

  4. Crystallization and preliminary crystallographic studies of human septin 1 with site-directed mutations

    PubMed Central

    Hu, Hao; Yu, Wen-bo; Li, Shu-xing; Ding, Xiang-ming; Yu, Long; Bi, Ru-Chang

    2006-01-01

    Septin 1 is a member of an evolutionarily conserved family of GTP-binding and filament-forming proteins named septins, which function in diverse processes including cytokinasis, vesicle trafficking, apoptosis, remodelling of the cytoskeleton, infection, neurodegeneration and neoplasia. Human septin 1 has been expressed and purified, but suffers from severe aggregation. Studies have shown that septin 1 with site-directed mutations of five serine residues (Ser19, Ser206, Ser307, Ser312 and Ser315) has a much lower degree of aggregation and better structural homogeneity and that the mutations cause only slight perturbations in the secondary structure of septin 1. This septin 1 mutant was crystallized and diffraction data were collected to 2.5 Å resolution. The space group is P422, with unit-cell parameters a = b = 106.028, c = 137.852 Å. PMID:16511282

  5. Acetylene is an active-site-directed, slow-binding, reversible inhibitor of Azotobacter vinelandii hydrogenase

    SciTech Connect

    Hyman, M.R.; Arp, D.J.

    1987-10-06

    The inhibition of purified and membrane-bound hydrogenase from Azotobacter vinelandii by dihydrogen-free acetylene was investigated. The inhibition was a time-dependent process which exhibited first-order kinetics. Both H/sub 2/ and CO protected against the inhibition by acetylene. K/sub protect(app)/ values of 0.41 and 24 ..mu..M were derived for these gases, respectively. Both H/sub 2/-oxidizing activity and the tritium exchange capacity of the purified enzyme were inhibited at the same rate by acetylene. Removal of acetylene reversed the inhibition for both the purified and the membrane-associated form of the enzyme. The purified hydrogenases from both Rhizobium japonicum and Alcaligenes eutrophus H16 were also inhibited by acetylene in a time-dependent fashion. These findings suggest that acetylene is an active-site-directed, slow-binding, reversible inhibitor of some membrane-bound hydrogenases from aerobic bacteria.

  6. Active-Site-Directed Inhibitors of Prolyl Oligopeptidase Abolish Its Conformational Dynamics.

    PubMed

    López, Abraham; Herranz-Trillo, Fátima; Kotev, Martin; Gairí, Margarida; Guallar, Víctor; Bernadó, Pau; Millet, Oscar; Tarragó, Teresa; Giralt, Ernest

    2016-05-17

    Deciphering conformational dynamics is crucial for understanding the biological functions of proteins and for designing compounds targeting them. In particular, providing an accurate description of microsecond-millisecond motions opens the opportunity for regulating protein-protein interactions (PPIs) by modulating the dynamics of one interacting partner. Here we analyzed the conformational dynamics of prolyl oligopeptidase (POP) and the effects of active-site-directed inhibitors on the dynamics. We used an integrated structural biology approach based on NMR spectroscopy and SAXS experiments complemented by MD simulations. We found that POP is in a slow equilibrium in solution between open and closed conformations, and that inhibitors effectively abolished this equilibrium by stabilizing the enzyme in the closed conformation. PMID:26918396

  7. Bromodeoxyuridine mutagenesis in mammalian cells is related to deoxyribonucleotide pool imbalance.

    PubMed Central

    Ashman, C R; Davidson, R L

    1981-01-01

    The relationship between bromodeoxyuridine (BrdUrd) mutagenesis in mammalian cells and the effects of BrdUrd on deoxyribonucleoside triphosphate pools was analyzed. It was found that the exposure of Syrian hamster melanoma cells to mutagenic concentrations of BrdUrd resulted in the formation of a large bromodeoxyuridine triphosphate (BrdUTP) pool, which remained at a high level for several days. In contrast, the size of the deoxycytidine triphosphate (dCTP) pool dropped rapidly after the addition of BrdUrd, reached a minimum at about 6 h, and then expanded gradually to nearly its original level over the next 3 days. The addition of lower concentrations of BrdUrd, which had less of a mutagenic effect, resulted in the formation of a smaller BrdUTP pool and a slightly smaller drop in the dCTP pool. When a high concentration of deoxycytidine was added at the same time as a normally mutagenic concentration of BrdUrd, the drop in the dCTP pool was prevented, as was BrdUrd mutagenesis. In all of these experiments, mutagenesis was related to the ratio of BrdUTP to dCTP in the cells. In addition, it was shown that mutagenesis occurred primarily during the first 24 h of BrdUrd exposure, when the BrdUTP/dCTP ratio was at its highest level. It appears that there is a critical ratio of BrdUTP to dCTP that must be attained for high levels of mutagenesis to occur and that the extent of mutagenesis is related to the ratio of the BrdUrd and dCTP pools. PMID:6965099

  8. Nonbinding Site-Directed Mutants of Transferrin Binding Protein B Exhibit Enhanced Immunogenicity and Protective Capabilities

    PubMed Central

    Frandoloso, Rafael; Martínez-Martínez, Sonia; Calmettes, Charles; Fegan, Jamie; Costa, Estela; Curran, Dave; Yu, Rong-hua; Gutiérrez-Martín, César B.; Rodríguez-Ferri, Elías F.; Moraes, Trevor F.

    2014-01-01

    Host-adapted Gram-negative bacterial pathogens from the Pasteurellaceae, Neisseriaceae, and Moraxellaceae families normally reside in the upper respiratory or genitourinary tracts of their hosts and rely on utilizing iron from host transferrin (Tf) for growth and survival. The surface receptor proteins that mediate this critical iron acquisition pathway have been proposed as ideal vaccine targets due to the critical role that they play in survival and disease pathogenesis in vivo. In particular, the surface lipoprotein component of the receptor, Tf binding protein B (TbpB), had received considerable attention as a potential antigen for vaccines in humans and food production animals but this has not translated into the series of successful vaccine products originally envisioned. Preliminary immunization experiments suggesting that host Tf could interfere with development of the immune response prompted us to directly address this question with site-directed mutant proteins defective in binding Tf. Site-directed mutants with dramatically reduced binding of porcine transferrin and nearly identical structure to the native proteins were prepared. A mutant Haemophilus parasuis TbpB was shown to induce an enhanced B-cell and T-cell response in pigs relative to native TbpB and provide superior protection from infection than the native TbpB or a commercial vaccine product. The results indicate that binding of host transferrin modulates the development of the immune response against TbpBs and that strategies designed to reduce or eliminate binding can be used to generate superior antigens for vaccines. PMID:25547790

  9. The effect of adaptive mutagenesis on genetic variation at a linked, neutral locus

    SciTech Connect

    Colby, C.; Williams, S.M.

    1995-07-01

    Based on recent studies in single-celled organisms, it has been argued that a fitness benefit associated with a mutation will increase the probability of that mutation occurring. This increase is independent of mutation rates at other loci and is called adaptive mutagenesis. We modeled the effect of adaptive mutagenesis on populations of haploid organisms with adaptive mutation rates ranging from 0 to 1 x 10{sup -5}. Allele frequencies at the selected locus and a neutral linked locus were tracked. We also observed the amount of linkage disequilibrium during the selective sweep and the final heterozygosity after the sweep. The presence of adaptive mutagenesis increases the number of genetic backgrounds carrying the new fitter allele, making the outcomes more representative of the population before the selection. Therefore, more neutral genetic variation is preserved in simulations with adaptive mutagenesis than in those without it due to hitchhiking. Since adaptive mutagensis is time-dependent, it can generate mutants when other mechanisms of mutation cannot. In addition, adaptive mutagenesis has the potential to confound both phylogeny construction and the detection of natural selection from patterns of nucleotide variation. 27 refs., 4 figs.

  10. Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice.

    PubMed

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2015-08-01

    The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and the tissues used for transformation and subsequent mutagenesis differed, hence the reported frequencies of targeted mutagenesis cannot be compared directly. Here, we evaluated mutation frequency in rice using different Cas9 and/or gRNA expression cassettes under standardized experimental conditions. We introduced Cas9 and gRNA expression cassettes separately or sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences. Mutation frequencies differed significantly depending on the Cas9 expression cassette used. In addition, a gRNA driven by the OsU6 promoter was superior to one driven by the OsU3 promoter. Using an all-in-one expression vector harboring the best combined Cas9/gRNA expression cassette resulted in a much improved frequency of targeted mutagenesis in rice calli, and bi-allelic mutant plants were produced in the T0 generation. The approach presented here could be adapted to optimize the construction of Cas9/gRNA cassettes for genome editing in a variety of plants. PMID:26188471

  11. A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing.

    PubMed

    Stiffler, Michael A; Subramanian, Subu K; Salinas, Victor H; Ranganathan, Rama

    2016-01-01

    Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 β-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to β-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place. PMID:27403811

  12. Mutagenesis and phenotyping resources in zebrafish for studying development and human disease.

    PubMed

    Varshney, Gaurav Kumar; Burgess, Shawn Michael

    2014-03-01

    The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts. PMID:24162064

  13. Discovery of novel STAT3 small molecule inhibitors via in silico site-directed fragment-based drug design.

    PubMed

    Yu, Wenying; Xiao, Hui; Lin, Jiayuh; Li, Chenglong

    2013-06-13

    Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been validated as an attractive therapeutic target for cancer therapy. To stop both STAT3 activation and dimerization, a viable strategy is to design inhibitors blocking its SH2 domain phosphotyrosine binding site that is responsible for both actions. A new fragment-based drug design (FBDD) strategy, in silico site-directed FBDD, was applied in this study. A designed novel compound, 5,8-dioxo-6-(pyridin-3-ylamino)-5,8-dihydronaphthalene-1-sulfonamide (LY5), was confirmed to bind to STAT3 SH2 by fluorescence polarization assay. In addition, four out of the five chosen compounds have IC50 values lower than 5 μM for the U2OS cancer cells. 8 (LY5) has an IC50 range in 0.5-1.4 μM in various cancer cell lines. 8 also suppresses tumor growth in an in vivo mouse model. This study has demonstrated the utility of this approach and could be used to other drug targets in general. PMID:23651330

  14. A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.

    PubMed

    Magnani, Francesca; Serrano-Vega, Maria J; Shibata, Yoko; Abdul-Hussein, Saba; Lebon, Guillaume; Miller-Gallacher, Jennifer; Singhal, Ankita; Strege, Annette; Thomas, Jennifer A; Tate, Christopher G

    2016-08-01

    The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes ∼6-12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of ∼300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an (125)I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies. PMID:27466713

  15. Rational and random mutagenesis of firefly luciferase to identify an efficient emitter of red bioluminescence

    NASA Astrophysics Data System (ADS)

    Branchini, Bruce R.; Southworth, Tara L.; Khattak, Neelum F.; Murtiashaw, Martha H.; Fleet, Sarah E.

    2004-06-01

    Firefly luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a bioluminescence reporter of gene expression. One particularly exciting application is in the area of in vivo bioluminescence imaging. Our interest is in developing improved reagents by identifying Photinus pyralis luciferase mutants that efficiently emit red bioluminescence. In this way, the proven advantages of the P. pyralis protein can be combined with the potential advantages of a red-shifted emitter. Using site-directed mutagenesis techniques, we have identified many mutants emitting red bioluminescence. Unfortunately, these enzymes generally have significantly decreased bioluminescence activity. Interestingly, we discovered a mutation, Ile351Ala, that produced a moderate 16 nm red-shift, while maintaining excellent bioluminescence activity. We then undertook a random mutagenesis approach to identify luciferase mutants that emit further red-shifted bioluminescence with minimal loss of activity. Libraries of mutants were created using an error-prone PCR method and the Ile351Ala luciferase mutant as the template DNA. The libraries were screened by in vivo bacterial assays and the promising mutants were purified to enable accurate determination of bioluminescence emission spectra and total bioluminescence activity. We will report the characterization results, including the identification of the randomly altered amino acids, of several mutants that catalyze bioluminescence with emission maxima of approximately 600 nm.

  16. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

    PubMed Central

    Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

    2016-01-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

  17. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants.

    PubMed

    Hehle, Verena K; Paul, Matthew J; Roberts, Victoria A; van Dolleweerd, Craig J; Ma, Julian K-C

    2016-04-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformedNicotiana tabacum Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy's 13 antibody heavy and light chain mutant combinations were expressed transiently inN. tabacumand demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.-Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

  18. Final report [DNA Repair and Mutagenesis - 1999

    SciTech Connect

    Walker, Graham C.

    2001-05-30

    The meeting, titled ''DNA Repair and Mutagenesis: Mechanism, Control, and Biological Consequences'', was designed to bring together the various sub-disciplines that collectively comprise the field of DNA Repair and Mutagenesis. The keynote address was titled ''Mutability Doth Play Her Cruel Sports to Many Men's Decay: Variations on the Theme of Translesion Synthesis.'' Sessions were held on the following themes: Excision repair of DNA damage; Transcription and DNA excision repair; UmuC/DinB/Rev1/Rad30 superfamily of DNA polymerases; Cellular responses to DNA damage, checkpoints, and damage tolerance; Repair of mismatched bases, mutation; Genome-instability, and hypermutation; Repair of strand breaks; Replicational fidelity, and Late-breaking developments; Repair and mutation in challenging environments; and Defects in DNA repair: consequences for human disease and aging.

  19. Expression and characterization of Pseudomonas aeruginosa cytochrome c-551 and two site-directed mutants: role of tryptophan 56 in the modulation of redox properties.

    PubMed Central

    Cutruzzolà, F; Ciabatti, I; Rolli, G; Falcinelli, S; Arese, M; Ranghino, G; Anselmino, A; Zennaro, E; Silvestrini, M C

    1997-01-01

    The gene coding for Pseudomonas aeruginosa cytochrome c-551 was expressed in Pseudomonas putida under aerobic conditions, using two different expression vectors; the more efficient proved to be pNM185, induced by m-toluate. Mature holo-(cytochrome c-551) was produced in high yield by this expression system, and was purified to homogeneity. Comparison of the recombinant wild-type protein with that purified from Ps. aeruginosa showed no differences in structural and functional properties. Trp56, an internal residue in cytochrome c-551, is located at hydrogen-bonding distance from haem propionate-17, together with Arg47. Ionization of propionate-17 was related to the observed pH-dependence of redox potential. The role of Trp56 in determining the redox properties of Ps. aeruginosa cytochrome c-551 was assessed by site-directed mutagenesis, by substitution with Tyr (W56Y) and Phe (W56F). The W56Y mutant is similar to the wild-type cytochrome. On the other hand, the W56F mutant, although similar to the wild-type protein in spectral properties and electron donation to azurin, is characterized by a weakening of the Fe-Met61 bond, as shown in the oxidized protein by the loss of the 695 nm band approx. 2 pH units below the wild-type. Moreover, in W56F, the midpoint potential and its pH-dependence are both different from the wild-type. These results are consistent with the hypothesis that hydrogen-bonding to haem propionate-17 is important in modulation of the redox properties of Ps. aeruginosa cytochrome c-551. PMID:9078240

  20. Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels.

    PubMed

    Basak, Sandip; Chatterjee, Soumili; Chakrapani, Sudha

    2016-01-01

    Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics. PMID:27403967

  1. pH-Dependent conformational changes in tear lipocalin by site-directed tryptophan fluorescence.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2010-01-26

    Tear lipocalin (TL), a major protein of human tears, binds a broad array of endogenous ligands. pH-dependent ligand binding in TL may have functional implications in tears. Previously, conformational selections of the AB and GH loops have been implicated in ligand binding by site-directed tryptophan fluorescence (SDTF). In this study, SDTF was applied to the AB and GH loops to investigate pH-driven conformational changes relevant to ligand binding. Both loops demonstrate significant but distinct conformational rearrangements over a wide pH range. In the low-pH transition, from 7.3 to 3.0, residues of the GH loop exhibit decreased solvent accessibilities. In acrylamide quenching experiments, the average quenching rate constant (k(q), accessibility parameter) of the residues in the GH loop is decreased approximately 38%, from 2.1 x 10(9) to 1.3 x 10(9) M(-1) s(-1). However, despite the significant changes in accessibilities for some residues in the AB loop, the average accessibility per residue remained unchanged (average k(q) = 1.2 M(-1) s(-1)). Accordingly, the low-pH transition induces conformational changes that reshuffle the accessibility profiles of the residues in the AB loop. A significant difference in the titration curves between the holo and apo forms of the W28 mutant suggests that the protonation states of the residues around position 28 modulate conformational switches of the AB loop relevant to ligand binding. PMID:20025287

  2. PH-dependent Conformational Changes in Tear Lipocalin by Site Directed Tryptophan Fluorescence

    PubMed Central

    Gasymov, Oktay K.; Abduragimov, Adil R.; Glasgow, Ben J.

    2010-01-01

    Tear lipocalin (TL), a major protein of human tears, binds a broad array of endogenous ligands. PH-dependent ligand binding in TL may have functional implications in tears. Previously, conformational selections of the loops AB and GH have been implicated in ligand binding by site-directed tryptophan fluorescence (SDTF). In this study, SDTF was applied on the loops AB and GH to investigate pH-driven conformational changes relevant to ligand binding. Both loops demonstrate significant but distinct conformational rearrangements over a wide pH range. In the low pH transition, from 7.3 to 3.0, residues of the loop GH show the decreased solvent accessibilities. In acrylamide quenching experiments, the average quenching rate constant (kq, accessibility parameter) of the residues in the loop GH is decreased about 38%, from 2.1×109 M−1s−1 to 1.3×109 M−1s−1. However, despite the significant changes in accessibilities for some residues in the loop AB, the average accessibility per residue remained unchanged (average kq= 1.2 M−1s−1). Accordingly, low pH transition induces conformational changes that reshuffle accessibility profiles of the residues in the loop AB. A significant difference in the titration curves between holo- and apo-forms of W28 mutant suggests that the protonation states of the residues around the position 28 modulate conformational switches of the loop AB relevant to ligand binding. PMID:20025287

  3. Nevada Test Site-Directed Research and Development FY 2010 Annual Report

    SciTech Connect

    Howard Bender, comp.

    2011-04-04

    This annual report of the Site-Directed Research and Development (SDRD) program represents the highly significant R&D accomplishments conducted during fiscal year 2010. This year was noteworthy historically, as the Nevada Test Site was renamed to the Nevada National Security Site (NNSS). This change not only recognizes how the site's mission has evolved, but also heralds a future of new challenges and opportunities for the NNSS. In many ways, since its inception in 2002, the SDRD program has helped shape that evolving mission. As we approach 2012, SDRD will also mark a milestone, having completed its first full decade of innovative R&D in support of the site and national security. The program continues to fund advanced science and technology development across traditional Department of Energy (DOE) nuclear security areas such as stockpile stewardship and non-proliferation while also supporting Department of Homeland Security (DHS) needs, and specialized work for government agencies like the Department of Defense (DoD) and others. The NNSS will also contribute technologies in the areas of treaty verification and monitoring, two areas of increasing importance to national security. Keyed to the NNSS's broadened scope, the SDRD program will continue to anticipate and advance R&D projects that will help the NNSS meet forthcoming challenges.

  4. Nevada National Security Site-Directed Research and Development FY 2011 Annual Report

    SciTech Connect

    Howard Bender, comp.

    2012-04-25

    This fiscal year 2011 annual report of the Site-Directed Research and Development program, the 10th anniversary edition, recognizes a full decade of innovative R&D accomplishments in support of the Nevada National Security Site (NNSS). Last year the NNSS itself was renamed to reflect a diversifying mission, and our R&D program has contributed significantly to shape emerging missions that will continue to evolve. New initiatives in stockpile stewardship science, nonproliferation, and treaty verification and monitoring have had substantial successes in FY 2011, and many more accomplishments are expected. SDRD is the cornerstone on which many of these initiatives rest. Historically supporting our main focus areas, SDRD is also building a solid foundation for new, and non-traditional, emerging national security missions. The program continues its charter to advance science and technology for a broad base of agencies including the U.S. Department of Energy (DOE), U.S. Department of Defense (DoD), U.S. Department of Homeland Security (DHS), and many others.

  5. A fully enzymatic method for site-directed spin labeling of long RNA

    PubMed Central

    Lebars, Isabelle; Vileno, Bertrand; Bourbigot, Sarah; Turek, Philippe; Wolff, Philippe; Kieffer, Bruno

    2014-01-01

    Site-directed spin labeling is emerging as an essential tool to investigate the structural and dynamical features of RNA. We propose here an enzymatic method, which allows the insertion of a paramagnetic center at a specific position in an RNA molecule. The technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5′-end by in vitro transcription with T7 RNA polymerase. The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product. This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA. The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR). This method enables various combinations of isotopic segmental labeling and spin labeling schemes, a strategy that will be of particular interest to investigate the structural and dynamical properties of large RNA complexes by NMR and EPR spectroscopies. PMID:24981512

  6. Sodium channel polypeptides in central nervous systems of various insects identified with site directed antibodies.

    PubMed

    Gordon, D; Moskowitz, H; Zlotkin, E

    1990-07-01

    Immunoprecipitation, radiophosphorylation and SDS-PAGE autoradiography enable the characterization of sodium channel polypeptides in the central nervous system of insects belonging to four phylogenetically distinct orders: grasshoppers, cockroaches, flies and moth larvae. It has been shown that the insect sodium channels: (1) Are recognized by the previously described (Gordon et al. (1988) Biochemistry 27, 7032-7038) site directed antibodies corresponding to a highly conserved segment linking the homologous domains III and IV in the vertebrate sodium channel alpha subunits. (2) Serve as substrates for phosphorylation by cAMP-dependent protein kinase. (3) Are devoid of disulfide linkage to smaller subunits unlike sodium channels in vertebrate brain. (4) Are glycoproteins as shown in the grasshopper by the decrease of apparent molecular weight following endoglycosidase F treatment and specific binding to the lectins concanavalin A and wheat germ agglutinin. (5) Reveal a diversity with regard to their (a) apparent molecular masses which range from 240 to 280 kDa and (b) V8 proteinase digestion phosphopeptides indicating either differences in the positioning of the enzymatic cleavage and/or phosphorylation sites. These results provide the first evidence for structural diversity of sodium channel subtypes among various insect orders and are compared to their mammalian counterparts. PMID:2165810

  7. Nevada Test Site-Directed Research, Development, and Demonstration. FY2005 report

    SciTech Connect

    Lewis, Will

    2006-09-01

    The Nevada Test Site-Directed Research, Development, and Demonstration (SDRD) program completed a very successful year of research and development activities in FY 2005. Fifty new projects were selected for funding this year, and five FY 2004 projects were brought to conclusion. The total funds expended by the SDRD program were $5.4 million, for an average per project cost of just under $100,000. Two external audits of SDRD accounting practices were conducted in FY 2005. Both audits found the program's accounting practices consistent with the requirements of DOE Order 413.2A, and one included the observation that the NTS contractor ''did an exceptional job in planning and executing year-start activities.'' Highlights for the year included: the filing of 18 invention disclosures for intellectual property generated by FY 2005 projects; programmatic adoption of 17 FY 2004 SDRD-developed technologies; participation in the tri-lab Laboratory Directed Research and Development (LDRD) and SDRD program review that was broadly attended by NTS, NNSA, LDRD, and U.S. Department of Homeland Security representatives; peer reviews of all FY 2005 projects; and the successful completion of 55 R&D projects, as presented in this report.

  8. Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation

    PubMed Central

    Kim, Soohyun; Kim, Hyori; Chung, Junho

    2016-01-01

    For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv), we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL) with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7) and two VH (H13 and H16) mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE) as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on cysteine is active and their binding activity is retained after the conjugation process. PMID:26764487

  9. Site-directed introduction of disulfide groups on antibodies for highly sensitive immunosensors.

    PubMed

    Acero Sánchez, Josep Ll; Fragoso, Alex; Joda, Hamdi; Suárez, Guillaume; McNeil, Calum J; O'Sullivan, Ciara K

    2016-07-01

    The interface between the sample and the transducer surface is critical to the performance of a biosensor. In this work, we compared different strategies for covalent self-assembly of antibodies onto bare gold substrates by introducing disulfide groups into the immunoglobulin structure, which acted as anchor molecules able to chemisorb spontaneously onto clean gold surfaces. The disulfide moieties were chemically introduced to the antibody via the primary amines, carboxylic acids, and carbohydrates present in its structure. The site-directed modification via the carbohydrate chains exhibited the best performance in terms of analyte response using a model system for the detection of the stroke marker neuron-specific enolase. SPR measurements clearly showed the potential for creating biologically active densely packed self-assembled monolayers (SAMs) in a one-step protocol compared to both mixed SAMs of alkanethiol compounds and commercial immobilization layers. The ability of the carbohydrate strategy to construct an electrochemical immunosensor was investigated using electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) transduction. Graphical Abstract Left: Functionalization strategies of bare gold substrates via direct bio-SAM using disulfide-containing antibody chemically modified via their primary amines (A), carbohydrates (B) and carboxylic acids (C). Right: Dependence of the peak height with NSE concentration at NSE21-CHO modified electrochemical immunosensor. Inset: Logarithmic calibration plot. PMID:27220524

  10. Pathogen corruption and site-directed recombination at a plant disease resistance gene cluster

    PubMed Central

    Nagy, Ervin D.; Bennetzen, Jeffrey L.

    2008-01-01

    The Pc locus of sorghum (Sorghum bicolor) determines dominant sensitivity to a host-selective toxin produced by the fungal pathogen Periconia circinata. The Pc region was cloned by a map-based approach and found to contain three tandemly repeated genes with the structures of nucleotide binding site–leucine-rich repeat (NBS–LRR) disease resistance genes. Thirteen independent Pc-to-pc mutations were analyzed, and each was found to remove all or part of the central gene of the threesome. Hence, this central gene is Pc. Most Pc-to-pc mutations were associated with unequal recombination. Eight recombination events were localized to different sites in a 560-bp region within the ∼3.7-kb NBS–LRR genes. Because any unequal recombination located within the flanking NBS–LRR genes would have removed Pc, the clustering of cross-over events within a 560-bp segment indicates that a site-directed recombination process exists that specifically targets unequal events to generate LRR diversity in NBS–LRR loci. PMID:18719093

  11. Intracavitary ligand distribution in tear lipocalin by site-directed tryptophan fluorescence.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2009-08-01

    Site-directed tryptophan fluorescence has been successfully used to determine the solution structure of tear lipocalin. Here, the technique is extended to measure the binding energy landscape. Single Trp mutants of tear lipocalin are bound to the native ligand and an analogue tagged with a quencher group to both populate and discriminate the excited protein states. Steady-state and time-resolved fluorescence quenching data reveal the intracavitary state of the ligand. The static components of fluorescence quenching identify the residues where nonfluorescence complexes form. An asymmetric distribution of the ligand within the cavity reflects the complex energy landscape of the excited protein states. These findings suggest that the excited protein states are not unique but consist of many substates. The roughness of the binding energy landscape is about 2.5kBT. The excited protein states originate primarily from conformational selections of loops AB and GH, a portal region. In contrast to static quenching, the dynamic components of fluorescence quenching by the ligand are relevant to both local side chain and ligand dynamics. Apparent bimolecular rate constants for collisional quenching of Trp by the nitroxide moiety are approximately 1 / 5 x 10(12) M(-1) s(-1). Estimations made for effective ligand concentrations establish actual rate constants on the order of 12 x 10(9) M(-1) s(-1). Prior to exit from the cavity of the protein, ligands explore binding sites in nanoseconds. Although microsecond fluctuations are rate-limiting processes in ligand binding for many proteins, accompanying nanosecond motion may be necessary for propagation of ligand binding. PMID:19586017

  12. Localization of ASV Integrase-DNA Contacts by Site-Directed Crosslinking and their Structural Analysis

    PubMed Central

    Merkel, George; Alexandratos, Jerry; Zhou, Dongwen; Bojja, Ravi S.; Satoh, Tadashi; Potapov, Mikhail; Kogon, Alex; Potapov, Viktor; Wlodawer, Alexander; Skalka, Anna Marie

    2011-01-01

    Background We applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins. Methodology/Results Photoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and amino-modified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate. Conclusion Analysis of our crosslinking results as well as published results of retroviral IN protein from other laboratories shows good agreement with the structure of PFV IN and derived ASV, HIV, and MuLV models for the core domain, but only partial agreement for the N- and C-terminal domains. These differences might be explained by structural variations and evolutionary selection for residues at alternate positions to perform analogous functions, and by methodological differences: i.e., a static picture of a particular assembly from crystallography vs

  13. Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

    SciTech Connect

    Couto, Sheila G.; Cristina Nonato, M.

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer EcDHODH is a membrane-associated enzyme and a promising target for drug design. Black-Right-Pointing-Pointer Enzyme's N-terminal extension is responsible for membrane association. Black-Right-Pointing-Pointer N-terminal works as a molecular lid regulating access to the protein interior. -- Abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

  14. Fluorometric method of quantitative cell mutagenesis

    DOEpatents

    Dolbeare, F.A.

    1980-12-12

    A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

  15. Fluorometric method of quantitative cell mutagenesis

    DOEpatents

    Dolbeare, Frank A.

    1982-01-01

    A method for assaying a cell culture for mutagenesis is described. A cell culture is stained first with a histochemical stain, and then a fluorescent stain. Normal cells in the culture are stained by both the histochemical and fluorescent stains, while abnormal cells are stained only by the fluorescent stain. The two stains are chosen so that the histochemical stain absorbs the wavelengths that the fluorescent stain emits. After the counterstained culture is subjected to exciting light, the fluorescence from the abnormal cells is detected.

  16. Nevada Test Site-Directed Research and Development, FY 2007 Report

    SciTech Connect

    Wil Lewis, editor

    2008-02-20

    The Nevada Test Site-Directed Research and Development (SDRD) program completed a very successful year of research and development activities in FY 2007. Twenty-nine new projects were selected for funding this year, and eight projects started in FY 2006 were brought to conclusion. The total funds expended by the SDRD program were $5.67 million, for an average per-project cost of $153 thousand. An external audit conducted in September 2007 verified that appropriate accounting practices were applied to the SDRD program. Highlights for the year included: programmatic adoption of 8 SDRD-developed technologies; the filing of 9 invention disclosures for innovation evolving from SDRD projects; participation in the tri-Lab Laboratory Directed Research and Development (LDRD) and SDRD Symposium that was broadly attended by Nevada Test Site (NTS), National Nuclear Security Administration (NNSA), LDRD, U.S. Department of Homeland Security (DHS), and U.S. Department of Defense (DoD) representatives; peer reviews of all FY 2007 projects; and the successful completion of 37 R&D projects, as presented in this report. In response to a company-wide call, authors throughout the NTS complex submitted 182 proposals for FY 2007 SDRD projects. The SDRD program has seen a dramatic increase in the yearly total of submitted proposals--from 69 in FY 2002 to 182 this year--while the number of projects funded has actually decreased from a program high of 57 in FY 2004. The overall effect of this trend has helped ensure an increasingly competitive program that benefited from a broader set of innovative ideas, making project selection both challenging and rewarding. Proposals were evaluated for technical merit, including such factors as innovation, probability of success, potential benefit, and mission applicability. Authors and reviewers benefited from the use of a shortfalls list entitled the 'NTS Technology Needs Assessment' that was compiled from NTS, National Weapons Laboratory (NWL), and

  17. Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments.

    PubMed

    Dmytruk, Kostyantyn V; Voronovsky, Andriy Y; Sibirny, Andriy A

    2006-09-01

    The feasibility of using random insertional mutagenesis to isolate mutants of the flavinogenic yeast Candida famata was explored. Mutagenesis was performed by transformation of the yeast with an integrative plasmid containing the Saccharomyces cerevisiae LEU2 gene as a selective marker. The addition of restriction enzyme together with the plasmid (restriction enzyme-mediated integration, REMI) increased the transformation frequency only slightly. Integration of the linearized plasmid occurred randomly in the C. famata genome. To investigate the potential of insertional mutagenesis, it was used for tagging genes involved in positive regulation of riboflavin synthesis in C. famata. Partial DNA sequencing of tagged genes showed that they were homologous to the S. cerevisiae genes RIB1, MET2, and SEF1. Intact orthologs of these genes isolated from Debaryomyces hansenii restored the wild phenotype of the corresponding mutants, i.e., the ability to overproduce riboflavin under iron limitation. The Staphylococcus aureus ble gene conferring resistance to phleomycin was used successfully in the study as a dominant selection marker for C. famata. The results obtained indicate that insertional mutagenesis is a powerful tool for tagging genes in C. famata. PMID:16770625

  18. N-Ethyl-N-Nitrosourea Mutagenesis: Boarding the Mouse Mutant Express

    PubMed Central

    Cordes, Sabine P.

