scholarly journals Mutation of High-Affinity Methionine Permease Contributes to Selenomethionyl Protein Production in Saccharomyces cerevisiae

2010 ◽  
Vol 76 (19) ◽  
pp. 6351-6359 ◽  
Author(s):  
Toshihiko Kitajima ◽  
Yasunori Chiba ◽  
Yoshifumi Jigami
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Methylotrophic Yeast ◽  
Culture Conditions ◽  
Crystallographic Analysis ◽  
Gene Encoding ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Affinity ◽  
Standard Culture ◽  
Resistant Mutants

ABSTRACT The production of selenomethionine (SeMet) derivatives of recombinant proteins allows phase determination by single-wavelength or multiwavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic proteins that require posttranslational modification, the toxic effects of SeMet often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast Pichia pastoris that is resistant to both SeMet and selenate and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to SeMet by the SMR-94 strain remains unclear. Here, we report the characterization of SeMet-resistant mutants of Saccharomyces cerevisiae and the identification of a mutant allele of the MUP1 gene encoding high-affinity methionine permease, which confers SeMet resistance. Although the total methionine uptake by the mup1 mutant (the SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into the overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture conditions, the mup1 mutant showed higher productivity of the SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude that the mup1 mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography.

scholarly journals Genome-wide screen of Saccharomyces cerevisiae null allele strains identifies genes involved in selenomethionine resistance

2008 ◽  
Vol 105 (46) ◽  
pp. 17682-17687 ◽  
Author(s):  
Jessica Bockhorn ◽  
Bharvi Balar ◽  
Dongming He ◽  
Eden Seitomer ◽  
Paul R. Copeland ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Null Allele ◽  
Cost Effective ◽  
Anomalous Dispersion ◽  
Gene Encoding ◽  
X Ray ◽  
X Ray Crystallography ◽  
Genome Wide ◽  
Cost Effective Method ◽  
Strain Collection

Selenomethionine (SeMet) is a potentially toxic amino acid, and yet it is a valuable tool in the preparation of labeled proteins for multiwavelength anomalous dispersion or single-wavelength anomalous dispersion phasing in X-ray crystallography. The mechanism by which high levels of SeMet exhibits its toxic effects in eukaryotic cells is not fully understood. Attempts to use Saccharomyces cerevisiae for the preparation of fully substituted SeMet proteins for X-ray crystallography have been limited. A screen of the viable S. cerevisiae haploid null allele strain collection for resistance to SeMet was performed. Deletion of the CYS3 gene encoding cystathionine gamma-lyase resulted in the highest resistance to SeMet. In addition, deletion of SSN