    2005-01-01

    In the mouse, random mutagenesis with N-ethyl-N-nitrosourea (ENU) has been used since the 1970s in forward mutagenesis screens. However, only in the last decade has ENU mutagenesis been harnessed to generate a myriad of new mouse mutations in large-scale genetic screens and focused, smaller efforts. The development of additional genetic tools, such as balancer chromosomes, refinements in genetic mapping strategies, and evolution of specialized assays, has allowed these screens to achieve new levels of sophistication. The impressive productivity of these screens has led to a deluge of mouse mutants that wait to be harnessed. Here the basic large- and small-scale strategies are described, as are the basics of screen design. Finally, and importantly, this review describes the mechanisms by which such mutants may be accessed now and in the future. Thus, this review should serve both as an overview of the power of forward mutagenesis in the mouse and as a resource for those interested in developing their own screens, adding onto existing efforts, or obtaining specific mouse mutants that have already been generated. PMID:16148305

  19. Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria

    PubMed Central

    Rodríguez-Rojas, Alexandro; Makarova, Olga; Müller, Uta; Rolff, Jens

    2015-01-01

    Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection. PMID:26430769

  20. Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria.

    PubMed

    Rodríguez-Rojas, Alexandro; Makarova, Olga; Müller, Uta; Rolff, Jens

    2015-10-01

    Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection. PMID:26430769

  1. Random mutagenesis strategies for construction of large and diverse clone libraries of mutated DNA fragments.

    PubMed

    Chusacultanachai, Sudsanguan; Yuthavong, Yongyuth

    2004-01-01

    The first important step toward a successful preparation of large and diverse DNA libraries with desired complexity is to select a suitable mutagenesis strategy. This chapter describes three different methods for random mutagenesis, the use of XL1-red cells, error-prone polymerase chain reaction (PCR), and degenerate oligonucleotides-Pfu (DOP). These mutagenesis strategies possess different benefits and pitfalls; thus, they are differentially useful for production of DNA libraries with different density and complexity. The use of XL1-red, an engineered Escherichia coli with DNA repair deficiency, is one of the simplest mutagenesis and requires no subcloning step. After plasmid encoding DNA of inter-est is transformed into the cells, the mutations are simply generated during each round of DNA replication. The mutation frequency of this method is reported to be 1 base change per 2000 nucleotides; however, it can be slightly increased by extending the culture period to allow the accumulation of more mutations. This strategy is suitable for generation of random mutations with low frequency in a large target DNA. Error-prone PCR is one of the most widely used random mutagenesis. During DNA amplification, misincorporation of nucleotides can be promoted by altering the nucleotide ratio and the concentration of divalent cations in the reaction. We discovered that, by adjusting template concentration, frequency of mutation could be controlled easily and a library with desired mutation rate could be obtained. Additionally, efficiency of subsequent cloning steps to insert the PCR product into plasmid DNA is also a key factor determining size and complexity of the libraries. DOP mutagenesis is a rapid and effective method for random mutagenesis of small DNA and peptides. This strategy uses two chemically synthesized degenerate oligonucleotides as primers. By controlling the positions and ratios of degenerate nucleotides used during oligonucleotide synthesis, it is possible to

  2. Mutagenesis as a Genetic Research Strategy

    PubMed Central

    Falk, Raphael

    2010-01-01

    Morgan's three students (Muller, Sturtevant, and Bridges) introduced reductionist empirical methods to the study of the chromosomal theory of heredity. Herman J. Muller concentrated on mutations, namely changes in the heterocatalytic properties of genes, without losing their autocatalytic (self-replication) properties. Experimental induction of mutations allowed quantitative analyses of genes' parameters, but hopes to deduce their chemicophysical character were never fulfilled. Once the model for DNA structure was proposed, the reductionist notions of mutation analysis were successfully applied to the molecular genes. However, it was soon realized that the concept of the particulate gene was inadequate. The more the molecular analysis of the genome advanced, the clearer it became that the entities of heredity must be conceived within systems' perspectives, for which special tools for handling large number of variables were developed. Analytic mutagenesis, however, continues to be a major strategy for the study of the cellular and chromosomal mechanisms that control mutation inductions. PMID:20713742

  3. Mutagenesis assays of human amniotic fluid

    SciTech Connect

    Everson, R.B.; Milne, K.L.; Warbuton, D.; McClamrock, H.D.; Buchanan, P.D.

    1985-01-01

    Extracts of amniocentesis samples from 144 women were tested for the presence of mutagenic substances using tester strain TA1538 in the Ames Salmonella/mammalian-microsome mutagenicity test. Because the volume of amniotic fluid in these samples was limited (generally less than 10 ml), the authors investigated modifications of this mutagenesis assay that could increase its ability to detect effects from small quantities of test material. Using mutagenicity in samples of urine from smokers as a model, it appeared that improved ability to detect small amounts of mutagen could be obtained by reducing volumes of media and reagents while keeping the amount of test sample constant. Tests of amniotic fluid extracts by this modified procedure showed small increases in revertants, about 50% above dimethylsulfoxide solvent control values. The increases suggest the presence of small amounts of mutagenic material in many of the amniotic fluid samples. At the doses employed, mutagenic activity in these samples was not associated with maternal smoking.

  4. Conformational dynamics of the active site loop of S-adenosylmethionine synthetase illuminated by site-directed spin labeling.

    PubMed

    Taylor, John C; Markham, George D

    2003-07-15

    S-adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase, methionine adenosyltransferase, a.k.a. MAT) is one of numerous enzymes that have a flexible polypeptide loop that moves to gate access to the active site in a motion that is closely coupled to catalysis. Crystallographic studies of this tetrameric enzyme have shown that the loop is closed in the absence of bound substrates. However, the loop must open to allow substrate binding and a variety of data indicate that the loop is closed during the catalytic steps. Previous kinetic studies indicate that during turnover loop motion occurs on a time scale of 10(-2)s, ca. 10-fold faster than chemical transformations and turnover. Site-directed spin labeling has been used to introduce nitroxide groups at two positions in the loop to illuminate how the motion of the loop is affected by substrate binding. The two loop mutants constructed, G105C and D107C, retain wild type levels of MAT activity; attachment of a methanethiosulfonate spin label to convert the cysteine to the "R1" residue reduced the k(cat) only for the labeled D107R1 form (7-fold). The K(m) value for methionine increased 2- to 4-fold for the cysteine mutants and 2- to 7-fold for the labeled proteins, whereas the K(m) for ATP was changed by at most 2-fold. EPR spectra for both labeled proteins are nearly identical and show the presence of two major spin label environments with rotational diffusion rates differing by approximately 10-fold; the slower rate is ca. 4-fold faster than the estimated protein rotational rate. The spectra are not altered by addition of substrates or products. At both positions the less mobile conformation constitutes ca. 65% of the total species, indicating an equilibrium that only slightly favors one form, that in which the label is more immobilized. The equilibrium constant that relates the two forms is comparable to the equilibrium constant of 1.5 for a conformational change that was previously deduced from the

  5. A novel method to identify nucleic acid binding sites in proteins by scanning mutagenesis: application to iron regulatory protein.

    PubMed Central

    Neupert, B; Menotti, E; Kühn, L C

    1995-01-01

    We describe a new procedure to identify RNA or DNA binding sites in proteins, based on a combination of UV cross-linking and single-hit chemical peptide cleavage. Site-directed mutagenesis is used to create a series of mutants with single Asn-Gly sequences in the protein to be analysed. Recombinant mutant proteins are incubated with their radiolabelled target sequence and UV irradiated. Covalently linked RNA- or DNA-protein complexes are digested with hydroxylamine and labelled peptides identified by SDS-PAGE and autoradiography. The analysis requires only small amounts of protein and is achieved within a relatively short time. Using this method we mapped the site at which human iron regulatory protein (IRP) is UV cross-linked to iron responsive element RNA to amino acid residues 116-151. Images PMID:7544459

  6. Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes.

    PubMed

    Burger, Alexa; Lindsay, Helen; Felker, Anastasia; Hess, Christopher; Anders, Carolin; Chiavacci, Elena; Zaugg, Jonas; Weber, Lukas M; Catena, Raul; Jinek, Martin; Robinson, Mark D; Mosimann, Christian

    2016-06-01

    CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient bi-allelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo. PMID:27130213

  7. Triplex technology in studies of DNA damage, DNA repair, and mutagenesis

    PubMed Central

    Mukherjee, Anirban; Vasquez, Karen M.

    2012-01-01

    Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. PMID:21501652

  8. Triplex technology in studies of DNA damage, DNA repair, and mutagenesis.

    PubMed

    Mukherjee, Anirban; Vasquez, Karen M

    2011-08-01

    Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. PMID:21501652

  9. The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences.

    PubMed

    Donnelly, M L; Hughes, L E; Luke, G; Mendoza, H; ten Dam, E; Gani, D; Ryan, M D

    2001-05-01

    The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins 'cleaving' at their own C termini. We have analysed this activity using artificial reporter polyprotein systems comprising green fluorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A to beta-glucuronidase (GUS) -- forming a single, long, open reading frame. Analysis of the distribution of radiolabel showed a high proportion of the in vitro translation products (approximately 90%) were in the form of the 'cleavage' products GUS and [GFP2A]. Alternative models have been proposed to account for the 'cleavage' activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect. To investigate the mechanism of this cleavage event constructs encoding site-directed mutant and naturally occurring '2A-like' sequences were used to program in vitro translation systems and the gel profiles analysed. Analysis of site-directed mutant 2A sequences showed that 'cleavage' occurred in constructs in which all the candidate nucleophilic residues were substituted -- with the exception of aspartate-12. This residue is not, however, conserved amongst all functional '2A-like' sequences. '2A-like' sequences were identified within insect virus polyproteins, the NS34 protein of type C rotaviruses, repeated sequences in Trypanosoma spp. and a eubacterial alpha-glucosiduronasesequence(Thermatoga maritima aguA). All of the 2A-like sequences analysed were active (to various extents), other than the eubacterial alpha-glucosiduronase 2A-like sequence. This method of control of protein biogenesis may well not, therefore, be confined to members of the PICORNAVIRIDAE: Taken together, these data provide additional evidence that neither FMDV 2A nor '2A-like' sequences are autoproteolytic elements. PMID:11297677

  10. Symposium on molecular and cellular mechanisms of mutagenesis

    SciTech Connect

    Not Available

    1981-01-01

    These proceedings contain abstracts only of the 21 papers presented at the Sympsoium. The papers dealt with molecular mechanisms of mutagenesis and cellular responses to chemical and physical mutagenic agents. (ERB)

  11. Mutagenesis during plant responses to UVB radiation.

    PubMed

    Holá, M; Vágnerová, R; Angelis, K J

    2015-08-01

    We tested an idea that induced mutagenesis due to unrepaired DNA lesions, here the UV photoproducts, underlies the impact of UVB irradiation on plant phenotype. For this purpose we used protonemal culture of the moss Physcomitrella patens with 50% of apical cells, which mimics actively growing tissue, the most vulnerable stage for the induction of mutations. We measured the UVB mutation rate of various moss lines with defects in DNA repair (pplig4, ppku70, pprad50, ppmre11), and in selected clones resistant to 2-Fluoroadenine, which were mutated in the adenosine phosphotrasferase gene (APT), we analysed induced mutations by sequencing. In parallel we followed DNA break repair and removal of cyclobutane pyrimidine dimers with a half-life τ = 4 h 14 min determined by comet assay combined with UV dimer specific T4 endonuclease V. We show that UVB induces massive, sequence specific, error-prone bypass repair that is responsible for a high mutation rate owing to relatively slow, though error-free, removal of photoproducts by nucleotide excision repair (NER). PMID:25542779

  12. Codon compression algorithms for saturation mutagenesis.

    PubMed

    Pines, Gur; Pines, Assaf; Garst, Andrew D; Zeitoun, Ramsey I; Lynch, Sean A; Gill, Ryan T

    2015-05-15

    Saturation mutagenesis is employed in protein engineering and genome-editing efforts to generate libraries that span amino acid design space. Traditionally, this is accomplished by using degenerate/compressed codons such as NNK (N = A/C/G/T, K = G/T), which covers all amino acids and one stop codon. These solutions suffer from two types of redundancy: (a) different codons for the same amino acid lead to bias, and (b) wild type amino acid is included within the library. These redundancies increase library size and downstream screening efforts. Here, we present a dynamic approach to compress codons for any desired list of amino acids, taking into account codon usage. This results in a unique codon collection for every amino acid to be mutated, with the desired redundancy level. Finally, we demonstrate that this approach can be used to design precise oligo libraries amendable to recombineering and CRISPR-based genome editing to obtain a diverse population with high efficiency. PMID:25303315

  13. Signature-tagged mutagenesis of Vibrio vulnificus

    PubMed Central

    YAMAMOTO, Mai; KASHIMOTO, Takashige; TONG, Ping; XIAO, Jianbo; SUGIYAMA, Michiko; INOUE, Miyuki; MATSUNAGA, Rie; HOSOHARA, Kohei; NAKATA, Kazue; YOKOTA, Kenji; OGUMA, Keiji; YAMAMOTO, Koichiro

    2015-01-01

    Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus. PMID:25755021

  14. Clonal Polymorphism of Borrelia burgdorferi Strain B31 MI: Implications for Mutagenesis in an Infectious Strain Background

    PubMed Central

    Elias, Abdallah F.; Stewart, Philip E.; Grimm, Dorothee; Caimano, Melissa J.; Eggers, Christian H.; Tilly, Kit; Bono, James L.; Akins, Darrin R.; Radolf, Justin D.; Schwan, Tom G.; Rosa, Patricia

    2002-01-01

    A major obstacle to studying the functions of particular gene products in the mouse-tick infectious cycle of Borrelia burgdorferi has been an inability to knock out genes in pathogenic strains. Here, we investigated conditions for site-directed mutagenesis in B31 MI, the low-passage-number, infectious B. burgdorferi strain whose genome was sequenced. We inactivated several plasmid and chromosomal genes in B31 MI and determined that clones carrying these mutations were not infectious for mice. However, we found extensive heterogeneity among clones and mutants derived from B31 MI based on colony phenotype, growth rate, plasmid content, protein profile, and transformability. Significantly, several B31 MI clones that were not subjected to mutagenesis but that lacked particular plasmids also exhibited defects at various stages in the infectious cycle. Therefore, the high degree of clonal polymorphism within B31 MI complicates the assessment of the contributions of individual genes to the observed phenotypes of the mutants. Our results indicate that B31 MI is not an appropriate strain background for genetic studies in infectious B. burgdorferi, and a well-defined isogenic clone is a prerequisite for targeted mutagenesis. To this end, we derived several wild-type clones from B31 MI that were infectious for mice, and gene inactivation was successful in one of these clones. Due to the instability of the genome with in vitro propagation, careful monitoring of plasmid content of derived mutants and complementation of inactivated genes will be crucial components of genetic studies with this pathogen. PMID:11895980

  15. Clonal polymorphism of Borrelia burgdorferi strain B31 MI: implications for mutagenesis in an infectious strain background.

    PubMed

    Elias, Abdallah F; Stewart, Philip E; Grimm, Dorothee; Caimano, Melissa J; Eggers, Christian H; Tilly, Kit; Bono, James L; Akins, Darrin R; Radolf, Justin D; Schwan, Tom G; Rosa, Patricia

    2002-04-01

    A major obstacle to studying the functions of particular gene products in the mouse-tick infectious cycle of Borrelia burgdorferi has been an inability to knock out genes in pathogenic strains. Here, we investigated conditions for site-directed mutagenesis in B31 MI, the low-passage-number, infectious B. burgdorferi strain whose genome was sequenced. We inactivated several plasmid and chromosomal genes in B31 MI and determined that clones carrying these mutations were not infectious for mice. However, we found extensive heterogeneity among clones and mutants derived from B31 MI based on colony phenotype, growth rate, plasmid content, protein profile, and transformability. Significantly, several B31 MI clones that were not subjected to mutagenesis but that lacked particular plasmids also exhibited defects at various stages in the infectious cycle. Therefore, the high degree of clonal polymorphism within B31 MI complicates the assessment of the contributions of individual genes to the observed phenotypes of the mutants. Our results indicate that B31 MI is not an appropriate strain background for genetic studies in infectious B. burgdorferi, and a well-defined isogenic clone is a prerequisite for targeted mutagenesis. To this end, we derived several wild-type clones from B31 MI that were infectious for mice, and gene inactivation was successful in one of these clones. Due to the instability of the genome with in vitro propagation, careful monitoring of plasmid content of derived mutants and complementation of inactivated genes will be crucial components of genetic studies with this pathogen. PMID:11895980

  16. Enhanced maltose production through mutagenesis of acceptor binding subsite +2 in Bacillus stearothermophilus maltogenic amylase.

    PubMed

    Sun, Yecheng; Duan, Xuguo; Wang, Lei; Wu, Jing

    2016-01-10

    Maltogenic amylases are used to decrease the maltotriose content of high maltose syrups. However, due to the interplay between the hydrolysis and transglycosylation activities of maltogenic amylases, the maltotriose contents of these syrups are still greater than that necessary for pure maltose preparation. In this study, the maltogenic amylase from Bacillus stearothermophilus was engineered to decrease its transglycosylation activity with the expectation that this would enhance maltose production. Site-directed mutagenesis was used to generate Trp 177 variants W177F, W177Y, W177L, W177N, and W177S. The transglycosylation activities of the mutant enzymes decreased as the hydrophilicity of the residue at position 177 increased. The mutant enzymes exhibited notable enhancements in maltose production, with a minimum of maltotriose contents of 0.2%, compared with 3.2% for the wild-type enzyme. Detailed characterization of the mutant enzymes suggests that the best of them, W177S, will deliver performance superior to that of the wild-type under industrial conditions. PMID:26597712

  17. The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach

    SciTech Connect

    Hoskins, J.T.; Zhou, Z.; Harding, P.A.

    2008-10-31

    A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser{sub 108/121}, HB-EGF-Cys/Ser{sub 116/132}, and HB-EGF-Cys/Ser{sub 134/143}) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with M{sub r} of 6.5, 21 and 24 kDa were observed from lysates of HB-EGF and each HB-EGF disulfide analogue. HB-EGF immunohistochemical analyses of each HB-EGF stable cell line demonstrated ubiquitous protein expression except HB-EGF-Cys/Ser{sub 108/121} and HB-EGF-Cys/Ser{sub 116/132} stable cell lines which exhibited accumulated expression immediately outside the nucleus. rHB-EGF, HB-EGF, and HB-EGF{sub 134/143} proteins competed with {sup 125}I-EGF in an A431 competitive binding assay, whereas HB-EGF-Cys/Ser{sub 108/121} and HB-EGF-Cys/Ser{sub 116/132} failed to compete. Each HB-EGF disulfide analogue lacked the ability to stimulate tyrosine phosphorylation of the 170 kDa EGFR. These results suggest that HB-EGF-Cys/Ser{sub 134/143} antagonizes EGFRs.

  18. Mutagenesis and functional analysis of the pore-forming toxin HALT-1 from Hydra magnipapillata.

    PubMed

    Liew, Yvonne Jing Mei; Soh, Wai Tuck; Jiemy, William Febry; Hwang, Jung Shan

    2015-02-01

    Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1-4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding. PMID:25654788

  19. Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata

    PubMed Central

    Liew, Yvonne Jing Mei; Soh, Wai Tuck; Jiemy, William Febry; Hwang, Jung Shan

    2015-01-01

    Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1–4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding. PMID:25654788

  20. Yellow fluorescent protein phiYFPv (Phialidium): structure and structure-based mutagenesis

    SciTech Connect

    Pletneva, Nadya V.; Pletnev, Vladimir Z. Souslova, Ekaterina; Chudakov, Dmitry M.; Lukyanov, Sergey; Martynov, Vladimir I.; Arhipova, Svetlena; Artemyev, Igor; Wlodawer, Alexander; Dauter, Zbigniew; Pletnev, Sergei

    2013-06-01

    The yellow fluorescent protein phiYFPv with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The yellow fluorescent protein phiYFPv (λ{sub em}{sup max} ≃ 537 nm) with improved folding has been developed from the spectrally identical wild-type phiYFP found in the marine jellyfish Phialidium. The latter fluorescent protein is one of only two known cases of naturally occurring proteins that exhibit emission spectra in the yellow–orange range (535–555 nm). Here, the crystal structure of phiYFPv has been determined at 2.05 Å resolution. The ‘yellow’ chromophore formed from the sequence triad Thr65-Tyr66-Gly67 adopts the bicyclic structure typical of fluorophores emitting in the green spectral range. It was demonstrated that perfect antiparallel π-stacking of chromophore Tyr66 and the proximal Tyr203, as well as Val205, facing the chromophore phenolic ring are chiefly responsible for the observed yellow emission of phiYFPv at 537 nm. Structure-based site-directed mutagenesis has been used to identify the key functional residues in the chromophore environment. The obtained results have been utilized to improve the properties of phiYFPv and its homologous monomeric biomarker tagYFP.

  1. Facilitating Structure-Function Studies of CFTR Modulator Sites with Efficiencies in Mutagenesis and Functional Screening.

    PubMed

    Molinski, Steven V; Ahmadi, Saumel; Hung, Maurita; Bear, Christine E

    2015-12-01

    There are nearly 2000 mutations in the CFTR gene associated with cystic fibrosis disease, and to date, the only approved drug, Kalydeco, has been effective in rescuing the functional expression of a small subset of these mutant proteins with defects in channel activation. However, there is currently an urgent need to assess other mutations for possible rescue by Kalydeco, and further, definition of the binding site of such modulators on CFTR would enhance our understanding of the mechanism of action of such therapeutics. Here, we describe a simple and rapid one-step PCR-based site-directed mutagenesis method to generate mutations in the CFTR gene. This method was used to generate CFTR mutants bearing deletions (p.Gln2_Trp846del, p.Ser700_Asp835del, p.Ile1234_Arg1239del) and truncation with polyhistidine tag insertion (p.Glu1172-3Gly-6-His*), which either recapitulate a disease phenotype or render tools for modulator binding site identification, with subsequent evaluation of drug responses using a high-throughput (384-well) membrane potential-sensitive fluorescence assay of CFTR channel activity within a 1 wk time frame. This proof-of-concept study shows that these methods enable rapid and quantitative comparison of multiple CFTR mutants to emerging drugs, facilitating future large-scale efforts to stratify mutants according to their "theratype" or most promising targeted therapy. PMID:26385858

  2. Influence of mutagenesis of Bacillus thuringiensis Cry1Aa toxin on larvicidal activity.

    PubMed

    Zhang, Chunyan; Xia, Liqiu; Ding, Xuezhi; Huang, Fan; Li, Huanfa; Sun, Yunjun; Yin, Jia

    2011-03-01

    Domain III of Bacillus thuringiensis Cry δ-endotoxins are considered to be related to the stability of the structure and avoidance of overdigestion by proteases. In this study, some residues of potential chymotrypsin and trypsin sites in Domain III of B. thuringiensis Cry1Aa were replaced individually with alanine by site-directed mutagenesis, in order to investigate their functional roles. Except F574A, all mutants F536A, R543A, F550A, F565A, R566A, F570A, F576A, F583A, and F590A were highly expressed the 130 kD protoxins at levels comparable to the wild-type tested by SDS-PAGE. In bioassays, F536A, R566A, and F590A increased toxicity against Spodoptera exigua Hüner larve by 20, 40, and 40%, respectively, as compared to the wild-type. F536A and F565A showed an increase of 6 and 10% in toxicity against Heliothis armigera Hubner than the wild-type. Toxicities of some mutants were altered greatly, and the same mutants were shown to have different toxicities against those two insects. Structural analyses showed that mutants R543A, F574A, F576A-affecting insecticidal activity might be relational to structural stability of toxin or decreased affinity for receptor binding. These results indicated that those residues were involved in the larvicidal activity of the Cry1Aa toxin. PMID:21082182

  3. Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas.

    PubMed

    Nault, Jean-Charles; Datta, Shalini; Imbeaud, Sandrine; Franconi, Andrea; Mallet, Maxime; Couchy, Gabrielle; Letouzé, Eric; Pilati, Camilla; Verret, Benjamin; Blanc, Jean-Frédéric; Balabaud, Charles; Calderaro, Julien; Laurent, Alexis; Letexier, Mélanie; Bioulac-Sage, Paulette; Calvo, Fabien; Zucman-Rossi, Jessica

    2015-10-01

    Hepatocellular carcinomas (HCCs) are liver tumors related to various etiologies, including alcohol intake and infection with hepatitis B (HBV) or C (HCV) virus. Additional risk factors remain to be identified, particularly in patients who develop HCC without cirrhosis. We found clonal integration of adeno-associated virus type 2 (AAV2) in 11 of 193 HCCs. These AAV2 integrations occurred in known cancer driver genes, namely CCNA2 (cyclin A2; four cases), TERT (telomerase reverse transcriptase; one case), CCNE1 (cyclin E1; three cases), TNFSF10 (tumor necrosis factor superfamily member 10; two cases) and KMT2B (lysine-specific methyltransferase 2B; one case), leading to overexpression of the target genes. Tumors with viral integration mainly developed in non-cirrhotic liver (9 of 11 cases) and without known risk factors (6 of 11 cases), suggesting a pathogenic role for AAV2 in these patients. In conclusion, AAV2 is a DNA virus associated with oncogenic insertional mutagenesis in human HCC. PMID:26301494

  4. Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids.

    PubMed

    Kim, Ki-Hyun; Nielsen, Peter E; Glazer, Peter M

    2007-01-01

    Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA-pcPNA duplexes but can bind to complementary DNA sequences by Watson-Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules. PMID:17977869

  5. Systematic Mutagenesis of the Escherichia coli Genome†

    PubMed Central

    Kang, Yisheng; Durfee, Tim; Glasner, Jeremy D.; Qiu, Yu; Frisch, David; Winterberg, Kelly M.; Blattner, Frederick R.

    2004-01-01

    A high-throughput method has been developed for the systematic mutagenesis of the Escherichia coli genome. The system is based on in vitro transposition of a modified Tn5 element, the Sce-poson, into linear fragments of each open reading frame. The transposon introduces both positive (kanamycin resistance) and negative (I-SceI recognition site) selectable markers for isolation of mutants and subsequent allele replacement, respectively. Reaction products are then introduced into the genome by homologous recombination via the λRed proteins. The method has yielded insertion alleles for 1976 genes during a first pass through the genome including, unexpectedly, a number of known and putative essential genes. Sce-poson insertions can be easily replaced by markerless mutations by using the I-SceI homing endonuclease to select against retention of the transposon as demonstrated by the substitution of amber and/or in-frame deletions in six different genes. This allows a Sce-poson-containing gene to be specifically targeted for either designed or random modifications, as well as permitting the stepwise engineering of strains with multiple mutations. The promiscuous nature of Tn5 transposition also enables a targeted gene to be dissected by using randomly inserted Sce-posons as shown by a lacZ allelic series. Finally, assessment of the insertion sites by an iterative weighted matrix algorithm reveals that these hyperactive Tn5 complexes generally recognize a highly degenerate asymmetric motif on one end of the target site helping to explain the randomness of Tn5 transposition. PMID:15262929

  6. Alkyl isocyanates as active site-directed inactivators of guinea pig liver transglutaminase.

    PubMed

    Gross, M; Whetzel, N K; Folk, J E

    1975-10-10

    Alkyl isocyanates are effective inactivators of guinea pig liver transglutaminase. Based on the specificity of the reaction the protection against inactivation by glutamine substrate, and the essential nature of calcium for the inactivation reaction, it is concluded that these reagents act as amide substrate analogs and, thus function in an active site-specific manner. Support for the contention that inactivation results from alkyl thiocarbamate ester formation through the single active site sulfhydryl group of the enzyme is (a) the loss of one free--SH group and the incorporation of 1 mol of reagent/mol of enzyme in the reaction, (b) similarity in chemical properties of the inactive enzyme derivative formed to those previously reported for another alkyl thiocarbamoylenzyme and an alkyl thiocarbamoylcysteine derivative, and (c) the finding that labeled peptides from digests of [methyl-14C]thiocarbamoyltransglutaminase and those from digests of iodoacetamide-inactivated enzyme occupy similar positions on peptide maps. Transglutaminase was found to be inactivated neither by urethan anlogs of its active ester substrates nor by urea analogs of its amide substrates. It is concluded on the basis of these findings that inactive carbamoylenzyme derivatives are formed only by direct addition of the transglutaminase active--SH group to the isocyanate C--N double bond, and not, like several serine active site enzymes, by nucleophilic displacement with urethan analogs of substrate, or by nucleophilic displacement with urea analogs of substrate. PMID:240837

  7. Transposon Mutagenesis of the Extremely Thermophilic Bacterium Thermus thermophilus HB27

    PubMed Central

    Carr, Jennifer F.; Gregory, Steven T.; Dahlberg, Albert E.

    2014-01-01

    Thermus thermophilus is an extremely thermophilic bacterium that grows between 50 °C and 80 °C and is an excellent model organism not only for understanding life at high temperature but also for its biotechnological and industrial applications. Multiple molecular capabilities are available including targeted gene inactivation and the use of shuttle plasmids that replicate in T. thermophilus and Escherichia coli; however the ability to disrupt gene function randomly by transposon insertion has not been developed. Here we report a detailed method of transposon mutagenesis of T. thermophilus HB27 based on the EZ-Tn5 system from Epicentre Biotechnologies. We were able to generate insertion mutations throughout the chromosome by in vitro transposition and transformation with mutagenized genomic DNA. We also report that an additional step, one that fills in single stranded gaps in donor DNA generated by the transposition reaction, was essential for successful mutagenesis. We anticipate that our method of transposon mutagenesis will enable further genetic development of T. thermophilus and may also be valuable for similar endeavors with other under-developed organisms. PMID:24948436

  8. Characterization of the functional role of a flexible loop in the alpha-subunit of tryptophan synthase from Salmonella typhimurium by rapid-scanning, stopped-flow spectroscopy and site-directed mutagenesis.

    PubMed

    Brzović, P S; Hyde, C C; Miles, E W; Dunn, M F

    1993-10-01

    The function of a flexible loop (loop 6) in the alpha-subunit from the tryptophan synthase alpha 2 beta 2 bienzyme complex has been investigated utilizing rapid-scanning (RSSF) and single-wavelength (SWSF) stopped-flow spectroscopies. Loop 6 is an extended sequence of residues which connects beta-strand 6 with alpha-helix 6 in the beta/alpha-barrel fold of the alpha-subunit. Substitution of Leu for Arg179 near the base of loop 6 does not significantly affect either the association of the alpha- and beta-subunits to form the bienzyme complex or the kinetics of the reaction of indole with L-serine (L-Ser) to form L-tryptophan (L-Trp), the process catalyzed by the wild-type beta-subunit [Kawasaki, H., Bauerle, R., Zon, G., Ahmed, S., & Miles, E. W. (1987) J. Biol. Chem. 262, 10678-10683]. However, the alpha-subunit-specific ligand glycerol phosphate (GP), which is an inhibitor of the wild-type beta-reaction, is a much less effective inhibitor of the alpha R179L-catalyzed beta-reaction. Equilibrium titration studies show that the affinity of GP for the alpha-site when either L-Ser or glycine is bound at the beta-site has been reduced by nearly 100- and 200-fold, respectively. SWSF analysis of the reaction of IGP and L-Ser to form L-Trp catalyzed by the bienzyme complex revealed a 15-fold reduction in the binding affinity of the alpha-site substrate 3-indole-D-glycerol 3'-phosphate (IGP) in the reaction catalyzed by the alpha R179L mutant as compared to the wild-type enzyme. These studies show that loop 6 is important both for ligand binding to the alpha-site and for the ligand-induced conformational transition of the alpha-subunit from an "open" to a "closed" structure. Modeling studies, based on extensive structural homology of the alpha-subunit with the glycolytic enzyme triosephosphate isomerase (TIM), predict that closure of loop 6 induced by ligand binding at the alpha-active site would effectively sequester the bound substrate from the solvent and trap indole, produced from the cleavage of IGP, within the confines of the bienzyme complex. This conformational transition would promote the diffusion of indole to the beta-active site via the interconnecting tunnel and would help ensure the close coordination of alpha- and beta-subunit catalytic activities. PMID:8399184

  9. Site-Directed Mutagenesis of IRX9, IRX9L and IRX14 Proteins Involved in Xylan Biosynthesis: Glycosyltransferase Activity Is Not Required for IRX9 Function in Arabidopsis

    PubMed Central

    Ren, Yanfang; Hansen, Sara Fasmer; Ebert, Berit; Lau, Jane; Scheller, Henrik Vibe

    2014-01-01

    Xylans constitute the main non-cellulosic polysaccharide in the secondary cell walls of plants. Several genes predicted to encode glycosyltransferases are required for the synthesis of the xylan backbone even though it is a homopolymer consisting entirely of β-1,4-linked xylose residues. The putative glycosyltransferases IRX9, IRX14, and IRX10 (or the paralogs IRX9L, IRX14L, and IRX10L) are required for xylan backbone synthesis in Arabidopsis. To investigate the function of IRX9, IRX9L, and IRX14, we identified amino acid residues known to be essential for catalytic function in homologous mammalian proteins and generated modified cDNA clones encoding proteins where these residues would be mutated. The mutated gene constructs were used to transform wild-type Arabidopsis plants and the irx9 and irx14 mutants, which are deficient in xylan synthesis. The ability of the mutated proteins to complement the mutants was investigated by measuring growth, determining cell wall composition, and microscopic analysis of stem cross-sections of the transgenic plants. The six different mutated versions of IRX9 and IRX9-L were all able to complement the irx9 mutant phenotype, indicating that residues known to be essential for glycosyltransferases function in homologous proteins are not essential for the biological function of IRX9/IRX9L. Two out of three mutated IRX14 complemented the irx14 mutant, including a mutant in the predicted catalytic amino acid. A IRX14 protein mutated in the substrate-binding DxD motif did not complement the irx14 mutant. Thus, substrate binding is important for IRX14 function but catalytic activity may not be essential for the function of the protein. The data indicate that IRX9/IRX9L have an essential structural function, most likely by interacting with the IRX10/IRX10L proteins, but do not have an essential catalytic function. Most likely IRX14 also has primarily a structural role, but it cannot be excluded that the protein has an important enzymatic activity. PMID:25118690

  10. Identification of essential residues in 2',3'-cyclic nucleotide 3'-phosphodiesterase. Chemical modification and site-directed mutagenesis to investigate the role of cysteine and histidine residues in enzymatic activity.

    PubMed

    Lee, J; Gravel, M; Gao, E; O'Neill, R C; Braun, P E

    2001-05-01

    2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP; EC ) catalyzes in vitro hydrolysis of 3'-phosphodiester bonds in 2',3'-cyclic nucleotides to produce 2'-nucleotides exclusively. N-terminal deletion mapping of the C-terminal two-thirds of recombinant rat CNP1 identified a region that possesses the catalytic domain, with further truncations abolishing activity. Proteolysis and kinetic analysis indicated that this domain forms a compact globular structure and contains all of the catalytically essential features. Subsequently, this catalytic fragment of CNP1 (CNP-CF) was used for chemical modification studies to identify amino acid residues essential for activity. 5,5'-Dithiobis-(2-nitrobenzoic acid) modification studies and kinetic analysis of cysteine CNP-CF mutants revealed the nonessential role of cysteines for enzymatic activity. On the other hand, modification studies with diethyl pyrocarbonate indicated that two histidines are essential for CNPase activity. Consequently, the only two conserved histidines, His-230 and His-309, were mutated to phenylalanine and leucine. All four histidine mutants had k(cat) values 1000-fold lower than wild-type CNP-CF, but K(m) values were similar. Circular dichroism studies demonstrated that the low catalytic activities of the histidine mutants were not due to gross changes in secondary structure. Taken together, these results demonstrate that both histidines assume critical roles for catalysis. PMID:11278504

  11. Structural requirements for assembly of dimeric IgA probed by site-directed mutagenesis of J chain and a cysteine residue of the alpha-chain CH2 domain.

    PubMed

    Krugmann, S; Pleass, R J; Atkin, J D; Woof, J M

    1997-07-01

    The structural features of J chain required for interaction with IgA in IgA dimer assembly were investigated by coexpression of wild-type and mutant forms of J chain with IgA1 in CHO cells. With wild-type J chain, a mixture of J chain-containing dimers and monomers was secreted. Substitution of Cys14 of J chain with Ser resulted in expression of only monomer IgA covalently associated with J chain. Similarly, mutation of Cys68 to Ser also resulted in expression predominantly of a monomer IgA-J chain species. These results suggest that Cys14 and Cys68 play critical roles in formation of J chain-containing IgA dimers, with each forming a disulfide bridge to an IgA monomer. Substitution of Asn48 with Ala, to prevent attachment of N-linked carbohydrate to J chain, also resulted in markedly reduced dimer assembly, suggesting a requirement for the sugar moiety in J chain function. We also mutated Cys311 on the C alpha2 domain of the IgA heavy chain to Ser. When coexpressed with wild-type J chain, this mutant was still capable of forming dimers, indicating that this residue was not involved in dimerization. Taken together, our results are consistent with an arrangement in which IgA monomers are linked end-to-end with J chain interposed. PMID:9200460

  12. Differences in the central nervous system distribution and pharmacology of the mouse 5-hydroxytryptamine-6 receptor compared with rat and human receptors investigated by radioligand binding, site-directed mutagenesis, and molecular modeling.

    PubMed

    Hirst, Warren D; Abrahamsen, Bjarke; Blaney, Frank E; Calver, Andrew R; Aloj, Lucia; Price, Gary W; Medhurst, Andrew D

    2003-12-01

    There is increasing evidence for a role of 5-hydroxytrypta-mine-6 (5-HT6) receptors in cognitive function. In the rat and human brain, 5-HT6 receptors are widely expressed and highly enriched in the basal ganglia. However, in the mouse brain, only very low levels of 5-HT6 receptor mRNA and receptor protein, measured by TaqMan reverse transcriptase-polymerase chain reaction and selective radioligand binding, could be detected, with no evidence of enrichment in the basal ganglia. The mouse receptor was cloned and transiently expressed in human embryonic kidney 293 cells to characterize its pharmacological profile. Despite significant sequence homology between human, rat, and mouse 5-HT6 receptors, the pharmacological profile of the mouse receptor was significantly different from the rat and human receptors. Four amino acid residues, conserved in rat and human and divergent in mouse receptors, were identified, and various mutant receptors were generated and their pharmacologies studied. Residues 188 (tyrosine in mouse, phenylalanine in rat and human) in transmembrane region 5 and 290 (serine in mouse, asparagine in rat and human) in transmembrane region 6 were identified as key amino acids responsible for the different pharmacological profiles. Molecular modeling of the receptor and docking of selective and nonselective compounds was undertaken to elucidate the ligand receptor interactions. The binding pocket was predicted to be different in the mouse compared with rat and human 5-HT6 receptors, and the models were in excellent agreement with the observed mutation results and have been used extensively in the design of further selective 5-HT6 antagonists. PMID:14645659

  13. Functional Differentiation of Antiporter-Like Polypeptides in Complex I; a Site-Directed Mutagenesis Study of Residues Conserved in MrpA and NuoL but Not in MrpD, NuoM, and NuoN

    PubMed Central

    Sperling, Eva; Górecki, Kamil; Drakenberg, Torbjörn

    2016-01-01

    It has long been known that the three largest subunits in the membrane domain (NuoL, NuoM and NuoN) of complex I are homologous to each other, as well as to two subunits (MrpA and MrpD) from a Na+/H+ antiporter, Mrp. MrpA and NuoL are more similar to each other and the same is true for MrpD and NuoN. This suggests a functional differentiation which was proven experimentally in a deletion strain model system, where NuoL could restore the loss of MrpA, but not that of MrpD and vice versa. The simplest explanation for these observations was that the MrpA and MrpD proteins are not antiporters, but rather single subunit ion channels that together form an antiporter. In this work our focus was on a set of amino acid residues in helix VIII, which are only conserved in NuoL and MrpA (but not in any of the other antiporter-like subunits.) and to compare their effect on the function of these two proteins. By combining complementation studies in B. subtilis and 23Na-NMR, response of mutants to high sodium levels were tested. All of the mutants were able to cope with high salt levels; however, all but one mutation (M258I/M225I) showed differences in the efficiency of cell growth and sodium efflux. Our findings showed that, although very similar in sequence, NuoL and MrpA seem to differ on the functional level. Nonetheless the studied mutations gave rise to interesting phenotypes which are of interest in complex I research. PMID:27391676

  14. 2012 MUTAGENESIS GORDON RESEARCH CONFERENCE, AUGUST 19-23, 2012

    SciTech Connect

    Demple, Bruce

    2012-08-23

    The delicate balance among cellular pathways that control mutagenic changes in DNA will be the focus of the 2012 Mutagenesis Gordon Research Conference. Mutagenesis is essential for evolution, while genetic stability maintains cellular functions in all organisms from microbes to metazoans. Different systems handle DNA lesions at various times of the cell cycle and in different places within the nucleus, and inappropriate actions can lead to mutations. While mutation in humans is closely linked to disease, notably cancers, mutational systems can also be beneficial. The conference will highlight topics of beneficial mutagenesis, including full establishment of the immune system, cell survival mechanisms, and evolution and adaptation in microbial systems. Equal prominence will be given to detrimental mutation processes, especially those involved in driving cancer, neurological diseases, premature aging, and other threats to human health. Provisional session titles include Branching Pathways in Mutagenesis; Oxidative Stress and Endogenous DNA Damage; DNA Maintenance Pathways; Recombination, Good and Bad; Problematic DNA Structures; Localized Mutagenesis; Hypermutation in the Microbial World; and Mutation and Disease.

  15. A Multiple-Labeling Strategy for Nonribosomal Peptide Synthetases Using Active-Site-Directed Proteomic Probes for Adenylation Domains.

    PubMed

    Ishikawa, Fumihiro; Suzuki, Takehiro; Dohmae, Naoshi; Kakeya, Hideaki

    2015-12-01

    Genetic approaches have greatly contributed to our understanding of nonribosomal peptide biosynthetic machinery; however, proteomic investigations are limited. Here, we developed a highly sensitive detection strategy for multidomain nonribosomal peptide synthetases (NRPSs) by using a multiple-labeling technique with active-site-directed probes for adenylation domains. When applied to gramicidin S-producing and -nonproducing strains of Aneurinibacillus migulanus (DSM 5759 and DSM 2895, respectively), the multiple technique sensitively detected an active multidomain NRPS (GrsB) in lysates obtained from the organisms. This functional proteomics method revealed an unknown inactive precursor (or other inactive form) of GrsB in the nonproducing strain. This method provides a new option for the direct detection, functional analysis, and high-resolution identification of low-abundance active NRPS enzymes in native proteomic environments. PMID:26467472

  16. Impact of Site-Directed Mutant Luciferase on Quantitative Green and Orange/Red Emission Intensities in Firefly Bioluminescence

    NASA Astrophysics Data System (ADS)

    Wang, Yu; Akiyama, Hidefumi; Terakado, Kanako; Nakatsu, Toru

    2013-08-01

    Firefly bioluminescence has attracted great interest because of its high quantum yield and intriguing modifiable colours. Modifications to the structure of the enzyme luciferase can change the emission colour of firefly bioluminescence, and the mechanism of the colour change has been intensively studied by biochemists, structural biologists, optical physicists, and quantum-chemistry theorists. Here, we report on the quantitative spectra of firefly bioluminescence catalysed by wild-type and four site-directed mutant luciferases. While the mutation caused different emission spectra, the spectra differed only in the intensity of the green component (λmax ~ 560 nm). In contrast, the orange (λmax ~ 610 nm) and red (λmax ~ 650 nm) components present in all the spectra were almost unaffected by the modifications to the luciferases and changes in pH. Our results reveal that the intensity of the green component is the unique factor that is influenced by the luciferase structure and other reaction conditions.

  17. CRISPR/Cas9 genome editing technique and its application in site-directed genome modification of animals.

    PubMed

    Jinwei, Zhou; Qipin, Xu; Jing, Yao; Shumin, Yu; Suizhong, Cao

    2015-10-01

    CRISPR/Cas system, which uses CRISPR RNAs (crRNAs) to guide Cas nuclease to silence invading nucleic acids, is self-defense system against exogenous virus or plasmid in bacteria and archaea. Through molecular modification, the typeⅡCRISPR/Cas system has become a highly efficient site-directed genome editing technique, which is simpler than zinc-finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs) and easier to be designed and applied. In this review, we summarize the evolutionary history of CRISPR/Cas9 system, the working principle and modification process of type Ⅱ CRISPR/Cas and its application in animal genome modification. We also analyze the existing problems and improvement program of the CRISPR/Cas9 system as well as its application prospect combined with successful cases, which may provide innovative perspectives on improving animal traits and establishing animal models of human diseases. PMID:26496753

  18. A Structural Study of Norovirus 3C Protease Specificity: Binding of a Designed Active Site-Directed Peptide Inhibitor†

    PubMed Central

    2010-01-01

    Noroviruses are the major cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.7 Å resolution. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, which is based on the most rapidly cleaved recognition sequence in the 200 kDa polyprotein substrate, reacts covalently through its propenyl ethyl ester group (X) with the active site nucleophile, Cys 139. The structure permits, for the first time, the identification of substrate recognition and binding groups in a noroviral 3C protease and thus provides important new information for the development of antiviral prophylactics. PMID:21128685

  19. Site-Directed RNA Editing in Vivo Can Be Triggered by the Light-Driven Assembly of an Artificial Riboprotein

    PubMed Central

    2015-01-01

    Site-directed RNA editing allows for the manipulation of RNA and protein function by reprogramming genetic information at the RNA level. For this we assemble artificial RNA-guided editases and demonstrate their transcript repair activity in cells and in developing embryos of the annelid Platynereis dumerilii. A hallmark of our assembly strategy is the covalent attachment of guideRNA and editing enzyme by applying the SNAP-tag technology, a process that we demonstrate here to be readily triggered by light in vitro, in mammalian cell culture, and also in P. dumerilii. Lacking both sophisticated chemistry and extensive genetic engineering, this technology provides a convenient route for the light-dependent switching of protein isoforms. The presented strategy may also serve as a blue-print for the engineering of addressable machineries that apply tailored nucleic acid analogues to manipulate RNA or DNA site-specifically in living organisms. PMID:26594902

  20. Mapping membrane protein backbone dynamics: a comparison of site-directed spin labeling with NMR 15N-relaxation measurements.

    PubMed

    Lo, Ryan H; Kroncke, Brett M; Solomon, Tsega L; Columbus, Linda

    2014-10-01

    The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR (15)N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R1 relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins. PMID:25296323

  1. Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics.

    PubMed

    Colson, Brett A; Thompson, Andrew R; Espinoza-Fonseca, L Michel; Thomas, David D

    2016-03-22

    We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron-electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. PMID:26908877

  2. Elucidating the design principles of photosynthetic electron-transfer proteins by site-directed spin labeling EPR spectroscopy.

    PubMed

    Ishara Silva, K; Jagannathan, Bharat; Golbeck, John H; Lakshmi, K V

    2016-05-01

    Site-directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy is a powerful tool to determine solvent accessibility, side-chain dynamics, and inter-spin distances at specific sites in biological macromolecules. This information provides important insights into the structure and dynamics of both natural and designed proteins and protein complexes. Here, we discuss the application of SDSL EPR spectroscopy in probing the charge-transfer cofactors in photosynthetic reaction centers (RC) such as photosystem I (PSI) and the bacterial reaction center (bRC). Photosynthetic RCs are large multi-subunit proteins (molecular weight≥300 kDa) that perform light-driven charge transfer reactions in photosynthesis. These reactions are carried out by cofactors that are paramagnetic in one of their oxidation states. This renders the RCs unsuitable for conventional nuclear magnetic resonance spectroscopy investigations. However, the presence of native paramagnetic centers and the ability to covalently attach site-directed spin labels in RCs makes them ideally suited for the application of SDSL EPR spectroscopy. The paramagnetic centers serve as probes of conformational changes, dynamics of subunit assembly, and the relative motion of cofactors and peptide subunits. In this review, we describe novel applications of SDSL EPR spectroscopy for elucidating the effects of local structure and dynamics on the electron-transfer cofactors of photosynthetic RCs. Because SDSL EPR Spectroscopy is uniquely suited to provide dynamic information on protein motion, it is a particularly useful method in the engineering and analysis of designed electron transfer proteins and protein networks. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson. PMID:26334844

  3. Site directed recombination

    DOEpatents

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  4. Graphene oxide can induce in vitro and in vivo mutagenesis

    NASA Astrophysics Data System (ADS)

    Liu, Yuanyuan; Luo, Yi; Wu, Jing; Wang, Yinsong; Yang, Xiaoying; Yang, Rui; Wang, Baiqi; Yang, Jinrong; Zhang, Ning

    2013-12-01

    Graphene oxide (GO) has attracted enormous interests due to its extraordinary properties. Recent studies have confirmed the cytotoxicity of GO, we further investigate its mutagenic potential in this study. The results showed that GO interfered with DNA replication and induced mutagenesis at molecular level. GO treatments at concentrations of 10 and 100 μg/mL altered gene expression patterns at cellular level, and 101 differentially expressed genes mediated DNA-damage control, cell apoptosis, cell cycle, and metabolism. Intravenous injection of GO at 4 mg/kg for 5 consecutive days clearly induced formation of micronucleated polychromic erythrocytes in mice, and its mutagenesis potential appeared to be comparable to cyclophosphamide, a classic mutagen. In conclusion, GO can induce mutagenesis both in vitro and in vivo, thus extra consideration is required for its biomedical applications.

  5. An algorithm for protein engineering: simulations of recursive ensemble mutagenesis.

    PubMed Central

    Arkin, A P; Youvan, D C

    1992-01-01

    An algorithm for protein engineering, termed recursive ensemble mutagenesis, has been developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Starting from partially randomized "wild-type" DNA sequences, a highly parallel search of sequence space for peptides fitting an experimenter's criteria is performed. Each iteration uses information gained from the previous rounds to search the space more efficiently. Simulations of the technique indicate that, under a variety of conditions, the algorithm can rapidly produce a diverse population of proteins fitting specific criteria. In the experimental analog, genetic selection or screening applied during recursive ensemble mutagenesis should force the evolution of an ensemble of mutants to a targeted cluster of related phenotypes. Images PMID:1502200

  6. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

    PubMed

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

    2015-12-18

    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

  7. The ClosTron: Mutagenesis in Clostridium refined and streamlined.

    PubMed

    Heap, John T; Kuehne, Sarah A; Ehsaan, Muhammad; Cartman, Stephen T; Cooksley, Clare M; Scott, Jamie C; Minton, Nigel P

    2010-01-01

    The recent development of the ClosTron Group II intron directed mutagenesis tool for Clostridium has advanced genetics in this genus, and here we present several significant improvements. We have shown how marker re-cycling can be used to construct strains with multiple mutations, demonstrated using FLP/FRT in Clostridium acetobutylicum; tested the capacity of the system for the delivery of transgenes to the chromosome of Clostridium sporogenes, which proved feasible for 1.0kbp transgenes in addition to a marker; and extended the host range of the system, constructing mutants in Clostridium beijerinckii and, for the first time, in a B1/NAP1/027 'epidemic' strain of Clostridium difficile. Automated intron design bioinformatics are now available free-of-charge at our website http://clostron.com; the out-sourced construction of re-targeted intron plasmids has become cost-effective as well as rapid; and the combination of constitutive intron expression with direct selection for intron insertions has made mutant isolation trivial. These developments mean mutants can now be constructed with very little time and effort for the researcher. Those who prefer to construct plasmids in-house are no longer reliant on a commercial kit, as a mixture of two new plasmids provides unlimited template for intron re-targeting by Splicing by Overlap Extension (SOE) PCR. The new ClosTron plasmids also offer blue-white screening and other options for identification of recombinant plasmids. The improved ClosTron system supersedes the prototype plasmid pMTL007 and the original method, and exploits the potential of Group II introns more fully. PMID:19891996

  8. Insertional mutagenesis using Tnt1 retrotransposon in potato

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potato is the third most important food crop in the world. However, genetics and genomics research of potato has lagged behind many major crop species due to its autotetraploidy and a highly heterogeneous genome. Insertional mutagenesis using T-DNA or transposable elements, which is available in sev...

  9. Methods for targetted mutagenesis in gram-positive bacteria

    SciTech Connect

    Yang, Yunfeng

    2014-05-27

    The present invention provides a method of targeted mutagenesis in Gram-positive bacteria. In particular, the present invention provides a method that effectively integrates a suicide integrative vector into a target gene in the chromosome of a Gram-positive bacterium, resulting in inactivation of the target gene.

  10. Coupled mutagenesis screens and genetic mapping in zebrafish.

    PubMed Central

    Rawls, John F; Frieda, Matthew R; McAdow, Anthony R; Gross, Jason P; Clayton, Chad M; Heyen, Candy K; Johnson, Stephen L

    2003-01-01

    Forward genetic analysis is one of the principal advantages of the zebrafish model system. However, managing zebrafish mutant lines derived from mutagenesis screens and mapping the corresponding mutations and integrating them into the larger collection of mutations remain arduous tasks. To simplify and focus these endeavors, we developed an approach that facilitates the rapid mapping of new zebrafish mutations as they are generated through mutagenesis screens. We selected a minimal panel of 149 simple sequence length polymorphism markers for a first-pass genome scan in crosses involving C32 and SJD inbred lines. We also conducted a small chemical mutagenesis screen that identified several new mutations affecting zebrafish embryonic melanocyte development. Using our first-pass marker panel in bulked-segregant analysis, we were able to identify the genetic map positions of these mutations as they were isolated in our screen. Rapid mapping of the mutations facilitated stock management, helped direct allelism tests, and should accelerate identification of the affected genes. These results demonstrate the efficacy of coupling mutagenesis screens with genetic mapping. PMID:12663538

  11. 2002 Gordon Research Conference on Mutagenesis. Final Progress Report

    SciTech Connect

    2002-08-02

    The Gordon Research Conference (GRC) on MUTAGENESIS was held at Bates College from 7/28/02 thru 8/2/02. The Conference was well-attended with 157 participants. The attendees represented the spectrum of endeavor in this field coming from academia, industry, and government laboratories, both U.S. and foreign scientists, senior researchers, young investigators, and students.

  12. Transposon mutagenesis of Bacteroides fragilis using a mariner transposon vector.

    PubMed

    Veeranagouda, Yaligara; Husain, Fasahath; Wexler, Hannah M

    2013-08-01

    The mariner transposon vector pYV07 was tested for use in the mutagenesis of Bacteroides fragilis 638R. The transposon vector efficiently generated mutants in B. fragilis 638R. The transposon disrupted genes were scattered throughout the genome of B. fragilis 638R. This method serves as a powerful tool to study B. fragilis. PMID:23664906

  13. CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans.

    PubMed

    Waaijers, Selma; Portegijs, Vincent; Kerver, Jana; Lemmens, Bennie B L G; Tijsterman, Marcel; van den Heuvel, Sander; Boxem, Mike

    2013-11-01

    The generation of genetic mutants in Caenorhabditis elegans has long relied on the selection of mutations in large-scale screens. Directed mutagenesis of specific loci in the genome would greatly speed up analysis of gene function. Here, we adapt the CRISPR/Cas9 system to generate mutations at specific sites in the C. elegans genome. PMID:23979586

  14. What Can a Micronucleus Teach? Learning about Environmental Mutagenesis

    ERIC Educational Resources Information Center

    Linde, Ana R.; Garcia-Vazquez, Eva

    2009-01-01

    The micronucleus test is widely employed in environmental health research. It can also be an excellent tool for learning important concepts in environmental health. In this article we present an inquiry-based laboratory exercise where students explore several theoretical and practical aspects of environmental mutagenesis employing the micronucleus…

  15. Favipiravir elicits antiviral mutagenesis during virus replication in vivo

    PubMed Central

    Arias, Armando; Thorne, Lucy; Goodfellow, Ian

    2014-01-01

    Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses. DOI: http://dx.doi.org/10.7554/eLife.03679.001 PMID:25333492

  16. GERM-LINE SPECIFIC FACTORS IN CHEMICAL MUTAGENESIS

    EPA Science Inventory

    Chemical mutagenesis test results ave not revealed evidence of germ-line specific mutagens. owever, conventional assays have indicated that there are male-female differences in mutagenic response, as well as quantitative/qualitative differences in induced mutations which depend u...

  17. Comparative Mutagenesis Studies of Retinal Release in Light-Activated Zebrafish Rhodopsin Using Fluorescence Spectroscopy.

    PubMed

    Morrow, J M; Chang, B S W

    2015-07-28

    Rhodopsin is the visual pigment responsible for initiating scotopic (dim-light) vision in vetebrates. Once activated by light, release of all-trans-retinal from rhodopsin involves hydrolysis of the Schiff base linkage, followed by dissociation of retinal from the protein moiety. This kinetic process has been well studied in model systems such as bovine rhodopsin, but not in rhodopsins from cold-blooded animals, where physiological temperatures can vary considerably. Here, we characterize the rate of retinal release from light-activated rhodopsin in an ectotherm, zebrafish (Danio rerio), demonstrating in a fluorescence assay that this process occurs more than twice as fast as bovine rhodopsin at similar temperatures in 0.1% dodecyl maltoside. Using site-directed mutagenesis, we found that differences in retinal release rates can be attributed to a series of variable residues lining the retinal channel in three key structural motifs: an opening in metarhodopsin II between transmembrane helix 5 (TM5) and TM6, in TM3 near E122, and in the "retinal plug" formed by extracellular loop 2 (EL2). The majority of these sites are more proximal to the β-ionone ring of retinal than the Schiff base, indicating their influence on retinal release is more likely due to steric effects during retinal dissociation, rather than alterations to Schiff base stability. An Arrhenius plot of zebrafish rhodopsin was consistent with this model, inferring that the activation energy for Schiff base hydrolysis is similar to that of bovine rhodopsin. Functional variation at key sites identified in this study is consistent with the idea that retinal release might be an adaptive property of rhodopsin in vertebrates. Our study is one of the few investigating a nonmammalian rhodopsin, which will help establish a better understanding of the molecular mechanisms contributing to vision in cold-blooded vertebrates. PMID:26098991

  18. Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus.

    PubMed

    Ma, Jun-Yan; Cao, Hai-Long; Tan, Hai-Dong; Hu, Xue-Jun; Liu, Wu-Jun; Du, Yu-Guang; Yin, Heng

    2016-01-01

    Inulinase is an enzyme that belongs to glycoside hydrolase family 32. It converts inulin into high-fructose syrups and fructoligosaccharides, both of which are widely used in pharmaceutical and food industries. In this study, the kcINU1 gene (GenBank accession number AF178979) encoding an exoinulinase was cloned from Kluyveromyces cicerisporus CBS4857 and expressed in Pichia pastoris X-33, yielding a maximum of 45.2 ± 0.6 U mL(-1) of inulinase activity of culture supernatant. The expressed inulinase was purified and characterized. The enzyme had an optimum temperature of 55 °C and an optimum pH of 4.5. It had a K m of 0.322 mM and a V max of 4317 μM min(-1) mg(-1) protein when inulin was used as a substrate. It retained nearly 90 % of the maximal activity after pre-incubation at 50 °C for 1 h or at pH ranging from 3.0 to 6.0 at 4 °C for 24 h, demonstrating that KcINU1 was stable at high temperature and low pH. Moreover, we constructed two KcINU1 mutants, Asp30Ala and Glu215Ala, by site-directed mutagenesis and confirmed via zymogram analysis that Asp-30 and Glu-215 of the enzyme were the catalytic active center. The present study has provided important information for understanding the catalytic mechanism of exoinulinase. PMID:26446826

  19. In vitro mutagenesis and functional expression in Escherichia coli of a cDNA encoding the catalytic domain of human DNA ligase I.

    PubMed Central

    Kodama, K; Barnes, D E; Lindahl, T

    1991-01-01

    Human cDNAs encoding fragments of DNA ligase I, the major replicative DNA ligase in mammalian cells, have been expressed as lacZ fusion proteins in Escherichia coli. A cDNA encoding the carboxyl-terminal catalytic domain of human DNA ligase I was able to complement a conditional-lethal DNA ligase mutation in E. coli as measured by growth of the mutant strain at the non-permissive temperature. Targeted deletions of the amino and carboxyl termini of the catalytic domain identified a minimum size necessary for catalytic function and a maximum size for optimal complementing activity in E. coli. The human cDNA was subjected to systematic site-directed mutagenesis in vitro and mutant polypeptides assayed for functional expression in the E. coli DNA ligase mutant. Such functional analysis of the active site of DNA ligase I identified specific residues required for the formation of an enzyme-adenylate reaction intermediate. Images PMID:1956768

  20. Tryptophan rotamer distribution revealed for the α-helix in tear lipocalin by site-directed tryptophan fluorescence.

    PubMed

    Gasymov, Oktay K; Abduragimov, Adil R; Glasgow, Ben J

    2012-11-15

    Rotamer libraries are a valuable tool for protein structure determination, modeling, and design. Site-directed tryptophan fluorescence (SDTF) was used in combination with the rotamer model for the fluorescence intensity decays to solve α-helical conformations of proteins in solution. Single Trp mutations located in an α-helical segment of human tear lipocalin were explored for structure assignment. Along with fluorescence λ(max) values, the rotamer model assignment of fluorescence lifetimes fits the backbone conformation. Typically, Trp fluorescence in proteins shows three lifetimes. However, for the α-helix, two lifetimes assigned to t and g(-) rotamers were satisfactory to describe Trp fluorescence intensity decays. The g(+) rotamer is not feasible in the α-helix due to steric restriction. Trp rotamer distributions obtained by fluorescence were compared with the rotamer library derived from X-ray crystallography data of proteins. The Trp rotamer distributions vary for solvent exposed and buried (tertiary interaction) sites. A new strategy using the rotamer distribution with SDTF (RD-SDTF) removes the limitation of regular SDTF and other labeling techniques, in which site-specific differences, e.g., accessibility, are presumed. The RD-SDTF technique does not rely on environmental differences of side chains and is able to detect α-helical structure where all side chains are exposed to solvent. Potentially, this technique is applicable to various proteins including membrane proteins, which are rich in α-helix motif. PMID:23088798

  1. Tryptophan Rotamer Distribution Revealed for the Alpha-Helix in Tear Lipocalin by Site Directed Tryptophan Fluorescence

    PubMed Central

    Gasymov, Oktay K.; Abduragimov, Adil R.; Glasgow, Ben J.

    2012-01-01

    Rotamer libraries are a valuable tool for protein structure determination, modeling and design. Site-directed tryptophan fluorescence (SDTF) was used in combination with the rotamer model for the fluorescence intensity decays to solve α-helical conformations of proteins in solution. Single Trp mutations located in an α-helical segment of human tear lipocalin were explored for structure assignment. Along with fluorescence λmax values, the rotamer model assignment of fluorescence lifetimes fits the backbone conformation. Typically Trp fluorescence in proteins shows three lifetimes. However, for the α-helix, two lifetimes assigned to t and g− rotamers were satisfactory to describe Trp fluorescence intensity decays. The g+ rotamer is not feasible in the α-helix due to steric restriction. Trp rotamer distributions obtained by fluorescence were compared with the rotamer library derived from X-ray crystallography data of proteins. The Trp rotamer distributions vary for solvent exposed and buried (tertiary interaction) sites. A new strategy using the rotamer distribution with SDTF (RD-SDTF) removes the limitation of regular SDTF and other labeling techniques, in which site specific differences, e.g. accessibility, are presumed. The RD-SDTF technique does not rely on environmental differences of side chains and is able to detect α-helical structure where all side chains are exposed to solvent. Potentially this technique is applicable to various proteins including membrane proteins, which are rich in α-helix motif. PMID:23088798

  2. Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy.

    PubMed Central

    Askew, G R; Doetschman, T; Lingrel, J B

    1993-01-01

    Sequential gene targeting was used to introduce point mutations into one alpha 2 isoform Na,K-ATPase homolog in mouse embryonic stem (ES) cells. In the first round of targeted replacement, the gene was tagged with selectable markers by insertion of a Neor/HSV-tk gene cassette, and this event was selected for by gain of neomycin (G418) resistance. In the second targeted replacement event, the tagged genomic sequence was exchanged with a vector consisting of homologous genomic sequences carrying five site-directed nucleotide substitutions. Embryonic stem cell clones modified by exchange with the mutation vector were selected for loss of the HSV-tk gene by resistance to ganciclovir. Candidate clones were further screened and identified by polymerase chain reaction and Southern blot analysis. By this strategy, the endogenous alpha 2 isoform Na,K-ATPase gene was altered to encode two other amino acids so that the enzyme is resistant to inhibition by cardiac glycosides while maintaining its transmembrane ion-pumping function. Since the initial tagging event and the subsequent mutation-exchange event are independent of one another, a tagged cell line can be used to generate a variety of mutant lines by exchange with various mutation vectors at the tagged locus. This method should be useful for testing specific mutations introduced into the genomes of tissue culture cells and animals and for developing animal models encompassing the mutational variability of known genetic disorders. Images PMID:8391633

  3. Site-Directed Conjugation of Antibodies to Apoferritin Nanocarrier for Targeted Drug Delivery to Prostate Cancer Cells.

    PubMed

    Dostalova, Simona; Cerna, Tereza; Hynek, David; Koudelkova, Zuzana; Vaculovic, Tomas; Kopel, Pavel; Hrabeta, Jan; Heger, Zbynek; Vaculovicova, Marketa; Eckschlager, Tomas; Stiborova, Marie; Adam, Vojtech

    2016-06-15

    Herein, we describe a novel approach for targeting of ubiquitous protein apoferritin (APO)-encapsulating doxorubicin (DOX) to prostate cancer using antibodies against prostate-specific membrane antigen (PSMA). The conjugation of anti-PSMA antibodies and APO was carried out using HWRGWVC heptapeptide, providing their site-directed orientation. The prostate-cancer-targeted and nontargeted nanocarriers were tested using LNCaP and HUVEC cell lines. A total of 90% of LNCaP cells died after treatment with DOX (0.25 μM) or DOX in nontargeted and prostate-cancer-targeted APO, proving that the encapsulated DOX toxicity for LNCaP cells remained the same. Free DOX showed higher toxicity for nonmalignant cells, whereas the toxicity was lower after treatment with the same dosage of APO-encapsulated DOX (APODOX) and even more in prostate-cancer-targeted APODOX. Hemolytic assay revealed exceptional hemocompatibility of the entire nanocarrier. The APO encapsulation mechanism ensures applicability using a wide variety of chemotherapeutic drugs, and the presented surface modification enables targeting to various tumors. PMID:27219717

  4. Combined mutagenesis and kinetics characterization of the bilin-binding GAF domain of the protein Slr1393 from the Cyanobacterium Synechocystis PCC6803.

    PubMed

    Xu, Xiu-Ling; Gutt, Alexander; Mechelke, Jonas; Raffelberg, Sarah; Tang, Kun; Miao, Dan; Valle, Lorena; Borsarelli, Claudio D; Zhao, Kai-Hong; Gärtner, Wolfgang

    2014-05-26

    The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (λmax =649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 μs, 390 μs, and 1.5 ms) for the red-to-green conversion, and 1.2 μs, 340 μs, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp. PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the green-absorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation. PMID:24764310

  5. Mutation induced extinction in finite populations: lethal mutagenesis and lethal isolation.

    PubMed

    Wylie, C Scott; Shakhnovich, Eugene I

    2012-01-01

    Reproduction is inherently risky, in part because genomic replication can introduce new mutations that are usually deleterious toward fitness. This risk is especially severe for organisms whose genomes replicate "semi-conservatively," e.g. viruses and bacteria, where no master copy of the genome is preserved. Lethal mutagenesis refers to extinction of populations due to an unbearably high mutation rate (U), and is important both theoretically and clinically, where drugs can extinguish pathogens by increasing their mutation rate. Previous theoretical models of lethal mutagenesis assume infinite population size (N). However, in addition to high U, small N can accelerate extinction by strengthening genetic drift and relaxing selection. Here, we examine how the time until extinction depends jointly on N and U. We first analytically compute the mean time until extinction (τ) in a simplistic model where all mutations are either lethal or neutral. The solution motivates the definition of two distinct regimes: a survival phase and an extinction phase, which differ dramatically in both how τ scales with N and in the coefficient of variation in time until extinction. Next, we perform stochastic population-genetics simulations on a realistic fitness landscape that both (i) features an epistatic distribution of fitness effects that agrees with experimental data on viruses and (ii) is based on the biophysics of protein folding. More specifically, we assume that mutations inflict fitness penalties proportional to the extent that they unfold proteins. We find that decreasing N can cause phase transition-like behavior from survival to extinction, which motivates the concept of "lethal isolation." Furthermore, we find that lethal mutagenesis and lethal isolation interact synergistically, which may have clinical implications for treating infections. Broadly, we conclude that stably folded proteins are only possible in ecological settings that support sufficiently large populations

  6. piggyBac-based insertional mutagenesis in the presence of stably integrated P elements in Drosophila.

    PubMed

    Hacker, Udo; Nystedt, Sverker; Barmchi, Mojgan Padash; Horn, Carsten; Wimmer, Ernst A

    2003-06-24

    P element-mediated mutagenesis has been used to disrupt an estimated 25% of genes essential for Drosophila adult viability. Mutation of all genes in the fly genome, however, poses a problem, because P elements show significant hotspots of integration. In addition, advanced screening scenarios often require the use of P element-based tools like the generation of germ-line mosaics using FLP recombinase-mediated recombination or gene misexpression using the UAS/Gal4 system. These techniques are P element-based and can therefore not be combined with the use of P elements as mutagenic agents. To circumvent these limitations, we have developed an insertional mutagenesis system using non-P element transposons. An enhanced yellow fluorescent protein-marked piggyBac-based mutator element was mobilized by a piggyBac specific transposase source expressed from a Hermes-based jump-starter transposon marked with enhanced cyan fluorescent protein. In a pilot screen, we have generated 798 piggyBac insertions on FRT bearing third chromosomes of which 9% have sustained a putatively piggyBac-related lethal hit. The FRTs present on the target chromosome remained stably integrated during the screen and could subsequently be used to generate germ-line clones associated with maternal and zygotic phenotypes. PCR-based analysis of insertion loci shows that 57% of the insertions are in genes for which no P element insertions have been reported. Our data demonstrate the potential of this technique to facilitate the quest for saturation mutagenesis of the Drosophila genome. The system is Drosophila nonspecific and potentially applicable in a broad spectrum of nonmodel organisms. PMID:12802016

  7. Structural and functional characterization of mutants of recombinant single-chain urokinase-type plasminogen activator obtained by site-specific mutagenesis of Lys158, Ile159 and Ile160.

    PubMed

    Lijnen, H R; Nelles, L; Van Hoef, B; Demarsin, E; Collen, D

    1988-11-15

    Single-chain urokinase-type plasminogen activator (scu-PA) is converted to urokinase by hydrolysis of the Lys158-Ile159 peptide bond. Site-directed mutagenesis of Lys158 to Gly or Glu yields plasmin-resistant mutants with a 10-20-fold reduced catalytic efficiency for the activation of plasminogen [Nelles et al. (1987) J. Biol. Chem. 262, 5682-5689]. In the present study, we have further evaluated the enzymatic properties of derivatives of recombinant scu-PA (rscu-PA), produced by site-directed mutagenesis of Lys158, Ile159 or Ile160, in order to obtain additional information on the structure/function relations underlying the enzymatic properties of the single- and two-chain u-PA moieties. [Arg158]rscu-PA (rscu-PA with Lys158 substituted with Arg) appeared to be indistinguishable from wild-type rscu-PA with respect to plasminogen-activating potential (catalytic efficiency k2/Km = 0.21 mM-1 s-1 versus 0.64 mM-1 s-1), conversion to active two-chain urokinase by plasmin (k2/Km = 0.13 microM-1 s-1 versus 0.28 microM-1 s-1), as well as its specific activity (48,000 IU/mg as compared to 60,000 IU/mg) and its fibrinolytic potential in a plasma medium (50% lysis in 2 h with 2.8 micrograms/ml versus 2.1 micrograms/ml). [Pro159]rscu-PA (Ile159 substituted with Pro) and [Gly159]rscu-PA (Ile159 converted to Gly) are virtually inactive towards plasminogen (k2/Km less than 0.004 mM-1 s-1). They are however converted to inactive two-chain derivatives by plasmin following cleavage of the Arg156-Phe157 peptide bond in [Pro159]rscu-PA and of the Lys158-Gly159 peptide bond in [Gly159]rscu-PA. [Gly158,Lys160]rscu-PA (with Lys158 converted to Gly and Ile160 to Lys) has a low catalytic efficiency towards plasminogen both as a single-chain form (k2/Km = 0.012 mM-1 s-1) and as the two-chain derivative (k2/Km = 0.13 mM-1 s-1) generated by cleavage of both the Arg156-Phe157 and/or the Lys160-Gly161 peptide bonds by plasmin. These findings suggest that the enzymatic properties of rscu-PA are

  8. Functional studies of the gene slr2049 from Synechocystis sp. PCC6803 and its site-directed mutation.

    PubMed

    Liu, Bingjun; Chen, Sili; Zhang, Lei

    2015-06-01

    Phycobiliprotein is a homologous family of light-harvesting chromoproteins existing in cyanobacteria, red algae and cryptophytes. Phycobiliprotein is made up of phycobilin and its corresponding apophycobiliprotein, and they are covalently linked by the thioether bond with the bilin lyase. Using the software BLAST, we have found gene slr2049 in Synechocystis sp. PCC6803 homologous to the biliprotein lyase gene cpeS. This paper investigates the protein expressed by gene slr2049 to find the enzymatic activity characteristics. We cloned slr2049 and its related genes cpcB, ho1, and pcyA which are linked with the synthesis of phycocyanin. Special amino acid mutagenesis was performed on slr2049 to construct eight mutants slr2049 (H21S), slr2049 (L23S), slr2049 (A24S), slr2049 (F25S), slr2049 (W72L), slr2049 (G84S), slr2049 (R107S) and slr2049 (Y124S). These mutants were ligated with vectors pEDFDuet-1 and pET-23a to construct pCDF-cpcB-slr2049 wild-type, pCDF-cpcB-slr2049 mutants and pET-ho1-pcyA, for the purpose of protein expression and analysis. The results showed that the wild-type and mutants slr2049 (H21S), slr2049 (L23S), slr2049 (F25S), slr2049 (W72L), slr2049 (G84S), and slr2049 (Y124S) can catalyze CpcB to couple on PCB correctly and the products have unique spectral characteristics. However mutants slr2049 (A24S) and slr2049 (R107S) have no spectral characteristics. Thus, it is suggested that alanine at position 24 and arginine at position 107 are the active sites. PMID:25791490

  9. Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin

    PubMed Central

    Lerch, Michael T.; Horwitz, Joseph; McCoy, John; Hubbell, Wayne L.

    2013-01-01

    Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875–85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate. PMID:24248390

  10. Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron-electron resonance.

    PubMed

    Lerch, Michael T; Yang, Zhongyu; Brooks, Evan K; Hubbell, Wayne L

    2014-04-01

    The dominance of a single native state for most proteins under ambient conditions belies the functional importance of higher-energy conformational states (excited states), which often are too sparsely populated to allow spectroscopic investigation. Application of high hydrostatic pressure increases the population of excited states for study, but structural characterization is not trivial because of the multiplicity of states in the ensemble and rapid (microsecond to millisecond) exchange between them. Site-directed spin labeling in combination with double electron-electron resonance (DEER) provides long-range (20-80 Å) distance distributions with angstrom-level resolution and thus is ideally suited to resolve conformational heterogeneity in an excited state populated under high pressure. DEER currently is performed at cryogenic temperatures. Therefore, a method was developed for rapidly freezing spin-labeled proteins under pressure to kinetically trap the high-pressure conformational ensemble for subsequent DEER data collection at atmospheric pressure. The methodology was evaluated using seven doubly-labeled mutants of myoglobin designed to monitor selected interhelical distances. For holomyoglobin, the distance distributions are narrow and relatively insensitive to pressure. In apomyoglobin, on the other hand, the distributions reveal a striking conformational heterogeneity involving specific helices in the pressure range of 0-3 kbar, where a molten globule state is formed. The data directly reveal the amplitude of helical fluctuations, information unique to the DEER method that complements previous rate determinations. Comparison of the distance distributions for pressure- and pH-populated molten globules shows them to be remarkably similar despite a lower helical content in the latter. PMID:24707053

  11. Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron–electron resonance

    PubMed Central

    Lerch, Michael T.; Yang, Zhongyu; Brooks, Evan K.; Hubbell, Wayne L.

    2014-01-01

    The dominance of a single native state for most proteins under ambient conditions belies the functional importance of higher-energy conformational states (excited states), which often are too sparsely populated to allow spectroscopic investigation. Application of high hydrostatic pressure increases the population of excited states for study, but structural characterization is not trivial because of the multiplicity of states in the ensemble and rapid (microsecond to millisecond) exchange between them. Site-directed spin labeling in combination with double electron–electron resonance (DEER) provides long-range (20–80 Å) distance distributions with angstrom-level resolution and thus is ideally suited to resolve conformational heterogeneity in an excited state populated under high pressure. DEER currently is performed at cryogenic temperatures. Therefore, a method was developed for rapidly freezing spin-labeled proteins under pressure to kinetically trap the high-pressure conformational ensemble for subsequent DEER data collection at atmospheric pressure. The methodology was evaluated using seven doubly-labeled mutants of myoglobin designed to monitor selected interhelical distances. For holomyoglobin, the distance distributions are narrow and relatively insensitive to pressure. In apomyoglobin, on the other hand, the distributions reveal a striking conformational heterogeneity involving specific helices in the pressure range of 0–3 kbar, where a molten globule state is formed. The data directly reveal the amplitude of helical fluctuations, information unique to the DEER method that complements previous rate determinations. Comparison of the distance distributions for pressure- and pH-populated molten globules shows them to be remarkably similar despite a lower helical content in the latter. PMID:24707053

  12. Site-directed spin labeling electron paramagnetic resonance study of the ORF1 protein from a mouse L1 retrotransposon

    PubMed Central

    Januszyk, Kurt; Fleissner, Mark R; Atchabahian, Lara; Shieh, Fa-Kuen; Altenbach, Christian; Martin, Sandra L; Guo, Feng; Hubbell, Wayne L; Clubb, Robert T

    2011-01-01

    Long interspersed nuclear element-1 is a highly abundant mammalian retrotransposon that comprises 17% of the human genome. L1 retrotransposition requires the protein encoded by open reading frame-1 (ORF1p), which binds single-stranded RNA with high affinity and functions as a nucleic acid chaperone. ORF1p has been shown to adopt a homo-trimeric, asymmetric dumbbell-shaped structure. However, its atomic-level structure and mechanism of RNA binding remains poorly understood. Here, we report the results of a site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) study of 27 residues within the RNA binding region of the full-length protein. The EPR data are compatible with the large RNA binding lobe of ORF1p containing a RNA recognition motif (RRM) domain and a carboxyl-terminal domain (CTD) that are predicted from crystallographic and NMR studies of smaller fragments of the protein. Interestingly, the EPR data indicate that residues in strands β3 and β4 of the RRM are structurally unstable, compatible with the previously observed sensitivity of this region to proteolysis. Affinity measurements and RNA-dependent EPR spectral changes map the RNA binding site on ORF1p to residues located in strands β3 and β4 of the RRM domain and to helix α1 of the CTD. Complementary in vivo studies also identify residues within the RRM domain that are required for retrotransposition. We propose that in the context of the full-length trimeric protein these distinct surfaces are positioned adjacent to one another providing a continuous surface that may interact with nucleic acids. PMID:21563223

  13. Motions of the Substrate Recognition Duplex in a Group I Intron Assessed by Site-Directed Spin Labeling

    SciTech Connect

    Grant, Gian Paola G; Boyd, Nathan; Herschlag, Daniel; Qin, Peter Z

    2009-03-11

    The Tetrahymena group I intron recognizes its oligonucleotide substrate in a two-step process. First, a substrate recognition duplex, called the P1 duplex, is formed. The P1 duplex then docks into the prefolded ribozyme core by forming tertiary contacts. P1 docking controls both the rate and the fidelity of substrate cleavage and has been extensively studied as a model for the formation of RNA tertiary structure. However, previous work has been limited to studying millisecond or slower motions. Here we investigated nanosecond P1 motions in the context of the ribozyme using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A nitroxide spin label (R5a) was covalently attached to a specific site of the substrate oligonucleotide, the labeled substrate was bound to a prefolded ribozyme to form the P1 duplex, and X-band EPR spectroscopy was used to monitor nitroxide motions in the 0.1-50 ns regime. Using substrates that favor the docked or the undocked states, it was established that R5a was capable of reporting P1 duplex motions. Using R5a-labeled substrates it was found that the J1/2 junction connecting P1 to the ribozyme core controls nanosecond P1 mobility in the undocked state. This may account for previous observations that J1/2 mutations weaken substrate binding and give rise to cryptic cleavage. This study establishes the use of SDSL to probe nanosecond dynamic behaviors of individual helices within large RNA and RNA/protein complexes. This approach may help in understanding the relationship between RNA structure, dynamics, and function.

  14. Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin.

    PubMed

    Lerch, Michael T; Horwitz, Joseph; McCoy, John; Hubbell, Wayne L

    2013-12-01

    Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875-85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate. PMID:24248390

  15. Functional consequences of site-directed mutation of conserved histidyl residues of the bacterial luciferase alpha subunit.

    PubMed

    Xin, X; Xi, L; Tu, S C

    1991-11-26

    The available sequences for the different bacterial luciferases reveal five conserved histidyl residues at positions 44, 45, 82, 224, and 285 of the alpha subunit. Ten variants of Vibrio harveyi luciferase were obtained by selective site-directed mutations of these five histidines. The essentiality of alpha His44 and alpha His45 was indicated by 4-7 orders of magnitude of bioluminescence activity reductions resulting from the substitution of either histidine by alanine (alpha H44A or alpha H45A), aspartate (alpha H44D or alpha H45D), or lysine (alpha H45K). Moreover, alpha H44A and alpha H45A were distinct from the native luciferase in thermal stabilities. Mutations at the other three positions also resulted in activity reductions ranging from a fewfold to 3 orders of magnitude. Despite these widely different bioluminescence light outputs, mutated luciferases exhibited, in nonturnover in vitro assays, light emission decay rates mostly similar to that of the native luciferase using octanal, decanal, or dodecanal as a substrate. This is attributed to a similarity in the catalytic rate constants of the light-emitting pathway for the native and mutated luciferases, but various mutated luciferases suffer in different degrees from competing dark reaction(s). In accord with this interpretation, the bioluminescence activities of mutated luciferases showed a general parallel with the relative stabilities of their 4a-hydroperoxyflavin intermediate species. Furthermore, the drastically reduced bioluminescence activities for luciferases with the alpha His44 or alpha His45 substituted by aspartate, alanine, or lysine were accompanied by little or no activities for consuming the aldehyde substrate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1958663

  16. Site-Directed Mutations and the Polymorphic Variant Ala160Thr in the Human Thromboxane Receptor Uncover a Structural Role for Transmembrane Helix 4

    PubMed Central

    Chakraborty, Raja; Pydi, Sai Prasad; Gleim, Scott; Dakshinamurti, Shyamala; Hwa, John; Chelikani, Prashen

    2012-01-01

    The human thromboxane A2 receptor (TP), belongs to the prostanoid subfamily of Class A GPCRs and mediates vasoconstriction and promotes thrombosis on binding to thromboxane (TXA2). In Class A GPCRs, transmembrane (TM) helix 4 appears to be a hot spot for non-synonymous single nucleotide polymorphic (nsSNP) variants. Interestingly, A160T is a novel nsSNP variant with unknown structure and function. Additionally, within this helix in TP, Ala1604.53 is highly conserved as is Gly1644.57. Here we target Ala1604.53 and Gly1644.57 in the TP for detailed structure-function analysis. Amino acid replacements with smaller residues, A160S and G164A mutants, were tolerated, while bulkier beta-branched replacements, A160T and A160V showed a significant decrease in receptor expression (Bmax). The nsSNP variant A160T displayed significant agonist-independent activity (constitutive activity). Guided by molecular modeling, a series of compensatory mutations were made on TM3, in order to accommodate the bulkier replacements on TM4. The A160V/F115A double mutant showed a moderate increase in expression level compared to either A160V or F115A single mutants. Thermal activity assays showed decrease in receptor stability in the order, wild type>A160S>A160V>A160T>G164A, with G164A being the least stable. Our study reveals that Ala1604.53 and Gly1644.57 in the TP play critical structural roles in packing of TM3 and TM4 helices. Naturally occurring mutations in conjunction with site-directed replacements can serve as powerful tools in assessing the importance of regional helix-helix interactions. PMID:22272267

  17. Site-directed mutations and the polymorphic variant Ala160Thr in the human thromboxane receptor uncover a structural role for transmembrane helix 4.

    PubMed

    Chakraborty, Raja; Pydi, Sai Prasad; Gleim, Scott; Dakshinamurti, Shyamala; Hwa, John; Chelikani, Prashen

    2012-01-01

    The human thromboxane A2 receptor (TP), belongs to the prostanoid subfamily of Class A GPCRs and mediates vasoconstriction and promotes thrombosis on binding to thromboxane (TXA2). In Class A GPCRs, transmembrane (TM) helix 4 appears to be a hot spot for non-synonymous single nucleotide polymorphic (nsSNP) variants. Interestingly, A160T is a novel nsSNP variant with unknown structure and function. Additionally, within this helix in TP, Ala160(4.53) is highly conserved as is Gly164(4.57). Here we target Ala160(4.53) and Gly164(4.57) in the TP for detailed structure-function analysis. Amino acid replacements with smaller residues, A160S and G164A mutants, were tolerated, while bulkier beta-branched replacements, A160T and A160V showed a significant decrease in receptor expression (Bmax). The nsSNP variant A160T displayed significant agonist-independent activity (constitutive activity). Guided by molecular modeling, a series of compensatory mutations were made on TM3, in order to accommodate the bulkier replacements on TM4. The A160V/F115A double mutant showed a moderate increase in expression level compared to either A160V or F115A single mutants. Thermal activity assays showed decrease in receptor stability in the order, wild type>A160S>A160V>A160T>G164A, with G164A being the least stable. Our study reveals that Ala160(4.53) and Gly164(4.57) in the TP play critical structural roles in packing of TM3 and TM4 helices. Naturally occurring mutations in conjunction with site-directed replacements can serve as powerful tools in assessing the importance of regional helix-helix interactions. PMID:22272267

  18. Assessing induced folding within the intrinsically disordered C-terminal domain of the Henipavirus nucleoproteins by site-directed spin labeling EPR spectroscopy.

    PubMed

    Martinho, Marlène; Habchi, Johnny; El Habre, Zeina; Nesme, Léo; Guigliarelli, Bruno; Belle, Valérie; Longhi, Sonia

    2013-01-01

    This work aims at characterizing structural transitions within the intrinsically disordered C-terminal domain of the nucleoprotein (NTAIL) from the Nipah and Hendra viruses, two recently emerged pathogens gathered within the Henipavirus genus. To this end, we used site-directed spin labeling combined with electron paramagnetic resonance spectroscopy to investigate the α-helical-induced folding that Henipavirus NTAIL domains undergo in the presence of the C-terminal X domain of the phosphoprotein (PXD). For each NTAIL protein, six positions located within four previously proposed molecular recognition elements (MoREs) were targeted for spin labeling, with three of these positions (475, 481, and 487) falling within the MoRE responsible for binding to PXD (Box3). A detailed analysis of the impact of the partner protein on the labeled NTAIL variants revealed a dramatic modification in the environment of the spin labels grafted within Box3, with the observed modifications supporting the formation of an induced α-helix within this region. In the free state, the slightly lower mobility of the spin labels grafted within Box3 as compared to the other positions suggests the existence of a transiently populated α-helix, as already reported for measles virus (MeV) NTAIL. Comparison with the well-characterized MeV NTAIL-PXD system, allowed us to validate the structural models of Henipavirus NTAIL-PXD complexes that we previously proposed. In addition, this study highlighted a few notable differences between the Nipah and Hendra viruses. In particular, the observation of composite spectra for the free form of the Nipah virus NTAIL variants spin labeled in Box3 supports conformational heterogeneity of this partly pre-configured α-helix, with the pre-existence of stable α-helical segments. Altogether these results provide insights into the molecular mechanisms of the Henipavirus NTAIL-PXD binding reaction. PMID:22881220

  19. Conformational Flexibility and Dynamics of the Internal Loop and Helical Regions of the Kink-Turn Motif in the Glycine Riboswitch by Site-Directed Spin-Labeling.

    PubMed

    Esquiaqui, Jackie M; Sherman, Eileen M; Ye, Jing-Dong; Fanucci, Gail E

    2016-08-01

    Site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy provides a means for a solution state description of site-specific dynamics and flexibility of large RNAs, facilitating our understanding of the effects of environmental conditions such as ligands and ions on RNA structure and dynamics. Here, the utility and capability of EPR line shape analysis and distance measurements to monitor and describe site-specific changes in the conformational dynamics of internal loop nucleobases as well as helix-helix interactions of the kink-turn motif in the Vibrio cholerae (VC) glycine riboswitch that occur upon sequential K(+)-, Mg(2+)-, and glycine-induced folding were explored. Spin-labels were incorporated into the 232-nucleotide sequence via splinted ligation strategies. Thiouridine nucleobase labeling within the internal loop reveals unambiguous differential dynamics for two successive sites labeled, with varied rates of motion reflective of base flipping and base stacking. EPR-based distance measurements for nitroxide spin-labels incorporated within the RNA backbone in the helical regions of the kink-turn motif are reflective of helical formation and tertiary interaction induced by ion stabilization. In both instances, results indicate that the structural formation of the kink-turn motif in the VC glycine riboswitch can be stabilized by 100 mM K(+) where the conformational flexibility of the kink-turn motif is not further tightened by subsequent addition of divalent ions. Although glycine binding is likely to induce structural and dynamic changes in other regions, SDSL indicates no impact of glycine binding on the local dynamics or structure of the kink-turn motif as investigated here. Overall, these results demonstrate the ability of SDSL to interrogate site-specific base dynamics and packing of helices in large RNAs and demonstrate ion-induced stability of the kink-turn fold of the VC riboswitch. PMID:27427937

  20. Double mutagenesis of a positive charge cluster in the ligand-binding site of the ferric enterobactin receptor, FepA.

    PubMed

    Newton, S M; Allen, J S; Cao, Z; Qi, Z; Jiang, X; Sprencel, C; Igo, J D; Foster, S B; Payne, M A; Klebba, P E

    1997-04-29

    Siderophores and colicins enter bacterial cells through TonB-dependent outer membrane proteins. Using site-directed substitution mutagenesis, we studied ligand recognition by a prototypic Escherichia coli siderophore receptor, FepA, that binds the iron chelate ferric enterobactin and colicins B and D. These genetic experiments identified a common binding site for two of the three ligands, containing multiple positive charges, within cell surface residues of FepA. Elimination of single residues in this region did not impair the adsorption or transport of ferric enterobactin, but double mutagenesis in the charge cluster identified amino acids (Arg-286 and Arg-316) that participate in siderophore binding and function in FepA-mediated killing by colicins B and D. Ferric enterobactin binding, furthermore, prevented covalent modification of FepA within this domain by either a fluorescent probe or an arginine-specific reagent, corroborating the involvement of this site in ligand recognition. These results identify, for the first time, residues in a TonB-dependent outer membrane protein that participate in ligand binding. They also explain the competition between ferric enterobactin and the colicins on the bacterial cell surface: all three ligands interact with the same arginine residues within FepA during their penetration through the outer membrane. PMID:9114029

  1. Insertional mutagenesis by transposable elements in the mammalian genome.

    PubMed

    Amariglio, N; Rechavi, G

    1993-01-01

    Several mammalian repetitive transposable genetic elements were characterized in recent years, and their role in mutagenesis is delineated in this review. Two main groups have been described: elements with symmetrical termini such as the murine IAP sequences and the human THE 1 elements and elements characterized by a poly-A rich tail at the 3' end such as the SINE and LINE sequences. The characteristic property of such mobile elements to spread and integrate in the host genome leads to insertional mutagenesis. Both germline and somatic mutations have been documented resulting from the insertion of the various types of mammalian repetitive transposable genetic elements. As foreseen by Barbara McClintock, such genetic events can cause either the activation or the inactivation of specific genes, resulting in their identification via an altered phenotype. Several disease states, such as hemophilia and cancer, are the result of this apparent aspect of genome instability. PMID:8385004

  2. European Community research on environmental mutagenesis and carcinogenesis.

    PubMed Central

    Sors, A I

    1993-01-01

    Within the 12 Member States of the European Community (EC), environmental policy is now formulated primarily at Community level. As a result, the EC has important regulatory responsibilities for the protection of workers, consumers, and the general public from risks that may arise from environmental chemicals, foremost among them potential carcinogens and mutagens. An important part of EC environmental research and development is intended to provide a scientific basis for these regulations as well as increasing understanding of the basic mechanisms involved in environmental carcinogenesis and mutagenesis. This paper contains a brief introduction to EC environment policy and research, followed by an overview of EC chemicals control activities that are of particular relevance to the research and development program. Community-level research on environmental mutagenesis and carcinogenesis is then reviewed in some detail, including the achievements of recent projects, the scientific content of the current program, and perspectives for the future. PMID:8143645

  3. Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis

    SciTech Connect

    Wang, Weina; Hellinga, Homme W.; Beese, Lorena S.

    2012-05-10

    Even though high-fidelity polymerases copy DNA with remarkable accuracy, some base-pair mismatches are incorporated at low frequency, leading to spontaneous mutagenesis. Using high-resolution X-ray crystallographic analysis of a DNA polymerase that catalyzes replication in crystals, we observe that a C {center_dot} A mismatch can mimic the shape of cognate base pairs at the site of incorporation. This shape mimicry enables the mismatch to evade the error detection mechanisms of the polymerase, which would normally either prevent mismatch incorporation or promote its nucleolytic excision. Movement of a single proton on one of the mismatched bases alters the hydrogen-bonding pattern such that a base pair forms with an overall shape that is virtually indistinguishable from a canonical, Watson-Crick base pair in double-stranded DNA. These observations provide structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis, a long-standing concept that has been difficult to demonstrate directly.

  4. Antiviral Strategies Based on Lethal Mutagenesis and Error Threshold.

    PubMed

    Perales, Celia; Domingo, Esteban

    2016-01-01

    The concept of error threshold derived from quasispecies theory is at the basis of lethal mutagenesis, a new antiviral strategy based on the increase of virus mutation rate above an extinction threshold. Research on this strategy is justified by several inhibitor-escape routes that viruses utilize to ensure their survival. Successive steps in the transition from an organized viral quasispecies into loss of biologically meaningful genomic sequences are dissected. The possible connections between theoretical models and experimental observations on lethal mutagenesis are reviewed. The possibility of using combination of virus-specific mutagenic nucleotide analogues and broad-spectrum, non-mutagenic inhibitors is evaluated. We emphasize the power that quasispecies theory has had to stimulate exploration of new means to combat pathogenic viruses. PMID:26294225

  5. Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases

    PubMed Central

    Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

    2015-01-01

    Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9), hold tremendous potential for applications in the clinical setting to treat genetic diseases or prevent infectious diseases. However, because the accuracy of DNA recognition by these nucleases is not always perfect, off-target mutagenesis may result in undesirable adverse events in treated patients such as cellular toxicity or tumorigenesis. Therefore, designing nucleases and analyzing their activity must be carefully evaluated to minimize off-target mutagenesis. Furthermore, rigorous genomic testing will be important to ensure the integrity of nuclease modified cells. In this review, we provide an overview of available nuclease designing platforms, nuclease engineering approaches to minimize off-target activity, and methods to evaluate both on- and off-target cleavage of CRISPR-Cas9. PMID:26501275

  6. Minimizing off-Target Mutagenesis Risks Caused by Programmable Nucleases.

    PubMed

    Ishida, Kentaro; Gee, Peter; Hotta, Akitsu

    2015-01-01

    Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeats associated protein-9 (CRISPR-Cas9), hold tremendous potential for applications in the clinical setting to treat genetic diseases or prevent infectious diseases. However, because the accuracy of DNA recognition by these nucleases is not always perfect, off-target mutagenesis may result in undesirable adverse events in treated patients such as cellular toxicity or tumorigenesis. Therefore, designing nucleases and analyzing their activity must be carefully evaluated to minimize off-target mutagenesis. Furthermore, rigorous genomic testing will be important to ensure the integrity of nuclease modified cells. In this review, we provide an overview of available nuclease designing platforms, nuclease engineering approaches to minimize off-target activity, and methods to evaluate both on- and off-target cleavage of CRISPR-Cas9. PMID:26501275

  7. Transcriptional mutagenesis: causes and involvement in tumor development

    PubMed Central

    Brégeon, Damien; Doetsch, Paul W.

    2013-01-01

    The majority of normal cells in a human do not multiply continuously but are quiescent and devote most of their energy to gene transcription. When DNA damages in the transcribed strand of an active gene are bypassed by an RNA polymerase, they can miscode at the damaged site and produce mutant transcripts. This process known as transcriptional mutagenesis can lead to the production of mutant proteins that could be important in tumor development. PMID:21346784

  8. Nitrosoguanidine and ultraviolet light mutagenesis in Eudorina elegans (chlorophyceae)

    SciTech Connect

    Toby, A.L.; Kemp, C.L.

    1980-06-01

    Reversion of an acetate requiring strain and the induction of sectored colonies are used to establish optimal conditions for nitrosoguanidine and ultraviolet light mutagenesis in Eudorina elegans Ehrenberg. Nitrosoguanidine is more effective in causing reversion of the acetate requiring strain and inducing auxotrophs. Morphogenetic mutants are more readily induced by ultraviolet light. The effectiveness of ultraviolet light as a mutagen is cell cycle dependent whereas the mutagenic action of nitrosoguanidine is not.

  9. A Chemical Mutagenesis Screen Identifies Mouse Models with ERG Defects.

    PubMed

    Charette, Jeremy R; Samuels, Ivy S; Yu, Minzhong; Stone, Lisa; Hicks, Wanda; Shi, Lan Ying; Krebs, Mark P; Naggert, Jürgen K; Nishina, Patsy M; Peachey, Neal S

    2016-01-01

    Mouse models provide important resources for many areas of vision research, pertaining to retinal development, retinal function and retinal disease. The Translational Vision Research Models (TVRM) program uses chemical mutagenesis to generate new mouse models for vision research. In this chapter, we report the identification of mouse models for Grm1, Grk1 and Lrit3. Each of these is characterized by a primary defect in the electroretinogram. All are available without restriction to the research community. PMID:26427409

  10. Metal mutagenesis in transgenic Chinese hamster cell lines.

    PubMed Central

    Klein, C B; Kargacin, B; Su, L; Cosentino, S; Snow, E T; Costa, M

    1994-01-01

    Metals are toxic agents for which genotoxic effects are often difficult to demonstrate. To study metal mutagenesis, we have used two stable hprt/gpt+ transgenic cell lines that were derived from Chinese hamster V79 cells. Both the G12 and G10 cell lines are known to be very sensitive to clastogens such as X-rays and bleomycin, with the mutagenic response of the integrated xanthine guanine phosphoribosyl transferase (gpt) gene in G10 usually exceeding that of the same gene in the transgenic G12 cells. In studies with carcinogenic insoluble nickel compounds, a high level of mutagenesis was found at the gpt locus of G12 cells but not at the endogenous hypoxanthine phosphoribosyl transferase (hprt) locus of V79 cells. We have since demonstrated the similar recovery of a high frequency of viable G12 mutants with other insoluble nickel salts including nickel oxides (black and green). The relative mutant yield for the insoluble nickel compounds (G12 > G10) is the opposite of that obtained with nonmetal clastogens (G10 > G12). In the G12 cells, nickel mutagenesis may be related to the integration of the gpt sequence into a heterochromatic region of the genome. For some of the insoluble nickel compounds, significant inhibition of both cytotoxicity and mutant yield resulted when the G12 cells were pretreated with vitamin E. In comparison with the nickel studies, the mutagenic responses to chromium compounds in these cell lines were not as dramatic. Mutagenesis of the gpt target could not be demonstrated with other metals such as mercury or vanadium. PMID:7843139

  11. Similarity between class A and class B G-protein-coupled receptors exemplified through calcitonin gene-related peptide receptor modelling and mutagenesis studies

    PubMed Central

    Vohra, Shabana; Taddese, Bruck; Conner, Alex C.; Poyner, David R.; Hay, Debbie L.; Barwell, James; Reeves, Philip J.; Upton, Graham J. G.; Reynolds, Christopher A.

    2013-01-01

    Modelling class B G-protein-coupled receptors (GPCRs) using class A GPCR structural templates is difficult due to lack of homology. The plant GPCR, GCR1, has homology to both class A and class B GPCRs. We have used this to generate a class A–class B alignment, and by incorporating maximum lagged correlation of entropy and hydrophobicity into a consensus score, we have been able to align receptor transmembrane regions. We have applied this analysis to generate active and inactive homology models of the class B calcitonin gene-related peptide (CGRP) receptor, and have supported it with site-directed mutagenesis data using 122 CGRP receptor residues and 144 published mutagenesis results on other class B GPCRs. The variation of sequence variability with structure, the analysis of polarity violations, the alignment of group-conserved residues and the mutagenesis results at 27 key positions were particularly informative in distinguishing between the proposed and plausible alternative alignments. Furthermore, we have been able to associate the key molecular features of the class B GPCR signalling machinery with their class A counterparts for the first time. These include the [K/R]KLH motif in intracellular loop 1, [I/L]xxxL and KxxK at the intracellular end of TM5 and TM6, the NPXXY/VAVLY motif on TM7 and small group-conserved residues in TM1, TM2, TM3 and TM7. The equivalent of the class A DRY motif is proposed to involve Arg2.39, His2.43 and Glu3.46, which makes a polar lock with T6.37. These alignments and models provide useful tools for understanding class B GPCR function. PMID:23235263

  12. Highly Efficient Targeted Mutagenesis in Mice Using TALENs

    PubMed Central

    Panda, Sudeepta Kumar; Wefers, Benedikt; Ortiz, Oskar; Floss, Thomas; Schmid, Bettina; Haass, Christian; Wurst, Wolfgang; Kühn, Ralf

    2013-01-01

    Targeted mouse mutants are instrumental for the analysis of gene function in health and disease. We recently provided proof-of-principle for the fast-track mutagenesis of the mouse genome, using transcription activator-like effector nucleases (TALENs) in one-cell embryos. Here we report a routine procedure for the efficient production of disease-related knockin and knockout mutants, using improved TALEN mRNAs that include a plasmid-coded poly(A) tail (TALEN-95A), circumventing the problematic in vitro polyadenylation step. To knock out the C9orf72 gene as a model of frontotemporal lobar degeneration, TALEN-95A mutagenesis induced sequence deletions in 41% of pups derived from microinjected embryos. Using TALENs together with mutagenic oligodeoxynucleotides, we introduced amyotrophic lateral sclerosis patient-derived missense mutations in the fused in sarcoma (Fus) gene at a rate of 6.8%. For the simple identification of TALEN-induced mutants and their progeny we validate high-resolution melt analysis (HRMA) of PCR products as a sensitive and universal genotyping tool. Furthermore, HRMA of off-target sites in mutant founder mice revealed no evidence for undesired TALEN-mediated processing of related genomic sequences. The combination of TALEN-95A mRNAs for enhanced mutagenesis and of HRMA for simplified genotyping enables the accelerated, routine production of new mouse models for the study of genetic disease mechanisms. PMID:23979585

  13. PAX5 is a tumor suppressor in mouse mutagenesis models of acute lymphoblastic leukemia

    PubMed Central

    Dang, Jinjun; Wei, Lei; de Ridder, Jeroen; Su, Xiaoping; Rust, Alistair G.; Roberts, Kathryn G.; Payne-Turner, Debbie; Cheng, Jinjun; Ma, Jing; Qu, Chunxu; Wu, Gang; Song, Guangchun; Huether, Robert G.; Schulman, Brenda; Janke, Laura; Zhang, Jinghui; Downing, James R.; van der Weyden, Louise; Adams, David J.

    2015-01-01

    Alterations of genes encoding transcriptional regulators of lymphoid development are a hallmark of B-progenitor acute lymphoblastic leukemia (B-ALL) and most commonly involve PAX5, encoding the DNA-binding transcription factor paired-box 5. The majority of PAX5 alterations in ALL are heterozygous, and key PAX5 target genes are expressed in leukemic cells, suggesting that PAX5 may be a haploinsufficient tumor suppressor. To examine the role of PAX5 alterations in leukemogenesis, we performed mutagenesis screens of mice heterozygous for a loss-of-function Pax5 allele. Both chemical and retroviral mutagenesis resulted in a significantly increased penetrance and reduced latency of leukemia, with a shift to B-lymphoid lineage. Genomic profiling identified a high frequency of secondary genomic mutations, deletions, and retroviral insertions targeting B-lymphoid development, including Pax5, and additional genes and pathways mutated in ALL, including tumor suppressors, Ras, and Janus kinase-signal transducer and activator of transcription signaling. These results show that in contrast to simple Pax5 haploinsufficiency, multiple sequential alterations targeting lymphoid development are central to leukemogenesis and contribute to the arrest in lymphoid maturation characteristic of ALL. This cross-species analysis also validates the importance of concomitant alterations of multiple cellular growth, signaling, and tumor suppression pathways in the pathogenesis of B-ALL. PMID:25855603

  14. An inducible tool for random mutagenesis in Aspergillus niger based on the transposon Vader.

    PubMed

    Paun, Linda; Nitsche, Benjamin; Homan, Tim; Ram, Arthur F; Kempken, Frank

    2016-07-01

    The ascomycete Aspergillus niger is widely used in the biotechnology, for instance in producing most of the world's citric acid. It is also known as a major food and feed contaminant. While generation of gene knockouts for functional genomics has become feasible in ku70 mutants, analyzing gene functions or metabolic pathways remains a laborious task. An unbiased transposon-based mutagenesis approach may aid this process of analyzing gene functions by providing mutant libraries in a short time. The Vader transposon is a non-autonomous DNA-transposon, which is activated by the homologous tan1-transposase. However, in the most commonly used lab strain of A. niger (N400 strain and derivatives), we found that the transposase, encoded by the tan1 gene, is mutated and inactive. To establish a Vader transposon-based mutagenesis system in the N400 background, we expressed the functional transposase of A. niger strain CBS 513.88 under the control of an inducible promoter based on the Tet-on system, which is activated in the presence of the antibiotic doxycycline (DOX). Increasing amounts of doxycycline lead to higher Vader excision frequencies, whereas little to none activity of Vader was observed without addition of doxycycline. Hence, this system appears to be suitable for producing stable mutants in the A. niger N400 background. PMID:27003267

  15. Involvement of a joker mutation in a polymerase-independent lethal mutagenesis escape mechanism.

    PubMed

    Agudo, Rubén; de la Higuera, Ignacio; Arias, Armando; Grande-Pérez, Ana; Domingo, Esteban

    2016-07-01

    We previously characterized a foot-and-mouth disease virus (FMDV) with three amino acid replacements in its polymerase (3D) that conferred resistance to the mutagenic nucleoside analogue ribavirin. Here we show that passage of this mutant in the presence of high ribavirin concentrations resulted in selection of viruses with the additional replacement I248T in 2C. This 2C substitution alone (even in the absence of replacements in 3D) increased FMDV fitness mainly in the presence of ribavirin, prevented an incorporation bias in favor of A and U associated with ribavirin mutagenesis, and conferred the ATPase activity of 2C decreased sensitivity to ribavirin-triphosphate. Since in previous studies we described that 2C with I248T was selected under different selective pressures, this replacement qualifies as a joker substitution in FMDV evolution. The results have identified a role of 2C in nucleotide incorporation, and have unveiled a new polymerase-independent mechanism of virus escape to lethal mutagenesis. PMID:27136067

  16. A mutagenesis-derived broad-spectrum disease resistance locus in wheat.

    PubMed

    Campbell, Jackie; Zhang, Hongtao; Giroux, Michael J; Feiz, Leila; Jin, Yue; Wang, Meinan; Chen, Xianming; Huang, Li

    2012-07-01

    Wheat leaf rust, stem rust, stripe rust, and powdery mildew caused by the fungal pathogens Puccinia triticina, P. graminis f. sp. tritici, P. striiformis f. sp. tritici, and Blumeria graminis f. sp. tritici, respectively, are destructive diseases of wheat worldwide. Breeding durable disease resistance cultivars rely largely on continually introgressing new resistance genes, especially the genes with different defense mechanisms, into adapted varieties. Here, we describe a new resistance gene obtained by mutagenesis. The mutant, MNR220 (mutagenesis-derived new resistance), enhances resistance to three rusts and powdery mildew, with the characteristics of delayed disease development at the seedling stage and completed resistance at the adult plant stage. Genetic analysis demonstrated that the resistance in MNR220 is conferred by a single semidominant gene mapped on the short arm of chromosome 2B. Gene expression profiling of several pathogenesis-related genes indicated that MNR220 has an elevated and rapid pathogen-induced response. In addition to its potential use in breeding for resistance to multiple diseases, high-resolution mapping and cloning of the disease resistance locus in MNR220 may lead to a better understanding of the regulation of defense responses in wheat. PMID:22446929

  17. Implementation of a large-scale ENU mutagenesis program: towards increasing the mouse mutant resource.

    PubMed

    Nolan, P M; Peters, J; Vizor, L; Strivens, M; Washbourne, R; Hough, T; Wells, C; Glenister, P; Thornton, C; Martin, J; Fisher, E; Rogers, D; Hagan, J; Reavill, C; Gray, I; Wood, J; Spurr, N; Browne, M; Rastan, S; Hunter, J; Brown, S D

    2000-07-01

    Systematic approaches to mouse mutagenesis will be vital for future studies of gene function. We have begun a major ENU mutagenesis program incorporating a large genome-wide screen for dominant mutations. Progeny of ENU-mutagenized mice are screened for visible defects at birth and weaning, and at 5 weeks of age by using a systematic and semi-quantitative screening protocol-SHIRPA. Following this, mice are screened for abnormal locomotor activity and for deficits in prepulse inhibition of the acoustic startle response. Moreover, in the primary screen, blood is collected from mice and subjected to a comprehensive clinical biochemical analysis. Subsequently, secondary and tertiary screens of increasing complexity can be used on animals demonstrating deficits in the primary screen. Frozen sperm is archived from all the male mice passing through the screen. In addition, tail tips are stored for DNA. Overall, the program will provide an extensive new resource of mutant and phenotype data to the mouse and human genetics communities at large. The challenge now is to employ the expanding mouse mutant resource to improve the mutant map of the mouse. An improved mutant map of the mouse will be an important asset in exploiting the growing gene map of the mouse and assisting with the identification of genes underlying novel mutations-with consequent benefits for the analysis of gene function and the identification of novel pathways. PMID:10886012

  18. Improving isopropanol tolerance and production of Clostridium beijerinckii DSM 6423 by random mutagenesis and genome shuffling.

    PubMed

    Gérando, H Máté de; Fayolle-Guichard, F; Rudant, L; Millah, S K; Monot, F; Ferreira, Nicolas Lopes; López-Contreras, A M

    2016-06-01

    Random mutagenesis and genome shuffling was applied to improve solvent tolerance and isopropanol/butanol/ethanol (IBE) production in the strictly anaerobic bacteria Clostridium beijerinckii DSM 6423. Following chemical mutagenesis with N-methyl-N-nitro-N-nitrosoguanidine (NTG), screening of putatively improved strains was done by submitting the mutants to toxic levels of inhibitory chemicals or by screening for their tolerance to isopropanol (>35 g/L). Suicide substrates, such as ethyl or methyl bromobutyrate or alcohol dehydrogenase inhibitors like allyl alcohol, were tested and, finally, 36 mutants were isolated. The fermentation profiles of these NTG mutant strains were characterized, and the best performing mutants were used for consecutive rounds of genome shuffling. Screening of strains with further enhancement in isopropanol tolerance at each recursive shuffling step was then used to spot additionally improved strains. Three highly tolerant strains were finally isolated and able to withstand up to 50 g/L isopropanol on plates. Even if increased tolerance to the desired end product was not always accompanied by higher production capabilities, some shuffled strains showed increased solvent titers compared to the parental strains and the original C. beijerinckii DSM 6423. This study confirms the efficiency of genome shuffling to generate improved strains toward a desired phenotype such as alcohol tolerance. This tool also offers the possibility of obtaining improved strains of Clostridium species for which targeted genetic engineering approaches have not been described yet. PMID:26852409

  19. Studies on the role of actin's N tau-methylhistidine using oligodeoxynucleotide-directed site-specific mutagenesis.

    PubMed

    Solomon, L R; Rubenstein, P A

    1987-08-15

    The primary structure of all actins except that isolated from Naegleria gruberi contains a unique N tau-methylhistidine (MeHis) at position 73. This modified residue has been implicated as possibly being important for the post-translational processing of actin's amino terminus, the binding of actin to DNase I, and in the polymerization of G-actin. We have investigated the potential role of MeHis in each of these processes by utilizing site-directed mutagenesis to change His-73 of skeletal muscle actin to Arg and Tyr. Wild type and mutant actins were synthesized in vivo, using non-muscle cells transfected with mutant cDNAs, and in vitro by translating mutant RNAs synthesized using SP6 RNA polymerase in a rabbit reticulocyte lysate. We have found that actins containing Arg or Tyr at position 73 undergo amino-terminal processing, bind to DNase I-agarose, and become incorporated into the cytoskeleton of a nonmuscle cell as efficiently as wild type actin. Furthermore, using an in vitro copolymerization assay we have found that although there is no difference between the Arg mutant and the wild type actins, the Tyr mutant has a slightly greater critical concentration for polymerization. These results show that MeHis is not absolutely required for any of these processes. PMID:3301854

  20. Changes in the catalytic properties of Pyrococcus furiosus thermostable amylase by mutagenesis of the substrate binding sites.

    PubMed

    Yang, Sung-Jae; Min, Byoung-Chul; Kim, Young-Wan; Jang, Sang-Mok; Lee, Byong-Hoon; Park, Kwan-Hwa

    2007-09-01

    Pyrococcus furiosus thermostable amylase (TA) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of four residues (His414, Gly415, Met439, and Asp440) in the function of P. furiosus TA by using site-directed mutagenesis and kinetic analysis. A variant form of P. furiosus TA containing two mutations (H414N and G415E) exhibited strongly enhanced alpha-(1,4)-transglycosylation activity, resulting in the production of a series of maltooligosaccharides that were longer than the initial substrates. In contrast, the variant enzymes with single mutations (H414N or G415E) showed a substrate preference similar to that of the wild-type enzyme. Other mutations (M439W and D440H) reversed the substrate preference of P. furiosus TA from CDs to maltooligosaccharides. Relative substrate preferences for maltoheptaose over beta-CD, calculated by comparing k(cat)/K(m) ratios, of 1, 8, and 26 for wild-type P. furiosus TA, P. furiosus TA with D440H, and P. furiosus TA with M439W and D440H, respectively, were found. Our results suggest that His414, Gly415, Met439, and Asp440 play important roles in substrate recognition and transglycosylation. Therefore, this study provides information useful in engineering glycoside hydrolase family 13 enzymes. PMID:17630303

  1. CRISPR/Cas9 mediated multiplex genome editing and heritable mutagenesis of BmKu70 in Bombyx mori

    PubMed Central

    Ma, Sanyuan; Chang, Jiasong; Wang, Xiaogang; Liu, Yuanyuan; Zhang, Jianduo; Lu, Wei; Gao, Jie; Shi, Run; Zhao, Ping; Xia, Qingyou

    2014-01-01

    CRISPR/Cas9, a bacterial adaptive immune system derived genome-editing technique, has become to be one of the most compelling topics in biotechnology. Bombyx mori is an economically important insect and a model organism for studying lepidopteran and arthropod biology. Here we reported highly efficient and multiplex genome editing in B. mori cell line and heritable site-directed mutagenesis of Bmku70, which is required for NHEJ pathway and also related to antigen diversity, telomere length maintenance and subtelomeric gene silencing, using CRISPR/Cas9 system. We established a simple and practicable method and obtained several Bmku70 knockout B. mori lines, and showed that the frequency of HR was increased in embryos of the Bmku70 knockout B. mori. The mutant lines obtained in this study could be a candidate genetic resource for efficient knock-in and fundamental research of DNA repair in B. mori. We also provided a strategy and procedure to perform heritable genome editing of target genes with no significant phenotype effect. PMID:24671069

  2. Mutagenesis of the La Crosse Virus glycoprotein supports a role for Gc (1066-1087) as the fusion peptide

    SciTech Connect

    Plassmeyer, Matthew L.; Soldan, Samantha S.; Stachelek, Karen M.; Roth, Susan M.; Martin-Garcia, Julio; Gonzalez-Scarano, Francisco . E-mail: scarano@mail.med.upenn.edu

    2007-02-20

    The La Crosse Virus (LACV) M segment encodes two glycoproteins (Gn and Gc), and plays a critical role in the neuropathogenesis of LACV infection as the primary determinant of neuroinvasion. A recent study from our group demonstrated that the region comprising the membrane proximal two-thirds of Gc, amino acids 860-1442, is critical in mediating LACV fusion and entry. Furthermore, computational analysis identified structural similarities between a portion of this region, amino acids 970-1350, and the E1 fusion protein of two alphaviruses: Sindbis virus and Semliki Forrest virus (SFV). Within the region 970-1350, a 22-amino-acid hydrophobic segment (1066-1087) is predicted to correlate structurally with the fusion peptides of class II fusion proteins. We performed site-directed mutagenesis of key amino acids in this 22-amino acid segment and determined the functional consequences of these mutations on fusion and entry. Several mutations within this hydrophobic domain affected glycoprotein expression to some extent, but all mutations either shifted the pH threshold of fusion below that of the wild-type protein, reduced fusion efficiency, or abrogated cell-to-cell fusion and pseudotype entry altogether. These results, coupled with the aforementioned computational modeling, suggest that the LACV Gc functions as a class II fusion protein and support a role for the region Gc 1066-1087 as a fusion peptide.

  3. Elucidation of Lipid Binding Sites on Lung Surfactant Protein A Using X-ray Crystallography, Mutagenesis, and Molecular Dynamics Simulations.

    PubMed

    Goh, Boon Chong; Wu, Huixing; Rynkiewicz, Michael J; Schulten, Klaus; Seaton, Barbara A; McCormack, Francis X

    2016-07-01

    Surfactant protein A (SP-A) is a collagenous C-type lectin (collectin) that is critical for pulmonary defense against inhaled microorganisms. Bifunctional avidity of SP-A for pathogen-associated molecular patterns (PAMPs) such as lipid A and for dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant membranes lining the air-liquid interface of the lung, ensures that the protein is poised for first-line interactions with inhaled pathogens. To improve our understanding of the motifs that are required for interactions with microbes and surfactant structures, we explored the role of the tyrosine-rich binding surface on the carbohydrate recognition domain of SP-A in the interaction with DPPC and lipid A using crystallography, site-directed mutagenesis, and molecular dynamics simulations. Critical binding features for DPPC binding include a three-walled tyrosine cage that binds the choline headgroup through cation-π interactions and a positively charged cluster that binds the phosphoryl group. This basic cluster is also critical for binding of lipid A, a bacterial PAMP and target for SP-A. Molecular dynamics simulations further predict that SP-A binds lipid A more tightly than DPPC. These results suggest that the differential binding properties of SP-A favor transfer of the protein from surfactant DPPC to pathogen membranes containing appropriate lipid PAMPs to effect key host defense functions. PMID:27324153

  4. Identification of catalytically relevant amino acids of the extracellular Serratia marcescens endonuclease by alignment-guided mutagenesis.

    PubMed Central

    Friedhoff, P; Gimadutdinow, O; Pingoud, A

    1994-01-01

    By sequence alignment of the extracellular Serratia marcescens nuclease with three related nucleases we have identified seven charged amino acid residues which are conserved in all four sequences. Six of these residues together with four other partially conserved His or Asp residues were changed to alanine by site-directed PCR-mediated mutagenesis using a variant of the nuclease gene in which the coding sequence of the signal peptide was replaced by the coding sequence for an N-terminal affinity tag [Met(His)6GlySer]. Four of the mutant proteins showed almost no reduction in nuclease activity but five displayed a 10- to 1000-fold reduction in activity and one (His110Ala) was inactive. Based upon these results it is suggested that the S.marcescens nuclease employs a mechanism in which His110 acts in concert with a Mg2+ ion and three carboxylates (Asp107, Glu148 and Glu232) as well as one or two basic amino acid residues (Arg108, Arg152). Images PMID:8078761

  5. Side chain mobility and ligand interactions of the G strand of tear lipocalins by site-directed spin labeling.

    PubMed

    Glasgow, B J; Gasymov, O K; Abduragimov, A R; Yusifov, T N; Altenbach, C; Hubbell, W L

    1999-10-12

    Side chain mobility, accessibility, and backbone motion were studied by site-directed spin labeling of sequential cysteine mutants of the G strand in tear lipocalins (TL). A nitroxide scan between residues 98 and 105 revealed the alternating periodicity of mobility and accessibility to NiEDDA and oxygen, characteristic of a beta-strand. Residue 99 was the most inaccessible to NiEDDA and oxygen. EPR spectra with the fast relaxing agent, K(3)Fe(CN)(6), exhibited two nitroxide populations for most residues. The motionally constrained population was relatively less accessible to K(3)Fe(CN)(6) because of dynamic tertiary contact, probably with side chain residues of adjacent strands. With increasing concentrations of sucrose, the spectral contribution of the immobile component was greater, indicating a larger population with tertiary contact. Increased concentrations of sucrose also resulted in a restriction of mobility of spin-labeled fatty acids which were bound within the TL cavity. The data suggest that sucrose enhanced ligand affinity by slowing the backbone motion of the lipocalin. The correlation time of an MTSL derivative (I) attached to F99C resulted in the lack of side chain motion and therefore reflects the overall rotation of the TL complex. The correlation time of F99C in tears (13.5 ns) was the same as that in buffer and indicates that TL exists as a dimer under native conditions. TL-spin-labeled ligand complexes have a shorter correlation time than the protein alone, indicating that the fatty acids are not rigidly anchored in the cavity, but move within the pocket. This segmental motion of the ligand was modulated by protein backbone fluctuations. Accessibility studies with oxygen and NiEDDA were performed to determine the orientation and depth of a series of fatty acid derivatives in the cavity of TL. Fatty acids are oriented with the hydrocarbon tail buried in the cavity and the carboxyl group oriented toward the mouth. In general, the mobility of the

  6. The manganese binding site of manganese peroxidase: Characterization of an Asp179Asn site-directed mutant protein

    SciTech Connect

    Kusters-van Someren, M.; Kishi, K.; Lundell, T.

    1995-08-22

    A site-directed mutant, D179N, in the gene encoding Phanerochaete chrysosporium manganese peroxidase isozyme 1 (mnp1), was created by overlap extension, using polymerase chain reaction. The mutant gene was expressed in P. chrysosporium under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The mutant manganese peroxidase (MnP) was purified, and its spectra and MW were very similar to those of the wild-type enzyme. Steady-state kinetic analysis of MnP D179N revealed that the K{sub m} for the substrate Mn{sup II} was {approximately}50-fold greater than the corresponding K{sub m} for the wild-type recombinant enzyme (3.7 mM versus {approximately}70 {mu}M). Likewise, the k{sub cat} value for Mn{sup II} oxidation of the mutant protein was only 1/265 of that for the wild-type enzyme. By comparison, the apparent K{sub m} for H{sub 2}O{sub 2} of MnP D179N was similar to the corresponding value of the wild-type MnP. The first-order rate constant for MnP D179N compound II reduction by Mn{sup II} was approximately 1/200 of that for the wild-type enzyme. The equilibrium dissociation constant (K{sub D}) for MnP D179N compound II reduction by Mn{sup II} was {approximately}100-fold greater than the K{sub D} for the wild-type compound II. In contrast, the second-order rate constant for p-cresol reduction of the mutant compound II was similar to that of the wild-type enzyme. These results also suggest that the mutation affects the binding of Mn{sup II} to the enzyme and, consequently, the rate of compound II reduction by Mn{sup II}. In contrast, the mutation apparently does not have a significant effect on H{sub 2}O{sub 2} cleavage during compound I formation or on p-cresol reduction of compound II. The results strongly suggest that Asp179 is one of the acidic amino acid ligands in the Mn{sup II} binding site of MnP. 53 refs., 7 figs., 3 tabs.

  7. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling

    PubMed Central

    Evans, Eric G.B.; Millhauser, Glenn L.

    2016-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain “K1,” and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron–electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite

  8. Sleeping Beauty Transposon Mutagenesis as a Tool for Gene Discovery in the NOD Mouse Model of Type 1 Diabetes

    PubMed Central

    Elso, Colleen M.; Chu, Edward P. F.; Alsayb, May A.; Mackin, Leanne; Ivory, Sean T.; Ashton, Michelle P.; Bröer, Stefan; Silveira, Pablo A.; Brodnicki, Thomas C.

    2015-01-01

    A number of different strategies have been used to identify genes for which genetic variation contributes to type 1 diabetes (T1D) pathogenesis. Genetic studies in humans have identified >40 loci that affect the risk for developing T1D, but the underlying causative alleles are often difficult to pinpoint or have subtle biological effects. A complementary strategy to identifying “natural” alleles in the human population is to engineer “artificial” alleles within inbred mouse strains and determine their effect on T1D incidence. We describe the use of the Sleeping Beauty (SB) transposon mutagenesis system in the nonobese diabetic (NOD) mouse strain, which harbors a genetic background predisposed to developing T1D. Mutagenesis in this system is random, but a green fluorescent protein (GFP)-polyA gene trap within the SB transposon enables early detection of mice harboring transposon-disrupted genes. The SB transposon also acts as a molecular tag to, without additional breeding, efficiently identify mutated genes and prioritize mutant mice for further characterization. We show here that the SB transposon is functional in NOD mice and can produce a null allele in a novel candidate gene that increases diabetes incidence. We propose that SB transposon mutagenesis could be used as a complementary strategy to traditional methods to help identify genes that, when disrupted, affect T1D pathogenesis. PMID:26438296

  9. Sleeping Beauty Transposon Mutagenesis as a Tool for Gene Discovery in the NOD Mouse Model of Type 1 Diabetes.

    PubMed

    Elso, Colleen M; Chu, Edward P F; Alsayb, May A; Mackin, Leanne; Ivory, Sean T; Ashton, Michelle P; Bröer, Stefan; Silveira, Pablo A; Brodnicki, Thomas C

    2015-12-01

    A number of different strategies have been used to identify genes for which genetic variation contributes to type 1 diabetes (T1D) pathogenesis. Genetic studies in humans have identified >40 loci that affect the risk for developing T1D, but the underlying causative alleles are often difficult to pinpoint or have subtle biological effects. A complementary strategy to identifying "natural" alleles in the human population is to engineer "artificial" alleles within inbred mouse strains and determine their effect on T1D incidence. We describe the use of the Sleeping Beauty (SB) transposon mutagenesis system in the nonobese diabetic (NOD) mouse strain, which harbors a genetic background predisposed to developing T1D. Mutagenesis in this system is random, but a green fluorescent protein (GFP)-polyA gene trap within the SB transposon enables early detection of mice harboring transposon-disrupted genes. The SB transposon also acts as a molecular tag to, without additional breeding, efficiently identify mutated genes and prioritize mutant mice for further characterization. We show here that the SB transposon is functional in NOD mice and can produce a null allele in a novel candidate gene that increases diabetes incidence. We propose that SB transposon mutagenesis could be used as a complementary strategy to traditional methods to help identify genes that, when disrupted, affect T1D pathogenesis. PMID:26438296

  10. Pollen tetrads in the detection of environmental mutagenesis

    SciTech Connect

    Mulcahy, D.L.

    1981-01-01

    Although pollen is a very sensitive indicator of environmental mutagenesis, it is also sensitive to nonmutagenic environmental stress. By analyzing pollen tetrads, rather than individual pollen grains, it is possible to distinguish between mutagenic and nonmutagenic influences. Another advantage of using pollen tetrads in mutagenicity studies is that it is possible to discriminate between pre- and post-pachytene mutations. This eliminates the mutant sector problem of a single mutational event giving rise to a large number of mutant cells. Methods of analyzing pollen tetrads are described.

  11. Chemical mutagenesis: an emerging issue for public health.

    PubMed Central

    Claxton, L D; Barry, P Z

    1977-01-01

    Chemical mutagens are recognized as prevalent in the environment and a potential threat to the health of future generations. This paper presents an overview of chemical mutagenesis as an issue for public health. Several problems in the determination of risk to human populations are discussed, including difficulties of extrapolating scientific data to humans, the latency period between exposure and recognizable genetic damage, and the large number of chemicals which must be tested. Test systems are described. Possibilities of control through federal regulation are discussed. PMID:911015

  12. KP-1212/1461, a nucleoside designed for the treatment of HIV by viral mutagenesis.

    PubMed

    Harris, Kevin S; Brabant, William; Styrchak, Sheila; Gall, Alexander; Daifuku, Richard

    2005-07-01

    We report the activities of a novel nucleoside analog against HIV. This nucleoside (KP-1212) is not a chain terminator but exerts its antiviral effects via mutagenesis of the viral genome. Serial passaging of HIV in the presence of KP-1212 causes an increase in the mutation rate of the virus leading to viral ablation. HIV strains resistant to KP-1212 have not yet been isolated. Quite to the contrary, virus treated with KP-1212 exhibited an increased sensitivity not only to KP-1212 but also to another nucleoside reverse transcriptase inhibitor (NRTI), zidovudine. HIV strains resistant to other NRTIs (e.g. zidovudine, lamivudine, stavudine, abacavir, etc.) exhibited no cross-resistance towards KP-1212. Multiple assays confirmed that KP-1212 has a favorable (low) genotoxicity profile when compared to some approved antiviral nucleosides. In addition, KP-1212 is not toxic to mitochondria nor does it exhibit any inhibitory effects on mitochondrial DNA synthesis. PMID:15890415

  13. Development of an inducible transposon system for efficient random mutagenesis in Clostridium acetobutylicum

    PubMed Central

    Zhang, Ying; Xu, Shu; Chai, Changsheng; Yang, Sheng; Jiang, Weihong; Minton, Nigel P.; Gu, Yang

    2016-01-01

    Clostridium acetobutylicum is an industrially important Gram-positive organism, which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner-based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP-based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A, thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum. PMID:27001972

  14. Development of an inducible transposon system for efficient random mutagenesis in Clostridium acetobutylicum.

    PubMed

    Zhang, Ying; Xu, Shu; Chai, Changsheng; Yang, Sheng; Jiang, Weihong; Minton, Nigel P; Gu, Yang

    2016-04-01

    Clostridium acetobutylicum is an industrially important Gram-positive organism, which is capable of producing economically important chemicals in the ABE (Acetone, Butanol and Ethanol) fermentation process. Renewed interests in the ABE process necessitate the availability of additional genetics tools to facilitate the derivation of a greater understanding of the underlying metabolic and regulatory control processes in operation through forward genetic strategies. In this study, a xylose inducible, mariner-based, transposon system was developed and shown to allow high-efficient random mutagenesis in the model strain ATCC 824. Of the thiamphenicol resistant colonies obtained, 91.9% were shown to be due to successful transposition of the catP-based mini-transposon element. Phenotypic screening of 200 transposon clones revealed a sporulation-defective clone with an insertion in spo0A, thereby demonstrating that this inducible transposon system can be used for forward genetic studies in C. acetobutylicum. PMID:27001972

  15. Structure-Based Mutagenesis Study of Hepatitis C Virus NS3 Helicase

    PubMed Central

    Lin, Chao; Kim, Joseph L.

    1999-01-01

    The NS3 protein of hepatitis C virus (HCV) is a bifunctional protein containing a serine protease in the N-terminal one-third, which is stimulated upon binding of the NS4A cofactor, and an RNA helicase in the C-terminal two-thirds. In this study, a C-terminal hexahistidine-tagged helicase domain of the HCV NS3 protein was expressed in Escherichia coli and purified to homogeneity by conventional chromatography. The purified HCV helicase domain has a basal ATPase activity, a polynucleotide-stimulated ATPase activity, and a nucleic acid unwinding activity and binds efficiently to single-stranded polynucleotide. Detailed characterization of the purified HCV helicase domain with regard to all four activities is presented. Recently, we published an X-ray crystallographic structure of a binary complex of the HCV helicase with a (dU)8 oligonucleotide, in which several conserved residues of the HCV helicase were shown to be involved in interactions between the HCV helicase and oligonucleotide. Here, site-directed mutagenesis was used to elucidate the roles of these residues in helicase function. Four individual mutations, Thr to Ala at position 269, Thr to Ala at position 411, Trp to Leu at position 501, and Trp to Ala at position 501, produced a severe reduction of RNA binding and completely abolished unwinding activity and stimulation of ATPase activity by poly(U), although the basal ATPase activity (activity in the absence of polynucleotide) of these mutants remained intact. Alanine substitution at Ser-231 or Ser-370 resulted in enzymes that were indistinguishable from wild-type HCV helicase with regard to all four activities. A mutant bearing Phe at Trp-501 showed wild-type levels of basal ATPase, unwinding activity, and single-stranded RNA binding activity. Interestingly, ATPase activity of this mutant became less responsive to stimulation by poly(U) but not to stimulation by other polynucleotides, such as poly(C). Given the conservation of some of these residues in

  16. Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis

    PubMed Central

    Ai, Yixin; Wu, Jing

    2016-01-01

    The sucrose isomerase of Serratia plymuthica AS9 (AS9 PalI) was expressed in Escherichia coli BL21(DE3) and characterized. The half-life of AS9 PalI was 20 min at 45°C, indicating that it was unstable. In order to improve its thermostability, six amino acid residues with higher B-factors were selected as targets for site-directed mutagenesis, and six mutants (E175N, K576D, K174D, G176D, S575D and N577K) were designed using the RosettaDesign server. The E175N and K576D mutants exhibited improved thermostability in preliminary experiments, so the double mutant E175N/K576D was constructed. These three mutants (E175N, K576D, E175N/K576D) were characterized in detail. The results indicate that the three mutants exhibit a slightly increased optimal temperature (35°C), compared with that of the wild-type enzyme (30°C). The mutants also share an identical pH optimum of 6.0, which is similar to that of the wild-type enzyme. The half-lives of the E175N, K576D and E175N/K576D mutants were 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme at 45°C, respectively. Kinetic studies showed that the Km values for the E175N, K576D and E175N/K576D mutants decreased by 6.6%, 2.0% and 11.0%, respectively, and their kcat/Km values increased by 38.2%, 4.2% and 19.4%, respectively, compared with those of the wild-type enzyme. After optimizing the conditions for isomaltulose production at 45°C, we found that the E175N, K576D and E175N/K576D mutants displayed slightly improved isomaltulose yields, compared with the wild-type enzyme. Therefore, the mutants produced in this study would be more suitable for industrial biosynthesis of isomaltulose. PMID:26886729

  17. Catalytic mechanism of a family 3 beta-glucosidase and mutagenesis study on residue Asp-247.

    PubMed Central

    Li, Y K; Chir, J; Chen, F Y

    2001-01-01

    A family 3 beta-glucosidase (EC 3.2.1.21) from Flavobacterium meningosepticum has been cloned and overexpressed. The mechanistic action of the enzyme was probed by NMR spectroscopy and kinetic investigations, including substrate reactivity, secondary kinetic isotope effects and inhibition studies. The stereochemistry of enzymic hydrolysis was identified as occurring with the retention of an anomeric configuration, indicating a double-displacement reaction. Based on the k(cat) values with a series of aryl glucosides, a Bronsted plot with a concave-downward shape was constructed. This biphasic behaviour is consistent with a two-step mechanism involving the formation and breakdown of a glucosyl-enzyme intermediate. The large Bronsted constant (beta=-0.85) for the leaving-group-dependent portion (pK(a) of leaving phenols >7) indicates substantial bond cleavage at the transition state. Secondary deuterium kinetic isotope effects with 2,4-dinitrophenyl beta-D-glucopyanoside, o-nitrophenyl beta-D-glucopyanoside and p-cyanophenyl beta-D-glucopyanoside as substrates were 1.17+/-0.02, 1.19+/-0.02 and 1.04+/-0.02 respectively. These results support an S(N)1-like mechanism for the deglucosylation step and an S(N)2-like mechanism for the glucosylation step. Site-directed mutagenesis was also performed to study essential amino acid residues. The activities (k(cat)/K(m)) of the D247G and D247N mutants were 30000- and 200000-fold lower respectively than that of the wild-type enzyme, whereas the D247E mutant retained 20% of wild-type activity. These results indicate that Asp-247 is an essential amino acid. It is likely that this residue functions as a nucleophile in the reaction. This conclusion is supported by the kinetics of the irreversible inactivation of the wild-type enzyme by conduritol-B-epoxide, compared with the much slower inhibition of the D247E mutant and the lack of irreversible inhibition of the D247G mutant. PMID:11311148

  18. Next-Generation Site-Directed Transgenesis in the Malaria Vector Mosquito Anopheles gambiae: Self-Docking Strains Expressing Germline-Specific phiC31 Integrase

    PubMed Central

    Meredith, Janet M.; Underhill, Ann; McArthur, Clare C.; Eggleston, Paul

    2013-01-01

    Diseases transmitted by mosquitoes have a devastating impact on global health and the situation is complicated due to difficulties with both existing control measures and the impact of climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. The Streptomyces phage phiC31 integrase system has been successfully adapted for site-directed transgene integration in a range of insects, thus overcoming many limitations due to size constraints and random integration associated with transposon-mediated transformation. Using this technology, we previously published the first site-directed transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 docking site at a defined genomic location. A second phase of genetic modification then achieved site-directed integration of an anti-malarial effector gene. In the current publication we report improved efficiency and utility of the phiC31 integrase system following the generation of Anopheles gambiae self-docking strains. Four independent strains, with docking sites at known locations on three different chromosome arms, were engineered to express integrase under control of the regulatory regions of the nanos gene from Anopheles gambiae. The resulting protein accumulates in the posterior oocyte to provide integrase activity at the site of germline development. Two self-docking strains, exhibiting significantly different levels of integrase expression, were assessed for site-directed transgene integration and found to demonstrate greatly improved survival and efficiency of transformation. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters to regulate their expression, enabling those offering maximum effect with minimum fitness cost to be identified

  19. Food additives

    MedlinePlus

    Food additives are substances that become part of a food product when they are added during the processing or making of that food. "Direct" food additives are often added during processing to: Add nutrients ...

  20. Food additives

    PubMed Central

    Spencer, Michael

    1974-01-01

    Food additives are discussed from the food technology point of view. The reasons for their use are summarized: (1) to protect food from chemical and microbiological attack; (2) to even out seasonal supplies; (3) to improve their eating quality; (4) to improve their nutritional value. The various types of food additives are considered, e.g. colours, flavours, emulsifiers, bread and flour additives, preservatives, and nutritional additives. The paper concludes with consideration of those circumstances in which the use of additives is (a) justified and (b) unjustified. PMID:4467857

  1. Breeding of New Strains of Mushroom by Basidiospore Chemical Mutagenesis

    PubMed Central

    Lee, Jia; Kang, Hyeon-Woo; Kim, Sang-Woo; Lee, Chang-Yun

    2011-01-01

    Chemical mutagenesis of basidiospores of Hypsizygus marmoreus generated new mushroom strains. The basidospores were treated with methanesulfonate methylester, an alkylating agent, to yield 400 mutant monokaryotic mycelia. Twenty fast-growing mycelia were selected and mated each other by hyphal fusion. Fifty out of the 190 matings were successful (mating rate of 26.3%), judged by the formation of clamp connections. The mutant dikaryons were cultivated to investigate their morphological and cultivation characteristics. Mutant strains No. 3 and No. 5 showed 10% and 6% increase in fruiting body production, respectively. Eight mutant strains showed delayed and reduced primordia formation, resulting in the reduced production yield with prolonged cultivation period. The number of the fruiting bodies of mutant No. 31, which displayed reduced primordial formation, was only 15, compared to the parental number of 65. Another interesting phenotype was a fruiting body with a flattened stipe and pileus. Dikaryons generated by mating with the mutant spore No. 14 produced flat fruiting bodies. Further molecular biological studies will provide details of the mechanism. This work shows that the chemical mutagenesis approach is highly utilizable in the development of mushroom strains as well as in the generation of resources for molecular genetic studies. PMID:22783115

  2. Mechanisms of Base Substitution Mutagenesis in Cancer Genomes

    PubMed Central

    Bacolla, Albino; Cooper, David N.; Vasquez, Karen M.

    2014-01-01

    Cancer genome sequence data provide an invaluable resource for inferring the key mechanisms by which mutations arise in cancer cells, favoring their survival, proliferation and invasiveness. Here we examine recent advances in understanding the molecular mechanisms responsible for the predominant type of genetic alteration found in cancer cells, somatic single base substitutions (SBSs). Cytosine methylation, demethylation and deamination, charge transfer reactions in DNA, DNA replication timing, chromatin status and altered DNA proofreading activities are all now known to contribute to the mechanisms leading to base substitution mutagenesis. We review current hypotheses as to the major processes that give rise to SBSs and evaluate their relative relevance in the light of knowledge acquired from cancer genome sequencing projects and the study of base modifications, DNA repair and lesion bypass. Although gene expression data on APOBEC3B enzymes provide support for a role in cancer mutagenesis through U:G mismatch intermediates, the enzyme preference for single-stranded DNA may limit its activity genome-wide. For SBSs at both CG:CG and YC:GR sites, we outline evidence for a prominent role of damage by charge transfer reactions that follow interactions of the DNA with reactive oxygen species (ROS) and other endogenous or exogenous electron-abstracting molecules. PMID:24705290

  3. Fitness Loss and Library Size Determination in Saturation Mutagenesis

    PubMed Central

    Nov, Yuval

    2013-01-01

    Saturation mutagenesis is a widely used directed evolution technique, in which a large number of protein variants, each having random amino acids in certain predetermined positions, are screened in order to discover high-fitness variants among them. Several metrics for determining the library size (the number of variants screened) have been suggested in the literature, but none of them incorporates the actual fitness of the variants discovered in the experiment. We present the results of an extensive simulation study, which is based on probabilistic models for protein fitness landscape, and which investigates how the result of a saturation mutagenesis experiment – the fitness of the best variant discovered – varies as a function of the library size. In particular, we study the loss of fitness in the experiment: the difference between the fitness of the best variant discovered, and the fitness of the best variant in variant space. Our results are that the existing criteria for determining the library size are conservative, so smaller libraries are often satisfactory. Reducing the library size can save labor, time, and expenses in the laboratory. PMID:23844158

  4. Establishment of a counter-selectable markerless mutagenesis system in Veillonella atypica.

    PubMed

    Zhou, Peng; Li, Xiaoli; Qi, Fengxia

    2015-05-01

    Using an alternative sigma factor ecf3 as target, we successfully established the first markerless mutagenesis system in the Veillonella genus. This system will be a valuable tool for mutagenesis of multiple genes for gene function analysis as well as for gene regulation studies in Veillonella. PMID:25771833

  5. A mouse chromosome 4 balancer ENU-mutagenesis screen isolates eleven lethal lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ENU-mutagenesis is a powerful technique to identify genes regulating mammalian development. To functionally annotate the distal region of mouse chromosome 4, we performed an ENU-mutagenesis screen using a balancer chromosome targeted to this region of the genome. We isolated 11 lethal lines that map...

  6. Sleeping Beauty transposon mutagenesis identifies genes that cooperate with mutant Smad4 in gastric cancer development.

    PubMed

    Takeda, Haruna; Rust, Alistair G; Ward, Jerrold M; Yew, Christopher Chin Kuan; Jenkins, Nancy A; Copeland, Neal G

    2016-04-01

    Mutations in SMAD4 predispose to the development of gastrointestinal cancer, which is the third leading cause of cancer-related deaths. To identify genes driving gastric cancer (GC) development, we performed a Sleeping Beauty (SB) transposon mutagenesis screen in the stomach of Smad4(+/-) mutant mice. This screen identified 59 candidate GC trunk drivers and a much larger number of candidate GC progression genes. Strikingly, 22 SB-identified trunk drivers are known or candidate cancer genes, whereas four SB-identified trunk drivers, including PTEN, SMAD4, RNF43, and NF1, are known human GC trunk drivers. Similar to human GC, pathway analyses identified WNT, TGF-β, and PI3K-PTEN signaling, ubiquitin-mediated proteolysis, adherens junctions, and RNA degradation in addition to genes involved in chromatin modification and organization as highly deregulated pathways in GC. Comparative oncogenomic filtering of the complete list of SB-identified genes showed that they are highly enriched for genes mutated in human GC and identified many candidate human GC genes. Finally, by comparing our complete list of SB-identified genes against the list of mutated genes identified in five large-scale human GC sequencing studies, we identified LDL receptor-related protein 1B (LRP1B) as a previously unidentified human candidate GC tumor suppressor gene. In LRP1B, 129 mutations were found in 462 human GC samples sequenced, and LRP1B is one of the top 10 most deleted genes identified in a panel of 3,312 human cancers. SB mutagenesis has, thus, helped to catalog the cooperative molecular mechanisms driving SMAD4-induced GC growth and discover genes with potential clinical importance in human GC. PMID:27006499

  7. Targeted Mutagenesis in Zebrafish Using Customized Zinc Finger Nucleases

    PubMed Central

    Foley, Jonathan E.; Maeder, Morgan L.; Pearlberg, Joseph; Joung, J. Keith; Peterson, Randall T.; Yeh, Jing-Ruey J.

    2009-01-01

    Zebrafish mutants have traditionally been obtained using random mutagenesis or retroviral insertions, methods that cannot be targeted to a specific gene and require laborious gene mapping and sequencing. Recently, we and others have shown that customized zinc finger nucleases (ZFNs) can introduce targeted frame-shift mutations with high efficiency, thereby enabling directed creation of zebrafish gene mutations. Here we describe a detailed protocol for constructing ZFN expression vectors, for generating and introducing ZFN-encoding RNAs into zebrafish embryos, and for identifying ZFN-generated mutations in targeted genomic sites. All of our vectors and methods are compatible with previously described Zinc Finger Consortium reagents for constructing engineered zinc finger arrays. Using these methods, zebrafish founders carrying targeted mutations can be identified within four months. PMID:20010934

  8. Mutagenesis and differentiation induction in mammalian cells by environmental chemicals

    SciTech Connect

    Friedman, J.; Huberman, E.

    1980-01-01

    These studies indicate that in agreement with the somatic mutation hypothesis, chemical carcinogens: (1) are mutagenic for mammalian cells as tested in the cell-mediated assay; (2) the degree of mutagenicity is correlated with their degree of carcinogenicity; (3) that at least in cases when analyzed carefully the metabolites responsible for mutagenesis are also responsible for initiating the carcinogenic event; and (4) that a cell organ type specificity can be established using the cell-mediated assay. Studies with HL-60 cells and HO melanoma cells and those of others suggest that tumor-promoting phorbol diesters can alter cell differentiation in various cell types and that the degree of the observed alteration in the differentiation properties may be related to the potency of the phorbol esters. Thus these and similar systems may serve as models for both studies and identification of certain types of tumor promoting agents. (ERB)

  9. CRISPR-Cas9 enables conditional mutagenesis of challenging loci

    PubMed Central

    Schick, Joel A.; Seisenberger, Claudia; Beig, Joachim; Bürger, Antje; Iyer, Vivek; Maier, Viola; Perera, Sajith; Rosen, Barry; Skarnes, William C.; Wurst, Wolfgang

    2016-01-01

    The International Knockout Mouse Consortium (IKMC) has produced a genome-wide collection of 15,000 isogenic targeting vectors for conditional mutagenesis in C57BL/6N mice. Although most of the vectors have been used successfully in murine embryonic stem (ES) cells, there remain a set of nearly two thousand genes that have failed to target even after several attempts. Recent attention has turned to the use of new genome editing technology for the generation of mutant alleles in mice. Here, we demonstrate how Cas9-assisted targeting can be combined with the IKMC targeting vector resource to generate conditional alleles in genes that have previously eluded targeting using conventional methods. PMID:27580957

  10. Environmental mutagenesis during the end-Permian ecological crisis.

    PubMed

    Visscher, Henk; Looy, Cindy V; Collinson, Margaret E; Brinkhuis, Henk; van Konijnenburg-van Cittert, Johanna H A; Kürschner, Wolfram M; Sephton, Mark A

    2004-08-31

    During the end-Permian ecological crisis, terrestrial ecosystems experienced preferential dieback of woody vegetation. Across the world, surviving herbaceous lycopsids played a pioneering role in repopulating deforested terrain. We document that the microspores of these lycopsids were regularly released in unseparated tetrads indicative of failure to complete the normal process of spore development. Although involvement of mutation has long been hinted at or proposed in theory, this finding provides concrete evidence for chronic environmental mutagenesis at the time of global ecological crisis. Prolonged exposure to enhanced UV radiation could account satisfactorily for a worldwide increase in land plant mutation. At the end of the Permian, a period of raised UV stress may have been the consequence of severe disruption of the stratospheric ozone balance by excessive emission of hydrothermal organohalogens in the vast area of Siberian Traps volcanism. PMID:15282373

  11. Radiosensitivity Parameters For Lethal Mutagenesis In Caenorhabditis Elegans

    SciTech Connect

    Cucinotta, F.A.; Wilson, J.W.; Katz, R.

    1994-01-01

    For the first time track structure theory has been applied to radiobiological effects in a living organism. Data for lethal mutagenesis in Caenorhabditis elegans, obtained after irradiation with nine different types of ions of atomic number 1-57 and gamma rays have yielded radiosensitivity parameters (E{sub 0}, sigma{sub 0}, Kappa, m = 68 Gy, 2.5 x 10(exp {minus}9) cm (exp 2), 750, 2) comparable with those found for the transformation of C3HT10 1/2 cells (180 Gy, 1.15 x 10(exp {minus}10) cm(exp 2), 750, 2) but remote from those (E{sub 0} and sigma{sub 0} = approx. 2 Gy, approx. 5 x 10(exp {minus}7) cm(exp 2)) for mammalian cell survival.

  12. Mutant fatty acid desaturase and methods for directed mutagenesis

    DOEpatents

    Shanklin, John; Whittle, Edward J.

    2008-01-29

    The present invention relates to methods for producing fatty acid desaturase mutants having a substantially increased activity towards substrates with fewer than 18 carbon atom chains relative to an unmutagenized precursor desaturase having an 18 carbon chain length specificity, the sequences encoding the desaturases and to the desaturases that are produced by the methods. The present invention further relates to a method for altering a function of a protein, including a fatty acid desaturase, through directed mutagenesis involving identifying candidate amino acid residues, producing a library of mutants of the protein by simultaneously randomizing all amino acid candidates, and selecting for mutants which exhibit the desired alteration of function. Candidate amino acids are identified by a combination of methods. Enzymatic, binding, structural and other functions of proteins can be altered by the method.

  13. CRISPR-Cas9 enables conditional mutagenesis of challenging loci.

    PubMed

    Schick, Joel A; Seisenberger, Claudia; Beig, Joachim; Bürger, Antje; Iyer, Vivek; Maier, Viola; Perera, Sajith; Rosen, Barry; Skarnes, William C; Wurst, Wolfgang

    2016-01-01

    The International Knockout Mouse Consortium (IKMC) has produced a genome-wide collection of 15,000 isogenic targeting vectors for conditional mutagenesis in C57BL/6N mice. Although most of the vectors have been used successfully in murine embryonic stem (ES) cells, there remain a set of nearly two thousand genes that have failed to target even after several attempts. Recent attention has turned to the use of new genome editing technology for the generation of mutant alleles in mice. Here, we demonstrate how Cas9-assisted targeting can be combined with the IKMC targeting vector resource to generate conditional alleles in genes that have previously eluded targeting using conventional methods. PMID:27580957

  14. Marker reconstitution mutagenesis: a simple and efficient reverse genetic approach.

    PubMed

    Tang, Xie; Huang, Junqi; Padmanabhan, Anup; Bakka, Kavya; Bao, Yun; Tan, Brenda Yuelin; Cande, W Zacheus; Balasubramanian, Mohan K

    2011-03-01

    A novel reverse genetic approach termed 'marker reconstitution mutagenesis' was designed to generate mutational allelic series in genes of interest. This approach consists of two simple steps which utilize two selective markers. First, using one selective marker, a partial fragment of another selective marker gene is inserted adjacently to a gene of interest by homologous recombination. Second, random mutations are introduced precisely into the gene of interest, together with the reconstitution of the latter selective marker by homologous recombination. This approach was successfully tested for several genes in the fission yeast Schizosaccharomyces pombe. It circumvents the problems encountered with other methods and should be adaptable to any organism that incorporates exogenous DNA by homologous recombination. PMID:21360732

  15. Environmental mutagenesis during the end-Permian ecological crisis

    PubMed Central

    Visscher, Henk; Looy, Cindy V.; Collinson, Margaret E.; Brinkhuis, Henk; van Konijnenburg-van Cittert, Johanna H. A.; Kürschner, Wolfram M.; Sephton, Mark A.

    2004-01-01

    During the end-Permian ecological crisis, terrestrial ecosystems experienced preferential dieback of woody vegetation. Across the world, surviving herbaceous lycopsids played a pioneering role in repopulating deforested terrain. We document that the microspores of these lycopsids were regularly released in unseparated tetrads indicative of failure to complete the normal process of spore development. Although involvement of mutation has long been hinted at or proposed in theory, this finding provides concrete evidence for chronic environmental mutagenesis at the time of global ecological crisis. Prolonged exposure to enhanced UV radiation could account satisfactorily for a worldwide increase in land plant mutation. At the end of the Permian, a period of raised UV stress may have been the consequence of severe disruption of the stratospheric ozone balance by excessive emission of hydrothermal organohalogens in the vast area of Siberian Traps volcanism. PMID:15282373

  16. Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice

    PubMed Central

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2016-01-01

    Recent reports of CRISPR- (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) mediated heritable mutagenesis in plants highlight the need for accuracy of the mutagenesis directed by this system. Off-target mutations are an important issue when considering functional gene analysis, as well as the molecular breeding of crop plants with large genome size, i.e. with many duplicated genes, and where the whole-genome sequence is still lacking. In mammals, off-target mutations can be suppressed by using Cas9 paired nickases together with paired guide RNAs (gRNAs). However, the performance of Cas9 paired nickases has not yet been fully assessed in plants. Here, we analyzed on- and off-target mutation frequency in rice calli and regenerated plants using Cas9 nuclease or Cas9 nickase with paired gRNAs. When Cas9 paired nickases were used, off-target mutations were fully suppressed in rice calli and regenerated plants. However, on-target mutation frequency also decreased compared with that induced by the Cas9 paired nucleases system. Since the gRNA sequence determines specific binding of Cas9 protein–gRNA ribonucleoproteins at the targeted sequence, the on-target mutation frequency of Cas9 paired nickases depends on the design of paired gRNAs. Our results suggest that a combination of gRNAs that can induce mutations at high efficiency with Cas9 nuclease should be used together with Cas9 nickase. Furthermore, we confirmed that a combination of gRNAs containing a one nucleotide (1 nt) mismatch toward the target sequence could not induce mutations when expressed with Cas9 nickase. Our results clearly show the effectiveness of Cas9 paired nickases in delivering on-target specific mutations. PMID:26936792

  17. Site-directed immobilization of a genetically engineered anti-methotrexate antibody via an enzymatically introduced biotin label significantly increases the binding capacity of immunoaffinity columns.

    PubMed

    Davenport, Kaitlynn R; Smith, Christopher A; Hofstetter, Heike; Horn, James R; Hofstetter, Oliver

    2016-05-15

    In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC). Then, two site-directed techniques were explored to increase column efficiency by immobilizing the antibody via its C-terminus, i.e., away from the antigen-binding site. In one approach, a tetra-lysine tail was added, and the antibody was immobilized onto DSC-activated POROS. In the second site-directed approach, the VHH was modified with the AviTag peptide, and a biotin-residue was enzymatically incorporated at the C-terminus using the biotin ligase BirA. The biotinylated antibody was subsequently immobilized onto NeutrAvidin-derivatized POROS. A comparison of the FAC analyses, which for all three columns showed excellent linearity (R(2)>0.999), revealed that both site-directed approaches yield better results than the random immobilization; the by far highest efficiency, however, was determined for the immunoaffinity column based on AviTag-biotinylated antibody. As proof of concept, all three columns were evaluated for quantification of MTX dissolved in phosphate buffered saline (PBS). Validation using UV-detection showed excellent linearity in the range of 0.04-12μM (R(2)>0.993). The lower limit of detection (LOD) and lower limit of quantification (LLOQ) were found to be independent of the immobilization strategy and were 40nM and 132nM, respectively. The intra- and inter-day precision was below 11.6%, and accuracy was between 90.7% and 112%. To the best of our knowledge, this is the first report of the AviTag-system in chromatography, and the first

  18. Food additives.

    PubMed

    Berglund, F

    1978-01-01

    The use of additives to food fulfils many purposes, as shown by the index issued by the Codex Committee on Food Additives: Acids, bases and salts; Preservatives, Antioxidants and antioxidant synergists; Anticaking agents; Colours; Emulfifiers; Thickening agents; Flour-treatment agents; Extraction solvents; Carrier solvents; Flavours (synthetic); Flavour enhancers; Non-nutritive sweeteners; Processing aids; Enzyme preparations. Many additives occur naturally in foods, but this does not exclude toxicity at higher levels. Some food additives are nutrients, or even essential nutritents, e.g. NaCl. Examples are known of food additives causing toxicity in man even when used according to regulations, e.g. cobalt in beer. In other instances, poisoning has been due to carry-over, e.g. by nitrate in cheese whey - when used for artificial feed for infants. Poisonings also occur as the result of the permitted substance being added at too high levels, by accident or carelessness, e.g. nitrite in fish. Finally, there are examples of hypersensitivity to food additives, e.g. to tartrazine and other food colours. The toxicological evaluation, based on animal feeding studies, may be complicated by impurities, e.g. orthotoluene-sulfonamide in saccharin; by transformation or disappearance of the additive in food processing in storage, e.g. bisulfite in raisins; by reaction products with food constituents, e.g. formation of ethylurethane from diethyl pyrocarbonate; by metabolic transformation products, e.g. formation in the gut of cyclohexylamine from cyclamate. Metabolic end products may differ in experimental animals and in man: guanylic acid and inosinic acid are metabolized to allantoin in the rat but to uric acid in man. The magnitude of the safety margin in man of the Acceptable Daily Intake (ADI) is not identical to the "safety factor" used when calculating the ADI. The symptoms of Chinese Restaurant Syndrome, although not hazardous, furthermore illustrate that the whole ADI

  19. Mutagenesis and Modeling To Predict Structural and Functional Characteristics of the Staphylococcus aureus MepA Multidrug Efflux Pump

    PubMed Central

    Schindler, Bryan D.; Patel, Diixa; Seo, Susan M.

    2013-01-01

    MepA is a multidrug and toxin extrusion (MATE) family protein and the only MATE protein encoded within the Staphylococcus aureus genome. Structural data for MATE proteins are limited to a single high-resolution example, NorM of Vibrio cholerae. Substitution mutations were created in MepA using gradient plates containing both a substrate and reserpine as an efflux pump inhibitor. Site-directed mutagenesis of plasmid-based mepA was used to reproduce these mutations, as well as unique or low-frequency mutations identified in mepA-overexpressing clinical strains, and to mutagenize conserved acidic residues. The effect of these changes on protein function was quantitated in a norA-disrupted host strain by susceptibility testing with and without inhibitors and by determining the proficiency of ethidium efflux. Up-function substitutions clustered in the carboxy half of MepA, near the cytoplasmic face of the protein. Repeated application of the same gradient plate conditions frequently reproduced identical substitution mutations, suggesting that individual residues are required for interaction with specific substrates. Acidic residues critical to protein function were identified in helices 4 and 5. In silico modeling revealed an outward-facing molecule, with helices 1, 2, 4, 7, 8, and 10 having contact with a central cavity that may represent a substrate translocation pathway. Functionally important residues within this cavity included S81, A161, M291, and A302. These data provide a critical starting point for understanding how MATE multidrug efflux proteins function and will be useful in refining crystallographic data when they are available. PMID:23175649

  20. Mechanism of Porcine Liver Xanthine Oxidoreductase Mediated N-Oxide Reduction of Cyadox as Revealed by Docking and Mutagenesis Studies

    PubMed Central

    Hao, Haihong; Dai, Menghong; Wang, Xu; Huang, Lingli; Liu, Zhenli; Yuan, Zonghui

    2013-01-01

    Xanthine oxidoreductase (XOR) is a cytoplasmic molybdenum-containing oxidoreductase, catalyzing both endogenous purines and exogenous compounds. It is suggested that XOR in porcine hepatocytes catalyzes the N-oxide reduction of quinoxaline 1,4-di-N-oxides (QdNOs). To elucidate the molecular mechanism underlying this metabolism, the cDNA of porcine XOR was cloned and heterologously expressed in Spodoptera frugiperda insect cells. The bovine XOR, showing sequence identity of 91% to porcine XOR, was employed as template for homology modeling. By docking cyadox, a representative compound of QdNOs, into porcine XOR model, eight amino acid residues, Gly47, Asn352, Ser360, Arg427, Asp430, Asp431, Ser1227 and Lys1230, were located at distances of less than 4Å to cyadox. Site-directed mutagenesis was performed to analyze their catalytic functions. Compared with wild type porcine XOR, G47A, S360P, D431A, S1227A, and K1230A displayed altered kinetic parameters in cyadox reduction, similarly to that in xanthine oxidation, indicating these mutations influenced electron-donating process of xanthine before subsequent electron transfer to cyadox to fulfill the N-oxide reduction. Differently, R427E and D430H, both located in the 424–434 loop, exhibited a much lower Km and a decreased Vmax respectively in cyadox reduction. Arg427 may be related to the substrate binding of porcine XOR to cyadox, and Asp430 is suggested to be involved in the transfer of electron to cyadox. This study initially reveals the possible catalytic mechanism of porcine XOR in cyadox metabolism, providing with novel insights into the structure-function relationship of XOR in the reduction of exogenous di-N-oxides. PMID:24040113

  1. [Improvement of the the thermostability of Penicillium expansum lipase by mutagenesis the random mutant ep8 at K55R].

    PubMed

    Cai, Shao-Li; Lin, Jun-Han; Wang, Cai-Mei; Lin, Lin

    2007-07-01

    In order to improve the thermostability of the Penicillium expansum Lipase (PEL), the lipase encoding genes was mutated by site-directed mutagenesis. A recombinant vector pAO815-ep8-K55R which contain double mutant genes was constructed by overlap extension PCR using the cDNA of a random-mutant lipase ep8 (a single site mutant) as the template and two special primers were used to generate another mutation site K55R. The recombinant vector was transformed into Pichia pastoris GS115 by electroporation and the recombinant mutant GS-pAO815-ep8- K55R can secret double-mutant lipase PEL-ep8-K55R-GS into the medium when it was induced by Methanol. The yield of the double-mutant lipase is 508 u/mL, which is 81% that of the wild type lipase PEL-GS (627 u/mL) and 55% that of random-mutant PEL-ep8-GS (924 u/mL). The specific activity of double-mutant lipase is 2309.1 u/mg, which is similar to random-mutant lipase PEL-ep8-GS and the wild type lipase PEL-GS. The optimum temperature of the double-mutant lipase is same with the wild type lipase PEL-GS and random-mutant lipase PEL-ep8-GS. While the Tm of the double-mutant lipase is 41.0 degrees C, 2.3 degrees C higher than the wild type lipase PEL-GS and 0.8% higher than the random-mutant lipase PEL-ep8-GS, indicating that the double-mutant lipase PEL-ep8-K55R-GS has higher thermostability. PMID:17822043

  2. Analysis of serpin inhibitory function by mutagenesis of ovalbumin and generation of chimeric ovalbumin/PAI-2 fusion proteins.

    PubMed

    McCarthy, B J; Worrall, D M

    1997-04-01

    Ovalbumin is a non-inhibitory serpin which lacks the ability to undergo the S --> R transition or conformational change. Amino acid residues in the hinge region (P11 to P14) of ovalbumin and other non-inhibitory serpins differ from the concensus sequence of this region of inhibitory serpins, and have been proposed to be responsible for lack of inhibitory properties, particularly the P14 charged residue. Site directed mutagenesis using PCR overlap extension was performed on these residues in ovalbumin to create a mutant with three amino acid changes, R340T, V342A and V343A. However analysis of the mutant recombinant ovalbumin with the consensus residues failed to show inhibitory activity or decreased stability, indicating that the hinge region alone is not responsible for lack of inhibition. A series of three fusion proteins were then constructed by replacing varying C-terminal regions of ovalbumin with the corresponding region of the inhibitory ov-serpin PAI-2 in order to further analyse serpin inhibitory function. Fusion proteins F1 and F2 contained approximately 16% and 35% PAI-2, respectively. This resulted in the replacing of structural features such as the reactive site loop, hinge region and beta sheet strands 5A and 6A. However both fusion proteins showed no inhibitory activity with the PAI-2 target protease urokinase (uPA) and no decrease in stability as analysed by transverse urea gradient (TUG) gels. The third chimeric fusion protein constructed (F3) contained 64% PAI-2 and did demonstrate inhibition of uPA, SDS-PAGE stable complex formation with uPA and increased instability on TUG gels. Structural differences between the inactive F2 and active F3 include the replacement of helix F and beta sheet strand 3A of ovalbumin with those of PAI-2, suggesting that these features may have a key role in serpin beta-sheet opening and inhibitory function. PMID:9126838

  3. Site-Directed Genome Knockout in Chicken Cell Line and Embryos Can Use CRISPR/Cas Gene Editing Technology

    PubMed Central

    Zuo, Qisheng; Wang, Yinjie; Cheng, Shaoze; Lian, Chao; Tang, Beibei; Wang, Fei; Lu, Zhenyu; Ji, Yanqing; Zhao, Ruifeng; Zhang, Wenhui; Jin, Kai; Song, Jiuzhou; Zhang, Yani; Li, Bichun

    2016-01-01

    The present study established an efficient genome editing approach for the construction of stable transgenic cell lines of the domestic chicken (Gallus gallus domesticus). Our objectives were to facilitate the breeding of high-yield, high-quality chicken strains, and to investigate gene function in chicken stem cells. Three guide RNA (gRNAs) were designed to knockout the C2EIP gene, and knockout efficiency was evaluated in DF-1 chicken fibroblasts and chicken ESCs using the luciferase single-strand annealing (SSA) recombination assay, T7 endonuclease I (T7EI) assay, and TA clone sequencing. In addition, the polyethylenimine-encapsulated Cas9/gRNA plasmid was injected into fresh fertilized eggs. At 4.5 d later, frozen sections of the embryos were prepared, and knockout efficiency was evaluated by the T7EI assay. SSA assay results showed that luciferase activity of the vector expressing gRNA-3 was double that of the control. Results of the T7EI assay and TA clone sequencing indicated that Cas9/gRNA vector-mediated gene knockdown efficiency was approximately 27% in both DF-1 cells and ESCs. The CRISPR/Cas9 vector was also expressed in chicken embryos, resulting in gene knockdown in three of the 20 embryos (gene knockdown efficiency 15%). Taken together, our results indicate that the CRISPR/Cas9 system can mediate stable gene knockdown at the cell and embryo levels in domestic chickens. PMID:27172204

  4. Site-Directed Genome Knockout in Chicken Cell Line and Embryos Can Use CRISPR/Cas Gene Editing Technology.

    PubMed

    Zuo, Qisheng; Wang, Yinjie; Cheng, Shaoze; Lian, Chao; Tang, Beibei; Wang, Fei; Lu, Zhenyu; Ji, Yanqing; Zhao, Ruifeng; Zhang, Wenhui; Jin, Kai; Song, Jiuzhou; Zhang, Yani; Li, Bichun

    2016-01-01

    The present study established an efficient genome editing approach for the construction of stable transgenic cell lines of the domestic chicken (Gallus gallus domesticus). Our objectives were to facilitate the breeding of high-yield, high-quality chicken strains, and to investigate gene function in chicken stem cells. Three guide RNA (gRNAs) were designed to knockout the C2EIP gene, and knockout efficiency was evaluated in DF-1 chicken fibroblasts and chicken ESCs using the luciferase single-strand annealing (SSA) recombination assay, T7 endonuclease I (T7EI) assay, and TA clone sequencing. In addition, the polyethylenimine-encapsulated Cas9/gRNA plasmid was injected into fresh fertilized eggs. At 4.5 d later, frozen sections of the embryos were prepared, and knockout efficiency was evaluated by the T7EI assay. SSA assay results showed that luciferase activity of the vector expressing gRNA-3 was double that of the control. Results of the T7EI assay and TA clone sequencing indicated that Cas9/gRNA vector-mediated gene knockdown efficiency was approximately 27% in both DF-1 cells and ESCs. The CRISPR/Cas9 vector was also expressed in chicken embryos, resulting in gene knockdown in three of the 20 embryos (gene knockdown efficiency 15%). Taken together, our results indicate that the CRISPR/Cas9 system can mediate stable gene knockdown at the cell and embryo levels in domestic chickens. PMID:27172204

  5. Potlining Additives

    SciTech Connect

    Rudolf Keller

    2004-08-10

    In this project, a concept to improve the performance of aluminum production cells by introducing potlining additives was examined and tested. Boron oxide was added to cathode blocks, and titanium was dissolved in the metal pool; this resulted in the formation of titanium diboride and caused the molten aluminum to wet the carbonaceous cathode surface. Such wetting reportedly leads to operational improvements and extended cell life. In addition, boron oxide suppresses cyanide formation. This final report presents and discusses the results of this project. Substantial economic benefits for the practical implementation of the technology are projected, especially for modern cells with graphitized blocks. For example, with an energy savings of about 5% and an increase in pot life from 1500 to 2500 days, a cost savings of $ 0.023 per pound of aluminum produced is projected for a 200 kA pot.

  6. Phosphazene additives

    SciTech Connect

    Harrup, Mason K; Rollins, Harry W

    2013-11-26

    An additive comprising a phosphazene compound that has at least two reactive functional groups and at least one capping functional group bonded to phosphorus atoms of the phosphazene compound. One of the at least two reactive functional groups is configured to react with cellulose and the other of the at least two reactive functional groups is configured to react with a resin, such as an amine resin of a polycarboxylic acid resin. The at least one capping functional group is selected from the group consisting of a short chain ether group, an alkoxy group, or an aryloxy group. Also disclosed are an additive-resin admixture, a method of treating a wood product, and a wood product.

  7. Association of elevated mutagenesis in the spleen with genetic susceptibility to induced plasmacytoma development in mice.

    PubMed

    Felix, K; Kelliher, K; Bornkamm, G W; Janz, S

    1998-04-15

    Using the phage lambdaLIZ-based transgenic in vivo mutagenesis assay, mean mutant rates were determined in the spleen of mice exposed to sustained oxidative stress and were found to be increased approximately 3-fold in plasmacytoma-susceptible BALB/c and C.D2-Idh1-Pep3 mice, but not in plasmacytoma-resistant DBA/2N mice. This finding suggests a correlation between the genetic susceptibility to inflammation-induced peritoneal plasmacytomagenesis and the phenotype of increased mutagenesis in lymphoid tissues, raising the possibility that plasmacytoma resistance genes may inhibit tumor development by minimizing oxidative mutagenesis in B cells. PMID:9563470

  8. Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

    PubMed Central

    da Silva, Luciana Madeira; Vandepas, Lauren; Bianco, Suzy D.C.

    2011-01-01

    The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in adulthood 1,2,3. Inactivating mutations in KISS1R identified in patients have been associated with iodiopathic hypogonadotropic hypogonadism (IHH) 1,2 and precocious puberty 4. Functional studies of these mutants are crucial for our understanding of the mechanisms underlying the regulation of reproduction by this receptor as well as those shaping the disease outcomes, which result from abnormal KISS1R signaling and function. However, the highly GC-rich sequence of the KISS1R gene makes it rather difficult to introduce mutations or amplify the gene encoding this receptor by PCR. Here we describe a method to introduce mutations of interest into this highly GC-rich sequence that has been used successfully to generate over a dozen KISS1R mutants in our laboratory. We have optimized the PCR conditions to facilitate the amplification of a range of KISS1R mutants that include substitutions, deletions or insertions in the KISS1R sequence. The addition of a PCR enhancer solution, as well as of a small percentage of DMSO were especially helpful to improve amplification. This optimized procedure may be useful for other GC-rich templates as well. The expression vector encoding the KISS1R is been used to characterize signaling and function of this receptor in order to understand how mutations may change KISS1R function and lead to the associated reproductive phenotypes. Accordingly, potential applications of KISS1R mutants generated by site-directed mutagenesis can be illustrated by many studies 1,4,5,6,7,8. As an example, the gain-of-function mutation in the KISS1R (Arg386Pro), which is associated with precocious puberty, has been shown to prolong responsiveness of the receptor to ligand stimulation 4 as well as to alter the rate of degradation of KISS1R 9. Interestingly, our studies indicate that KISS1R is degraded by the

  9. Simple sequence repeats and mucoid conversion: biased mucA mutagenesis in mismatch repair-deficient Pseudomonas aeruginosa.

    PubMed

    Moyano, Alejandro J; Smania, Andrea M

    2009-01-01

    In Pseudomonas aeruginosa, conversion to the mucoid phenotype marks the onset of an irreversible state of the infection in Cystic Fibrosis (CF) patients. The main pathway for mucoid conversion is mutagenesis of the mucA gene, frequently due to -1 bp deletions in a simple sequence repeat (SSR) of 5 Gs (G(5)-SSR(426)). We have recently observed that this mucA mutation is particularly accentuated in Mismatch Repair System (MRS)-deficient cells grown in vitro. Interestingly, previous reports have shown a high prevalence of hypermutable MRS-deficient strains occurring naturally in CF chronic lung infections. Here, we used mucA as a forward mutation model to systematically evaluate the role of G(5)-SSR(426) in conversion to mucoidy in a MRS-deficient background, with this being the first analysis combining SSR-dependent localized hypermutability and the acquisition of a particular virulence/persistence trait in P. aeruginosa. In this study, mucA alleles were engineered with different contents of G:C SSRs, and tested for their effect on the mucoid conversion frequency and mucA mutational spectra in a mutS-deficient strain of P. aeruginosa. Importantly, deletion of G(5)-SSR(426) severely reduced the emergence frequency of mucoid variants, with no preferential site of mutagenesis within mucA. Moreover, although mutagenesis in mucA was not totally removed, this was no longer the main pathway for mucoid conversion, suggesting that G(5)-SSR(426) biased mutations towards mucA. Mutagenesis in mucA was restored by the addition of a new SSR (C(6)-SSR(431)), and even synergistically increased when G(5)-SSR(426) and C(6)-SSR(431) were present simultaneously, with the mucA mutations being restricted to -1 bp deletions within any of both G:C SSRs. These results confirm a critical role for G(5)-SSR(426) enhancing the mutagenic process of mucA in MRS-deficient cells, and shed light on another mechanism, the SSR- localized hypermutability, contributing to mucoid conversion in P

  10. Retroviral Vectors for Analysis of Viral Mutagenesis and Recombination

    PubMed Central

    Rawson, Jonathan M.O.; Mansky, Louis M.

    2014-01-01

    Retrovirus population diversity within infected hosts is commonly high due in part to elevated rates of replication, mutation, and recombination. This high genetic diversity often complicates the development of effective diagnostics, vaccines, and antiviral drugs. This review highlights the diverse vectors and approaches that have been used to examine mutation and recombination in retroviruses. Retroviral vectors for these purposes can broadly be divided into two categories: those that utilize reporter genes as mutation or recombination targets and those that utilize viral genes as targets of mutation or recombination. Reporter gene vectors greatly facilitate the detection, quantification, and characterization of mutants and/or recombinants, but may not fully recapitulate the patterns of mutagenesis or recombination observed in native viral gene sequences. In contrast, the detection of mutations or recombination events directly in viral genes is more biologically relevant but also typically more challenging and inefficient. We will highlight the advantages and disadvantages of the various vectors and approaches used as well as propose ways in which they could be improved. PMID:25254386

  11. Oligonucleotide-directed mutagenesis for precision gene editing.

    PubMed

    Sauer, Noel J; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W

    2016-02-01

    Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide-directed mutagenesis (ODM), one of the many tools of Cibus' Rapid Trait Development System (RTDS(™) ) technology, offers a rapid, precise and non-transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide-mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed. PMID:26503400

  12. Genome-wide transposon mutagenesis in pathogenic Leptospira species.

    PubMed

    Murray, Gerald L; Morel, Viviane; Cerqueira, Gustavo M; Croda, Julio; Srikram, Amporn; Henry, Rebekah; Ko, Albert I; Dellagostin, Odir A; Bulach, Dieter M; Sermswan, Rasana W; Adler, Ben; Picardeau, Mathieu

    2009-02-01

    Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans. PMID:19047402

  13. Efficient Gene Transfer and Targeted Mutagenesis in Fusobacterium nucleatum

    PubMed Central

    Haake, Susan Kinder; Yoder, Sean; Gerardo, Sharon Hunt

    2006-01-01

    Fusobacterium nucleatum is a Gram-negative anaerobe important in dental biofilm ecology and infectious diseases with significant societal impact. The lack of efficient genetic systems has hampered molecular analyses in this microorganism. We previously reported construction of a shuttle plasmid, pHS17, using the native fusobacterial plasmid pFN1 and an erythromycin resistance cassette. However, the host range of pHS17 was restricted to F. nucleatum, ATCC 10953 and the transformation efficiency was limited. This study was undertaken to improve genetic systems for molecular analysis in F. nucleatum. We identified a second F. nucleatum strain, ATCC 23726, which is transformed with improved efficiency compared to ATCC 10953. Two novel second generation pFN1-based shuttle plasmids, pHS23 and pHS30, were developed and enable transformation of ATCC 23726 at 6.2 x 104 and 1.5 x 106 transformants/microgram of plasmid DNA, respectively. The transformation efficiency of pHS30, which harbors a catP gene conferring resistance to chloramphenicol, was more than 1,000-fold greater than that of pHS17. The improved transformation efficiency facilitated disruption of the chromosomal rnr gene using a suicide plasmid pHS19, the first demonstration of targeted mutagenesis in F. nucleatum. These results provide significant advances in the development of systems for molecular analysis in F. nucleatum. PMID:16115683

  14. Targeted mutagenesis using CRISPR/Cas system in medaka

    PubMed Central

    Ansai, Satoshi; Kinoshita, Masato

    2014-01-01

    ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-based RNA-guided endonuclease (RGEN) has recently emerged as a simple and efficient tool for targeted genome editing. In this study, we showed successful targeted mutagenesis using RGENs in medaka, Oryzias latipes. Somatic and heritable mutations were induced with high efficiency at the targeted genomic sequence on the DJ-1 gene in embryos that had been injected with the single guide RNA (sgRNA) transcribed by a T7 promoter and capped RNA encoding a Cas9 nuclease. The sgRNAs that were designed for the target genomic sequences without the 5′ end of GG required by the T7 promoter induced the targeted mutations. This suggests that the RGEN can target any sequence adjacent to an NGG protospacer adjacent motif (PAM) sequence, which occurs once every 8 bp. The off-target alterations at 2 genomic loci harboring double mismatches in the 18-bp targeting sequences were induced in the RGEN-injected embryos. However, we also found that the off-target effects could be reduced by lower dosages of sgRNA. Taken together, our results suggest that CRISPR/Cas-mediated RGENs may be an efficient and flexible tool for genome editing in medaka. PMID:24728957

  15. Analysis of HIV-2 Vpx by modeling and insertional mutagenesis

    SciTech Connect

    Mahnke, Lisa A. . E-mail: lmahnke@im.wustl.edu; Belshan, Michael; Ratner, Lee . E-mail: lratner@im.wustl.edu

    2006-04-25

    Vpx facilitates HIV-2 nuclear localization by a poorly understood mechanism. We have compared Vpx to an NMR structure HIV-1 Vpr in a central helical domain and probed regions of Vpx by insertional mutagenesis. A predicted loop between helices two and three appears to be unique, overlapping with a known novel nuclear localization signal. Overall, Vpx was found to be surprisingly flexible, tolerating a series of large insertions. We found that insertion within the polyproline-containing C-terminus destabilizes nuclear localization, whereas mutating a second helix in the central domain disrupts viral packaging. Other insertional mutants in the predicted loop and in a linker region between the central domain and the C-terminus may be useful as sites of intramolecular tags as they could be packaged adequately and retained preintegration complex associated integration activity in a serum starvation assay. An unexpected result was found within a previously defined nuclear localization motif near aa 71. This mutant retained robust nuclear localization in a GFP fusion assay and was competent for preintegration complex associated nuclear import. In summary, we have modeled helical content in Vpx and assessed potential sites of intramolecular tags which may prove useful for protein-protein interactions studies.

  16. Lethal Mutagenesis of HIV with Mutagenic Nucleoside Analogs

    NASA Astrophysics Data System (ADS)

    Loeb, Lawrence A.; Essigmann, John M.; Kazazi, Farhad; Zhang, Jue; Rose, Karl D.; Mullins, James I.

    1999-02-01

    The human immunodeficiency virus (HIV) replicates its genome and mutates at exceptionally high rates. As a result, the virus is able to evade immunological and chemical antiviral agents. We tested the hypothesis that a further increase in the mutation rate by promutagenic nucleoside analogs would abolish viral replication. We evaluated deoxynucleoside analogs for lack of toxicity to human cells, incorporation by HIV reverse transcriptase, resistance to repair when incorporated into the DNA strand of an RNA\\cdot DNA hybrid, and mispairing at high frequency. Among the candidates tested, 5-hydroxydeoxycytidine (5-OH-dC) fulfilled these criteria. In seven of nine experiments, the presence of this analog resulted in the loss of viral replicative potential after 9-24 sequential passages of HIV in human CEM cells. In contrast, loss of viral replication was not observed in 28 control cultures passaged in the absence of the nucleoside analog, nor with other analogs tested. Sequence analysis of a portion of the HIV reverse transcriptase gene demonstrated a disproportionate increase in G -> A substitutions, mutations predicted to result from misincorporation of 5-OH-dC into the cDNA during reverse transcription. Thus, "lethal mutagenesis" driven by the class of deoxynucleoside analogs represented by 5-OH-dC could provide a new approach to treating HIV infections and, potentially, other viral infections.

  17. Functional mapping of Cre recombinase by pentapeptide insertional mutagenesis.

    PubMed

    Petyuk, Vladislav; McDermott, Jeffrey; Cook, Malcolm; Sauer, Brian

    2004-08-27

    Cre is a site-specific recombinase from bacteriophage P1. It is a member of the tyrosine integrase family and catalyzes reciprocal recombination between specific 34-bp sites called loxP. To analyze the structure-function relationships of this enzyme, we performed large scale pentapeptide insertional mutagenesis to generate insertions of five amino acids at random positions in the protein. The high density of insertion mutations into Cre allowed us to identify an unexpected degree of functional tolerance to insertions into the 4-5 beta-hairpin and into the loop between helices J and K (both of which contact the DNA in the minor groove) and also into helix A. The phenotypes of the majority of inserts allowed us to confirm a variety of predictions made on the basis of sequence conservation, known three-dimensional structure, and proposed catalytic mechanism. In particular, most insertions into conserved regions or secondary structure elements inactivated Cre, and most insertions located in nonconserved, unstructured regions preserved Cre activity. Less expectedly, the non-conserved and poorly structured loops and linkers between helices A-B, E-F, and M-N did not tolerate insertions, thus identifying these as critical regions for recombinase activity. We purified and characterized in vitro several representatives of these "unexpected" Cre insertion mutants. The role of those regions in the recombination process is discussed. PMID:15218019

  18. Particulate matter inhibits DNA repair and enhances mutagenesis.

    PubMed

    Mehta, Manju; Chen, Lung-Chi; Gordon, Terry; Rom, William; Tang, Moon-Shong

    2008-12-01

    Exposure to ambient air pollution has been associated with adverse health effects including lung cancer. A recent epidemiology study has established that each 10 microg/m3 elevation in long-term exposure to average PM2.5 ambient concentration was associated with approximately 8% of lung cancer mortality. The underlying mechanisms of how PM contributes to lung carcinogenesis, however, remain to be elucidated. We have recently found that transition metals such as nickel and chromium and oxidative stress induced lipid peroxidation metabolites such as aldehydes can greatly inhibit nucleotide excision repair (NER) and enhance carcinogen-induced mutations. Because PM is rich in metal and aldehyde content and can induce oxidative stress, we tested the effect of PM on DNA repair capacity in cultured human lung cells using in vitro DNA repair synthesis and host cell reactivation assays. We found that PM greatly inhibits NER for ultraviolet (UV) light and benzo(a)pyrene diol epoxide (BPDE) induced DNA damage in human lung cells. We further demonstrated that PM exposure can significantly increase both spontaneous and UV-induced mutagenesis. These results together suggest that the carcinogenicity of PM may act through its combined effect on suppression of DNA repair and enhancement of DNA replication errors. PMID:18804180

  19. X-ray structure at 1.75 resolution of a norovirus 3C protease linked to an active site-directed peptide inhibitor

    SciTech Connect

    Cooper, Jon; Coates, Leighton; Hussey, Robert

    2010-01-01

    Noroviruses are recognized universally as the most important cause of human epidemic non-bacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.75 resolution, following initial MAD phasing with a selenomethionine derivative. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, based on a 3C protease cleavage recognition sequences in the 200kDa polyprotein substrate, reacts covalently through its propenylethylester group (X) with the active site nucleophile, Cys 139. The 3C protease-inhibitor structure permits, for the first time, the identification of substrate recognition and binding groups and provides important new information for the development of antiviral prophylactics.

  20. Site directed immobilization of glucose-6-phosphate dehydrogenase via thiol-disulfide interchange: influence on catalytic activity of cysteines introduced at different positions.

    PubMed

    Simons, J R; Mosisch, M; Torda, A E; Hilterhaus, L

    2013-08-10

    This study shows the effect of site-directed enzyme immobilization upon the enzyme activity of covalently bound glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Immobilization points were introduced at sterically accessible sites in order to control the protein's orientation and twice as much activity was recovered in comparison to conventionally immobilized enzyme. Immobilization of G6PDH via genetically engineered cysteine provided a simple, but effective method to control the immobilization process. G6PDH variants with cysteine close to the active center (L218C), close to the dimer interface (D205C) as well as far from the active center (D453C) showed changes in activity and the efficacy of immobilization. PMID:23770076

  1. Applications of CRISPR/Cas9 technology for targeted mutagenesis, gene replacement and stacking of genes in higher plants.

    PubMed

    Luo, Ming; Gilbert, Brian; Ayliffe, Michael

    2016-07-01

    Mutagenesis continues to play an essential role for understanding plant gene function and, in some instances, provides an opportunity for plant improvement. The development of gene editing technologies such as TALENs and zinc fingers has revolutionised the targeted mutation specificity that can now be achieved. The CRISPR/Cas9 system is the most recent addition to gene editing technologies and arguably the simplest requiring only two components; a small guide RNA molecule (sgRNA) and Cas9 endonuclease protein which complex to recognise and cleave a specific 20 bp target site present in a genome. Target specificity is determined by complementary base pairing between the sgRNA and target site sequence enabling highly specific, targeted mutation to be readily engineered. Upon target site cleavage, error-prone endogenous repair mechanisms produce small insertion/deletions at the target site usually resulting in loss of gene function. CRISPR/Cas9 gene editing has been rapidly adopted in plants and successfully undertaken in numerous species including major crop species. Its applications are not restricted to mutagenesis and target site cleavage can be exploited to promote sequence insertion or replacement by recombination. The multiple applications of this technology in plants are described. PMID:27146973

  2. Photo-Oxidative Stress-Driven Mutagenesis and Adaptive Evolution on the Marine Diatom Phaeodactylum tricornutum for Enhanced Carotenoid Accumulation

    PubMed Central

    Yi, Zhiqian; Xu, Maonian; Magnusdottir, Manuela; Zhang, Yuetuan; Brynjolfsson, Sigurdur; Fu, Weiqi

    2015-01-01

    Marine diatoms have recently gained much attention as they are expected to be a promising resource for sustainable production of bioactive compounds such as carotenoids and biofuels as a future clean energy solution. To develop photosynthetic cell factories, it is important to improve diatoms for value-added products. In this study, we utilized UVC radiation to induce mutations in the marine diatom Phaeodactylum tricornutum and screened strains with enhanced accumulation of neutral lipids and carotenoids. Adaptive laboratory evolution (ALE) was also used in parallel to develop altered phenotypic and biological functions in P. tricornutum and it was reported for the first time that ALE was successfully applied on diatoms for the enhancement of growth performance and productivity of value-added carotenoids to date. Liquid chromatography-mass spectrometry (LC-MS) was utilized to study the composition of major pigments in the wild type P. tricornutum, UV mutants and ALE strains. UVC radiated strains exhibited higher accumulation of fucoxanthin as well as neutral lipids compared to their wild type counterpart. In addition to UV mutagenesis, P. tricornutum strains developed by ALE also yielded enhanced biomass production and fucoxanthin accumulation under combined red and blue light. In short, both UV mutagenesis and ALE appeared as an effective approach to developing desired phenotypes in the marine diatoms via electromagnetic radiation-induced oxidative stress. PMID:26426027

  3. Photo-Oxidative Stress-Driven Mutagenesis and Adaptive Evolution on the Marine Diatom Phaeodactylum tricornutum for Enhanced Carotenoid Accumulation.

    PubMed

    Yi, Zhiqian; Xu, Maonian; Magnusdottir, Manuela; Zhang, Yuetuan; Brynjolfsson, Sigurdur; Fu, Weiqi

    2015-10-01

    Marine diatoms have recently gained much attention as they are expected to be a promising resource for sustainable production of bioactive compounds such as carotenoids and biofuels as a future clean energy solution. To develop photosynthetic cell factories, it is important to improve diatoms for value-added products. In this study, we utilized UVC radiation to induce mutations in the marine diatom Phaeodactylum tricornutum and screened strains with enhanced accumulation of neutral lipids and carotenoids. Adaptive laboratory evolution (ALE) was also used in parallel to develop altered phenotypic and biological functions in P. tricornutum and it was reported for the first time that ALE was successfully applied on diatoms for the enhancement of growth performance and productivity of value-added carotenoids to date. Liquid chromatography-mass spectrometry (LC-MS) was utilized to study the composition of major pigments in the wild type P. tricornutum, UV mutants and ALE strains. UVC radiated strains exhibited higher accumulation of fucoxanthin as well as neutral lipids compared to their wild type counterpart. In addition to UV mutagenesis, P. tricornutum strains developed by ALE also yielded enhanced biomass production and fucoxanthin accumulation under combined red and blue light. In short, both UV mutagenesis and ALE appeared as an effective approach to developing desired phenotypes in the marine diatoms via electromagnetic radiation-induced oxidative stress. PMID:26426027

  4. Site-directed Fluorescence Labeling Reveals a Revised N-terminal Membrane Topology and Functional Periplasmic Residues in the Escherichia coli Cell Division Protein FtsK*

    PubMed Central

    Berezuk, Alison M.; Goodyear, Mara; Khursigara, Cezar M.

    2014-01-01

    In Escherichia coli, FtsK is a large integral membrane protein that coordinates chromosome segregation and cell division. The N-terminal domain of FtsK (FtsKN) is essential for division, and the C terminus (FtsKC) is a well characterized DNA translocase. Although the function of FtsKN is unknown, it is suggested that FtsK acts as a checkpoint to ensure DNA is properly segregated before septation. This may occur through modulation of protein interactions between FtsKN and other division proteins in both the periplasm and cytoplasm; thus, a clear understanding of how FtsKN is positioned in the membrane is required to characterize these interactions. The membrane topology of FtsKN was initially determined using site-directed reporter fusions; however, questions regarding this topology persist. Here, we report a revised membrane topology generated by site-directed fluorescence labeling. The revised topology confirms the presence of four transmembrane segments and reveals a newly identified periplasmic loop between the third and fourth transmembrane domains. Within this loop, four residues were identified that, when mutated, resulted in the appearance of cellular voids. High resolution transmission electron microscopy of these voids showed asymmetric division of the cytoplasm in the absence of outer membrane invagination or visible cell wall ingrowth. This uncoupling reveals a novel role for FtsK in linking cell envelope septation events and yields further evidence for FtsK as a critical checkpoint of cell division. The revised topology of FtsKN also provides an important platform for future studies on essential interactions required for this process. PMID:25002583

  5. Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban.

    PubMed Central

    Ludlam, C F; Arkin, I T; Liu, X M; Rothman, M S; Rath, P; Aimoto, S; Smith, S O; Engelman, D M; Rothschild, K J

    1996-01-01

    Phospholamban is a 52-amino acid residue membrane protein that regulates Ca(2+)-ATPase activity in the sarcoplasmic reticulum of cardiac muscle cells. The hydrophobic C-terminal 28 amino acid fragment of phospholamban (hPLB) anchors the protein in the membrane and may form part of a Ca(2+)-selective ion channel. We have used polarized attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy along with site-directed isotope labeling to probe the local structure of hPLB. The frequency and dichroism of the amide I and II bands appearing at 1658 cm-1 and 1544 cm-1, respectively, show that dehydrated and hydrated hPLB reconstituted into dimyristoylphosphatidycholine bilayer membranes is predominantly alpha-helical and has a net transmembrane orientation. Specific local secondary structure of hPLB was probed by incorporating 13C at two positions in the protein backbone. A small band seen near 1614 cm-1 is assigned to the amide I mode of the 13C-labeled amide carbonyl group(s). The frequency and dichroism of this band indicate that residues 39 and 46 are alpha-helical, with an axial orientation that is approximately 30 degrees relative to the membrane normal. Upon exposure to 2H2O (D2O), 30% of the peptide amide groups in hPLB undergo a slow deuterium/hydrogen exchange. The remainder of the protein, including the peptide groups of Leu-39 and Leu-42, appear inaccessible to exchange, indicating that most of the hPLB fragment is embedded in the lipid bilayer. By extending spectroscopic characterization of PLB to include hydrated, deuterated as well as site-directed isotope-labeled hPLB films, our results strongly support models of PLB that predict the existence of an alpha-helical hydrophobic region spanning the membrane domain. PMID:8785331

  6. Structure-Based Mutagenesis of the Substrate-Recognition Domain of Nrdp1/FLRF Identifies the Binding Site for the Receptor Tyrosine Kinase ErbB3

    SciTech Connect

    Bouyain,S.; Leahy, D.

    2007-01-01

    The E3 ubiquitin ligase neuregulin receptor degrading protein 1 (Nrdp1) mediates the ligand-independent degradation of the epidermal growth factor receptor family member ErbB3/HER3. By regulating cellular levels of ErbB3, Nrdp1 influences ErbB3-mediated signaling, which is essential for normal vertebrate development. Nrdp1 belongs to the tripartite or RBCC (RING, B-box, coiled-coil) family of ubiquitin ligases in which the RING domain is responsible for ubiquitin ligation and a variable C-terminal region mediates substrate recognition. We report here the 1.95 A crystal structure of the C-terminal domain of Nrdp1 and show that this domain is sufficient to mediate ErbB3 binding. Furthermore, we have used site-directed mutagenesis to map regions of the Nrdp1 surface that are important for interacting with ErbB3 and mediating its degradation in transfected cells. The ErbB3-binding site localizes to a region of Nrdp1 that is conserved from invertebrates to vertebrates, in contrast to ErbB3, which is only found in vertebrates. This observation suggests that Nrdp1 uses a common binding site to recognize its targets in different species.

  7. Mutagenesis of charged residues in a conserved sequence in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

    PubMed Central

    Bertrand, L; Vertommen, D; Feytmans, E; Di Pietro, A; Rider, M H; Hue, L

    1997-01-01

    Arg-136, Glu-137, Arg-138 and Arg-139 are conserved in all sequences of the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Their role was studied by site-directed mutagenesis. All the mutations had little, if any, effect on fructose-2,6-bisphosphatase activity. Mutations of Arg-136 and Glu-137 into Ala caused only minor modifications of phosphofructo-2-kinase activity. In contrast, mutation of Arg138 into Ala increased 280-fold the Km for fructose 6-phosphate of phosphofructo-2-kinase. Mutation of Arg-139 into Ala resulted in decreases in phosphofructo-2-kinase Vmax/Km for MgATP and fructose 6-phosphate 600-fold and 5000-fold respectively. Mutation of Arg-139 into Lys and Gln increased the Km of phosphofructo-2-kinase for MgATP (20-fold and 25-fold respectively) and for fructose 6-phosphate (8-fold and 13-fold), and the IC50 for MgADP (30-fold and 50-fold) and for magnesium citrate (7-fold and 25-fold). However, these two mutations did not affect nucleotide binding, as measured by quenching of intrinsic fluorescence. The changes in kinetic properties induced by mutations could not be attributed to structural changes. It is proposed that Arg-138 is involved in fructose 6-phosphate binding and that Arg-139 is probably involved in the stabilization of the transition state and so participates in catalysis. PMID:9032444

  8. Derivation of a growth hormone gene cassette for goat by mutagenesis of the corresponding bovine construct and its expression in Pichia pastoris.

    PubMed

    Reyes-Ruíz, Jorge M; Ascacio-Martínez, Jorge A; Barrera-Saldaña, Hugo A

    2006-07-01

    Recombinant bovine growth hormone (rbGH), a 191-aa polypeptide that affects animal growth and lactation, has been used for several years to increase milk production in dairy cattle. It has also been used in goats (Capra hircus) instead of their own hormone (chGH), which is still not available in the market. Since both hormones differ in only one amino acid residue, a strategy based on PCR mediated site-directed mutagenesis, was used to convert the bGH expression cassette harbored by an integration plasmid for Pichia pastoris into a chGH. Transformation by homologous recombination of Pichia pastoris GS115 strain with the linearized new plasmid resulted in transformants that, upon fermentation and induction with methanol, secreted a band with the expected size and immunoreactivity for GH. Production of total proteins secreted into culture medium (50 ml) was 20 microg/ml, of which 60% was chGH as judged by densitometry in SDS-PAGE. Its biological activity was confirmed in vitro when 3T3 pre-adipocytes exposed to the induced culture medium differentiated into adipocytes in cell culture. PMID:16799765

  9. Protein mutagenesis with monodispersity-based quality probing: selective inactivation of p53 degradation and DNA-binding properties of HPV E6 oncoprotein.

    PubMed

    Ristriani, Tutik; Nominé, Yves; Laurent, Cécile; Weiss, Etienne; Travé, Gilles

    2002-12-01

    Interpretation of protein mutagenesis experiments requires the ability to distinguish functionally relevant mutations from mutations affecting the structure. When a protein is expressed soluble in bacteria, properly folded mutants are expected to remain soluble whereas misfolded mutants should form insoluble aggregates. However, this rule may fail for proteins fused to highly soluble carrier proteins. In a previous study, we analysed the biophysical status of HPV oncoprotein E6 fused to the C-terminus of maltose-binding protein (MBP) and found that misfolded E6 moieties fused to MBP formed soluble aggregates of high molecular weight. By contrast, preparations of properly folded E6 fused to MBP were monodisperse. Here, we have used this finding to evaluate the quality of 19 MBP-fused E6 site-directed mutants by using a light scattering assay performed in a fluorimeter. This assay guided us to rule out structurally defective mutants and to obtain functionally relevant E6 mutants selectively altered for two molecular activities: degradation of tumour suppressor p53 and DNA recognition. PMID:12460759

  10. Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development

    PubMed Central

    Liu, Chia-Lin; Hung, Hui-Chen; Lo, Shou-Chen; Chiang, Ching-Hui; Chen, I-Jung; Hsu, John T.-A.; Hou, Ming-Hon

    2016-01-01

    Nucleoprotein (NP) is the most abundant type of RNA-binding viral protein in influenza A virus–infected cells and is necessary for viral RNA transcription and replication. Recent studies demonstrated that influenza NP is a valid target for antiviral drug development. The surface of the groove, covered with numerous conserved residues between the head and body domains of influenza A NP, plays a crucial role in RNA binding. To explore the mechanism by which NP binds RNA, we performed a series of site-directed mutagenesis in the RNA-binding groove, followed by surface plasmon resonance (SPR), to characterize the interactions between RNA and NP. Furthermore, a role of Y148 in NP stability and NP-RNA binding was evaluated. The aromatic residue of Y148 was found to stack with a nucleotide base. By interrupting the stacking interaction between Y148 and an RNA base, we identified an influenza virus NP inhibitor, (E, E)-1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione; this inhibitor reduced the NP’s RNA-binding affinity and hindered viral replication. Our findings will be useful for the development of new drugs that disrupt the interaction between RNA and viral NP in the influenza virus. PMID:26916998

  11. Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example

    PubMed Central

    Katzberg, Michael; Skorupa-Parachin, Nàdia; Gorwa-Grauslund, Marie-Françoise; Bertau, Martin

    2010-01-01

    The synthesis of pharmaceuticals and catalysts more and more relies on enantiopure chiral building blocks. These can be produced in an environmentally benign and efficient way via bioreduction of prochiral ketones catalyzed by dehydrogenases. A productive source of these biocatalysts is the yeast Saccharomyces cerevisiae, whose genome also encodes a reductase catalyzing the sequential reduction of the γ-diketone 2,5-hexanedione furnishing the diol (2S,5S)-hexanediol and the γ-hydroxyketone (5S)-hydroxy-2-hexanone in high enantio- as well as diastereoselectivity (ee and de >99.5%). This enzyme prefers NADPH as the hydrogen donating cofactor. As NADH is more stable and cheaper than NADPH it would be more effective if NADH could be used in cell-free bioreduction systems. To achieve this, the cofactor binding site of the dehydrogenase was altered by site-directed mutagenesis. The results show that the rational approach based on a homology model of the enzyme allowed us to generate a mutant enzyme having a relaxed cofactor preference and thus is able to use both NADPH and NADH. Results obtained from other mutants are discussed and point towards the limits of rationally designed mutants. PMID:20480039

  12. Specificity inversion of Ochrobactrum anthropi D-aminopeptidase to a D,D-carboxypeptidase with new penicillin binding activity by directed mutagenesis.

    PubMed

    Delmarcelle, Michaël; Boursoit, Marie-Caroline; Filée, Patrice; Baurin, Stéphane Lucius; Frère, Jean-Marie; Joris, Bernard

    2005-09-01

    The serine penicillin-recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to beta-lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D-aminopeptidase and of the Streptomyces R61 D,D-carboxypeptidase/transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D-aminopeptidase lost its original activity in favor of a new penicillin-binding activity. D-aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild-type enzyme. By a second step site-directed mutagenesis, the specificity of the Ochrobactrum anthropi D-aminopeptidase was inverted to a D,D-carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin-recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin-recognizing proteins are discussed. PMID:16131658

  13. Multiple mutagenesis of non-universal serine codons of the Candida rugosa LIP2 gene and biochemical characterization of purified recombinant LIP2 lipase overexpressed in Pichia pastoris.

    PubMed Central

    Lee, Guan-Chiun; Lee, Li-Chiun; Sava, Vasyl; Shaw, Jei-Fu

    2002-01-01

    The 17 non-universal serine codons (CTG) in the Candida rugosa LIP2 gene have been converted into universal serine codons (TCT) by overlap extension PCR-based multiple site-directed mutagenesis. An active recombinant LIP2 lipase was overexpressed in Pichia pastoris and secreted into the culture medium. The recombinant LIP2 showed distinguishing catalytic activities when compared with recombinant LIP4 and commercial C. rugosa lipase. The purified enzyme showed optimum activity at pH 7 and a broad temperature optimum in the range 30-50 degrees C. The enzyme retained 80% of residual activity after being heated at 70 degrees C for 10 min. Recombinant LIP2 demonstrated high esterase activity towards long-chain (C12-C16) p-nitrophenyl esters. Tributyrin was the preferred substrate among all triacylglycerols tested for lipolysis. Among cholesteryl esters, LIP2 showed highest lipolytic activity towards cholesteryl laurate. The esterification of myristic acid with alcohols of various chain lengths showed that the long-chain n-octadecanol (C18) was the preferred substrate. In contrast, the esterification of n-propanol with fatty acids of various chain lengths showed that the short-chain butyric acid was the best substrate. From comparative modelling analysis, it appears that several amino acid substitutions resulting in greater hydrophobicity in the substrate-binding site might play an important role in the substrate specificity of LIP2. PMID:12020350

  14. Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development.

    PubMed

    Liu, Chia-Lin; Hung, Hui-Chen; Lo, Shou-Chen; Chiang, Ching-Hui; Chen, I-Jung; Hsu, John T-A; Hou, Ming-Hon

    2016-01-01

    Nucleoprotein (NP) is the most abundant type of RNA-binding viral protein in influenza A virus-infected cells and is necessary for viral RNA transcription and replication. Recent studies demonstrated that influenza NP is a valid target for antiviral drug development. The surface of the groove, covered with numerous conserved residues between the head and body domains of influenza A NP, plays a crucial role in RNA binding. To explore the mechanism by which NP binds RNA, we performed a series of site-directed mutagenesis in the RNA-binding groove, followed by surface plasmon resonance (SPR), to characterize the interactions between RNA and NP. Furthermore, a role of Y148 in NP stability and NP-RNA binding was evaluated. The aromatic residue of Y148 was found to stack with a nucleotide base. By interrupting the stacking interaction between Y148 and an RNA base, we identified an influenza virus NP inhibitor, (E, E)-1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione; this inhibitor reduced the NP's RNA-binding affinity and hindered viral replication. Our findings will be useful for the development of new drugs that disrupt the interaction between RNA and viral NP in the influenza virus. PMID:26916998

  15. Multiplex Conditional Mutagenesis Using Transgenic Expression of Cas9 and sgRNAs.

    PubMed

    Yin, Linlin; Maddison, Lisette A; Li, Mingyu; Kara, Nergis; LaFave, Matthew C; Varshney, Gaurav K; Burgess, Shawn M; Patton, James G; Chen, Wenbiao

    2015-06-01

    Determining the mechanism of gene function is greatly enhanced using conditional mutagenesis. However, generating engineered conditional alleles is inefficient and has only been widely used in mice. Importantly, multiplex conditional mutagenesis requires extensive breeding. Here we demonstrate a system for one-generation multiplex conditional mutagenesis in zebrafish (Danio rerio) using transgenic expression of both cas9 and multiple single guide RNAs (sgRNAs). We describe five distinct zebrafish U6 promoters for sgRNA expression and demonstrate efficient multiplex biallelic inactivation of tyrosinase and insulin receptor a and b, resulting in defects in pigmentation and glucose homeostasis. Furthermore, we demonstrate temporal and tissue-specific mutagenesis using transgenic expression of Cas9. Heat-shock-inducible expression of cas9 allows temporal control of tyr mutagenesis. Liver-specific expression of cas9 disrupts insulin receptor a and b, causing fasting hypoglycemia and postprandial hyperglycemia. We also show that delivery of sgRNAs targeting ascl1a into the eye leads to impaired damage-induced photoreceptor regeneration. Our findings suggest that CRISPR/Cas9-based conditional mutagenesis in zebrafish is not only feasible but rapid and straightforward. PMID:25855067

  16. Extinction of hepatitis C virus by ribavirin in hepatoma cells involves lethal mutagenesis.

    PubMed

    Ortega-Prieto, Ana M; Sheldon, Julie; Grande-Pérez, Ana; Tejero, Héctor; Gregori, Josep; Quer, Josep; Esteban, Juan I; Domingo, Esteban; Perales, Celia

    2013-01-01

    Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV. PMID:23976977

  17. Genome-Wide Transposon Mutagenesis in Saccharomyces cerevisiae and Candida albicans

    PubMed Central

    Xu, Tao; Bharucha, Nikë; Kumar, Anuj

    2016-01-01

    Transposon mutagenesis is an effective method for generating large sets of random mutations in target DNA, with applicability toward numerous types of genetic screens in prokaryotes, single-celled eukaryotes, and metazoans alike. Relative to methods of random mutagenesis by chemical/UV treatment, transposon insertions can be easily identified in mutants with phenotypes of interest. The construction of transposon insertion mutants is also less labor-intensive on a genome-wide scale than methods for targeted gene replacement, although transposon insertions are not precisely targeted to a specific residue, and thus coverage of the target DNA can be problematic. The collective advantages of transposon mutagenesis have been well demonstrated in studies of the budding yeast Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans, as transposon mutagenesis has been used extensively for phenotypic screens in both yeasts. Consequently, we present here protocols for the generation and utilization of transposon-insertion DNA libraries in S. cerevisiae and C. albicans. Specifically, we present methods for the large-scale introduction of transposon insertion alleles in a desired strain of S. cerevisiae. Methods are also presented for transposon mutagenesis of C. albicans, encompassing both the construction of the plasmid-based transposon-mutagenized DNA library and its introduction into a desired strain of Candida. In total, these methods provide the necessary information to implement transposon mutagenesis in yeast, enabling the construction of large sets of identifiable gene disruption mutations, with particular utility for phenotypic screening in nonstandard genetic backgrounds. PMID:21815095

  18. Exploring purine N7 interactions via atomic mutagenesis: The group I ribozyme as a case study

    PubMed Central

    Forconi, Marcello; Benz-Moy, Tara; Gleitsman, Kristin Rule; Ruben, Eliza; Metz, Clyde; Herschlag, Daniel

    2012-01-01

    Atomic mutagenesis has emerged as a powerful tool to unravel specific interactions in complex RNA molecules. An early extensive study of analogs of the exogenous guanosine nucleophile in group I intron self-splicing by Bass and Cech demonstrated structure–function relationships analogous to those seen for protein ligands and provided strong evidence for a well-formed substrate binding site made of RNA. Subsequent functional and structural studies have confirmed these interacting sites and extended our understanding of them, with one notable exception. Whereas 7-methyl guanosine did not affect reactivity in the original study, a subsequent study revealed a deleterious effect of the seemingly more conservative 7-deaza substitution. Here we investigate this paradox, studying these and other analogs with the more thoroughly characterized ribozyme derived from the Tetrahymena group I intron. We found that the 7-deaza substitution lowers binding by ∼20-fold, relative to the cognate exogenous guanosine nucleophile, whereas binding and reaction with 7-methyl and 8-aza-7-deaza substitutions have no effect. These and additional results suggest that there is no functionally important contact between the N7 atom of the exogenous guanosine and the ribozyme. Rather, they are consistent with indirect effects introduced by the N7 substitution on stacking interactions and/or solvation that are important for binding. The set of analogs used herein should be valuable in deciphering nucleic acid interactions and how they change through reaction cycles for other RNAs and RNA/protein complexes. PMID:22543863

  19. Transposon mutagenesis of Campylobacter jejuni identifies a bipartite energy taxis system required for motility.

    PubMed

    Hendrixson, D R; Akerley, B J; DiRita, V J

    2001-04-01

    Campylobacter jejuni constitutes the leading cause of bacterial gastroenteritis in the United States and a major cause of diarrhoea worldwide. Little is known about virulence mechanisms in this organism because of the scarcity of suitable genetic tools. We have developed an efficient system of in vitro transposon mutagenesis using a mariner-based transposon and purified mariner transposase. Through in vitro transposition of C. jejuni chromosomal DNA followed by natural transformation of the transposed DNA, large random transposon mutant libraries consisting of approximately 16 000 individual mutants were generated. The first genetic screen of C. jejuni using a transposon-generated mutant library identified 28 mutants defective for flagellar motility, one of the few known virulence determinants of this pathogen. We developed a second genetic system, which allows for the construction of defined chromosomal deletions in C. jejuni, and demonstrated the requirement of sigma28 and sigma54 for motility. In addition, we show that sigma28 is involved in the transcription of flaA and that sigma54 is required for transcription of three other flagellar genes, flaB and flgDE. We also identified two previously uncharacterized genes required for motility encoding proteins that we call CetA and CetB, which mediate energy taxis responses. Through our analysis of the Cet proteins, we propose a unique mechanism for sensing energy levels and mediating energy taxis in C. jejuni. PMID:11298288

  20. Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis.

    PubMed

    March, H Nikki; Rust, Alistair G; Wright, Nicholas A; ten Hoeve, Jelle; de Ridder, Jeroen; Eldridge, Matthew; van der Weyden, Louise; Berns, Anton; Gadiot, Jules; Uren, Anthony; Kemp, Richard; Arends, Mark J; Wessels, Lodewyk F A; Winton, Douglas J; Adams, David J

    2011-12-01

    The evolution of colorectal cancer suggests the involvement of many genes. To identify new drivers of intestinal cancer, we performed insertional mutagenesis using the Sleeping Beauty transposon system in mice carrying germline or somatic Apc mutations. By analyzing common insertion sites (CISs) isolated from 446 tumors, we identified many hundreds of candidate cancer drivers. Comparison to human data sets suggested that 234 CIS-targeted genes are also dysregulated in human colorectal cancers. In addition, we found 183 CIS-containing genes that are candidate Wnt targets and showed that 20 CISs-containing genes are newly discovered modifiers of canonical Wnt signaling. We also identified mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included those encoding members of the FGF signaling cascade. Some 70 genes had co-occurrence of CIS pairs, clustering into 38 sub-networks that may regulate tumor development. PMID:22057237

  1. An Assessment of Heavy Ion Irradiation Mutagenesis for Reverse Genetics in Wheat (Triticum aestivum L.)

    PubMed Central

    Fitzgerald, Timothy L.; Powell, Jonathan J.; Stiller, Jiri; Weese, Terri L.; Abe, Tomoko; Zhao, Guangyao; Jia, Jizeng; McIntyre, C. Lynne; Li, Zhongyi; Manners, John M.; Kazan, Kemal

    2015-01-01

    Reverse genetic techniques harnessing mutational approaches are powerful tools that can provide substantial insight into gene function in plants. However, as compared to diploid species, reverse genetic analyses in polyploid plants such as bread wheat can present substantial challenges associated with high levels of sequence and functional similarity amongst homoeologous loci. We previously developed a high-throughput method to identify deletions of genes within a physically mutagenized wheat population. Here we describe our efforts to combine multiple homoeologous deletions of three candidate disease susceptibility genes (TaWRKY11, TaPFT1 and TaPLDß1). We were able to produce lines featuring homozygous deletions at two of the three homoeoloci for all genes, but this was dependent on the individual mutants used in crossing. Intriguingly, despite extensive efforts, viable lines possessing homozygous deletions at all three homoeoloci could not be produced for any of the candidate genes. To investigate deletion size as a possible reason for this phenomenon, we developed an amplicon sequencing approach based on synteny to Brachypodium distachyon to assess the size of the deletions removing one candidate gene (TaPFT1) in our mutants. These analyses revealed that genomic deletions removing the locus are relatively large, resulting in the loss of multiple additional genes. The implications of this work for the use of heavy ion mutagenesis for reverse genetic analyses in wheat are discussed. PMID:25719507

  2. High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase.

    PubMed

    Gajula, Kiran S; Huwe, Peter J; Mo, Charlie Y; Crawford, Daniel J; Stivers, James T; Radhakrishnan, Ravi; Kohli, Rahul M

    2014-09-01

    Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9-11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function. PMID:25064858

  3. High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase

    PubMed Central

    Gajula, Kiran S.; Huwe, Peter J.; Mo, Charlie Y.; Crawford, Daniel J.; Stivers, James T.; Radhakrishnan, Ravi; Kohli, Rahul M.

    2014-01-01

    Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9–11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function. PMID:25064858

  4. Role of electrostatic potential in the in silico prediction of molecular bioactivation and mutagenesis.

    PubMed

    Ford, Kevin A

    2013-04-01

    Electrostatic potential (ESP) is a useful physicochemical property of a molecule that provides insights into inter- and intramolecular associations, as well as prediction of likely sites of electrophilic and nucleophilic metabolic attack. Knowledge of sites of metabolic attack is of paramount importance in DMPK research since drugs frequently fail in clinical trials due to the formation of bioactivated metabolites which are often difficult to measure experimentally due to their reactive nature and relatively short half-lives. Computational chemistry methods have proven invaluable in recent years as a means to predict and study bioactivated metabolites without the need for chemical syntheses, or testing on experimental animals. Additional molecular properties (heat of formation, heat of solvation and E(LUMO) - E(HOMO)) are discussed in this paper as complementary indicators of the behavior of metabolites in vivo. Five diverse examples are presented (acetaminophen, aniline/phenylamines, imidacloprid, nefazodone and vinyl chloride) which illustrate the utility of this multidimensional approach in predicting bioactivation, and in each case the predicted data agreed with experimental data described in the scientific literature. A further example of the usefulness of calculating ESP, in combination with the molecular properties mentioned above, is provided by an examination of the use of these parameters in providing an explanation for the sites of nucleophilic attack of the nucleic acid cytosine. Exploration of sites of nucleophilic attack of nucleic acids is important as adducts of DNA have the potential to result in mutagenesis. PMID:23323940

  5. Catalytic improvement and structural analysis of atrazine chlorohydrolase by site-saturation mutagenesis.

    PubMed

    Guo, Yuan; Zhao, Panjie; Zhang, Wenhao; Li, Xiaolong; Chen, Xiwen; Chen, Defu

    2016-07-01

    To improve the catalytic activity of atrazine chlorohydrolase (AtzA), amino acid residues involved in substrate binding (Gln71) and catalytic efficiency (Val12, Ile393, and Leu395) were targeted to generate site-saturation mutagenesis libraries. Seventeen variants were obtained through Haematococcus pluvialis-based screening, and their specific activities were 1.2-5.2-fold higher than that of the wild type. For these variants, Gln71 tended to be substituted by hydrophobic amino acids, Ile393 and Leu395 by polar ones, especially arginine, and Val12 by alanine, respectively. Q71R and Q71M significantly decreased the Km by enlarging the substrate-entry channel and affecting N-ethyl binding. Mutations at sites 393 and 395 significantly increased the kcat/Km, probably by improving the stability of the dual β-sheet domain and the whole enzyme, owing to hydrogen bond formation. In addition, the contradictory relationship between the substrate affinity improvement by Gln71 mutation and the catalytic efficiency improvement by the dual β-sheet domain modification was discussed. PMID:27010688

  6. Marinomonas mediterranea MMB-1 Transposon Mutagenesis: Isolation of a Multipotent Polyphenol Oxidase Mutant

    PubMed Central

    Solano, Francisco; Lucas-Elío, Patricia; Fernández, Eva; Sanchez-Amat, Antonio

    2000-01-01

    Marinomonas mediterranea is a melanogenic marine bacterium expressing a multifunctional polyphenol oxidase (PPO) able to oxidize substrates characteristic for laccases and tyrosinases, as well as produce a classical tyrosinase. A new and quick method has been developed for screening laccase activity in culture plates to detect mutants differentially affected in this PPO activity. Transposon mutagenesis has been applied for the first time to M. mediterranea by using different minitransposons loaded in R6K-based suicide delivery vectors mobilizable by conjugation. Higher frequencies of insertions were obtained by using mini-Tn10 derivatives encoding kanamycin or gentamycin resistance. After applying this protocol, a multifunctional PPO-negative mutant was obtained. By using the antibiotic resistance cassette as a marker, flanking regions were cloned. Then the wild-type gene was amplified by PCR and was cloned and sequenced. This is the first report on cloning and sequencing of a gene encoding a prokaryotic enzyme with laccase activity. The deduced amino acid sequence shows the characteristic copper-binding sites of other blue copper proteins, including fungal laccases. In addition, it shows some extra copper-binding sites that might be related to its multipotent enzymatic capability. PMID:10850991

  7. Structure-based design of combinatorial mutagenesis libraries

    PubMed Central

    Verma, Deeptak; Grigoryan, Gevorg; Bailey-Kellogg, Chris

    2015-01-01

    The development of protein variants with improved properties (thermostability, binding affinity, catalytic activity, etc.) has greatly benefited from the application of high-throughput screens evaluating large, diverse combinatorial libraries. At the same time, since only a very limited portion of sequence space can be experimentally constructed and tested, an attractive possibility is to use computational protein design to focus libraries on a productive portion of the space. We present a general-purpose method, called “Structure-based Optimization of Combinatorial Mutagenesis” (SOCoM), which can optimize arbitrarily large combinatorial mutagenesis libraries directly based on structural energies of their constituents. SOCoM chooses both positions and substitutions, employing a combinatorial optimization framework based on library-averaged energy potentials in order to avoid explicitly modeling every variant in every possible library. In case study applications to green fluorescent protein, β-lactamase, and lipase A, SOCoM optimizes relatively small, focused libraries whose variants achieve energies comparable to or better than previous library design efforts, as well as larger libraries (previously not designable by structure-based methods) whose variants cover greater diversity while still maintaining substantially better energies than would be achieved by representative random library approaches. By allowing the creation of large-scale combinatorial libraries based on structural calculations, SOCoM promises to increase the scope of applicability of computational protein design and improve the hit rate of discovering beneficial variants. While designs presented here focus on variant stability (predicted by total energy), SOCoM can readily incorporate other structure-based assessments, such as the energy gap between alternative conformational or bound states. PMID:25611189

  8. Germinal cell mutagenesis in specially designed maize genotypes.

    PubMed Central

    Plewa, M J; Wagner, E D

    1981-01-01

    We have used three inbreds of Zea mays in our in situ and laboratory studies in environmental mutagenesis. Inbred W22 plants homozygous for wx-C were used in a study to detect the possible mutagenic properties of 32 pesticides or combination of pesticides under modern agricultural conditions. The large numbers of pollen grains analyzed and the ease in detecting mutant pollen grains enabled us to treat the experimental plants with field recommended rates of pesticides. In a current study we are evaluating the possible mutagenicity of Chicago municipal sewage sludge. We are measuring the frequency of mutant pollen grains in inbred M14 at both the wx-C and wx-90 heteroalleles. These plants were exposed to various concentrations of municipal sewage sludge under field conditions. We have inbred Early-Early Synthetic for five generations and tested this inbred with known mutagens. Early-Early Synthetic is a rapidly maturing inbred growing from kernel to anthesis in approximately 4 weeks and attaining a height of approximately 50 cm. Plants of this inbred have been chronically treated with ethylmethanesulfonate (EMS) or maleic hydrazide (MH) under laboratory conditions and forward mutation at the wx locus was measured in the pollen grains. EMS and MH were mutagenic at concentrations of 1 microM and 10 nM, respectively. The concentrations of EMS and MH were calibrated in Early-Early Synthetic to a linear increase in the frequency of forward mutant pollen grains. The construction of a maize monitor for environmental mutagens is currently in progress. This assay will measure forward or reverse mutation at the wx locus in pollen grains, point mutation in somatic cells and will incorporate a cytogenetic endpoint in root-tip cells. Images FIGURE 2. FIGURE 3. FIGURE 5. PMID:6780335

  9. Synthetic approach to stop-codon scanning mutagenesis.

    PubMed

    Nie, Lihua; Lavinder, Jason J; Sarkar, Mohosin; Stephany, Kimberly; Magliery, Thomas J

    2011-04-27

    A general combinatorial mutagenesis strategy using common dimethoxytrityl-protected mononucleotide phosphoramidites and a single orthogonally protected trinucleotide phosphoramidite (Fmoc-TAG; Fmoc = 9-fluorenylmethoxycarbonyl) was developed to scan a gene with the TAG amber stop codon with complete synthetic control. In combination with stop-codon suppressors that insert natural (e.g., alanine) or unnatural (e.g., p-benzoylphenylalanine, Bpa) amino acids, a single DNA library can be used to incorporate different amino acids for diverse purposes. Here, we scanned TAG codons through part of the gene for a model four-helix bundle protein, Rop, which regulates the copy number of ColE1 plasmids. Alanine was incorporated into Rop for mapping its binding site using an in vivo activity screen, and subtle but important differences from in vitro gel-shift studies of Rop function are evident. As a test, Bpa was incorporated using a Phe14 amber mutant isolated from the scanning library. Surprisingly, Phe14Bpa-Rop is weakly active, despite the critical role of Phe14 in Rop activity. Bpa is a photoaffinity label unnatural amino acid that can form covalent bonds with adjacent molecules upon UV irradiation. Irradiation of Phe14Bpa-Rop, which is a dimer in solution like wild-type Rop, results in covalent dimers, trimers, and tetramers. This suggests that Phe14Bpa-Rop weakly associates as a tetramer in solution and highlights the use of Bpa cross-linking as a means of trapping weak and transient interactions. PMID:21452871

  10. Genetic Regulation of Charged Particle Mutagenesis in Human Cells

    NASA Technical Reports Server (NTRS)

    Kronenberg, Amy; Gauny, S.; Cherbonnel-Lasserre, C.; Liu, W.; Wiese, C.

    1999-01-01

    Our studies use a series of syngeneic, and where possible, isogenic human B-lymphoblastoid cell lines to assess the genetic factors that modulate susceptibility apoptosis and their impact on the mutagenic risks of low fluence exposures to 1 GeV Fe ions and 55 MeV protons. These ions are representative of the types of charged particle radiation that are of particular significance for human health in the space radiation environment. The model system employs cell lines derived from the male donor WIL-2. These cells have a single X chromosome and they are hemizygous for one mutation marker, hypoxanthine phosphoribosyltransferase (HPRT). TK6 and WTK1 cells were each derived from descendants of WIL-2 and were each selected as heterozygotes for a second mutation marker, the thymidine kinase (TK) gene located on chromosome 17q. The HPRT and TK loci can detect many different types of mutations, from single basepair substitutions up to large scale loss of heterozygosity (LOH). The single expressing copy of TK in the TK6 and WTKI cell lines is found on the same copy of chromosome 17, and this allele can be identified by a restriction fragment length polymorphism (RFLP) identified when high molecular weight DNA is digested by the SacI restriction endonuclease and hybridized against the cDNA probe for TK. A large series of polymorphic linked markers has been identified that span more than 60 cM of DNA (approx. 60 megabasepairs) and distinguish the copy of chromosome 17 bearing the initially active TK allele from the copy of chromosome 17 bearing the silent TK allele in both TK6 and WTKI cells. TK6 cells express normal p53 protein while WTKI cells express homozygous mutant p53. Expression of mutant p53 can increase susceptibility to x-ray-induced mutations. It's been suggested that the increased mutagenesis in p53 mutant cells might be due to reduced apoptosis.

  11. Radiation mutagenesis from molecular and genetic points of view

    SciTech Connect

    Chen, D.J.C.; Park, M.S.; Okinaka, R.T.; Jaberaboansari, A.

    1993-02-01

    An important biological effect of ionizing radiation on living organisms is mutation induction. Mutation is also a primary event in the etiology of cancer. The chain events, from induction of DNA damage by ionizing radiation to processing of these damages by the cellular repair/replication machinery, that lead to mutation are not well understood. The development of quantitative methods for measuring mutation-induction, such as the HPRT system, in cultured mammalian cells has provided an estimate of the mutagenic effects of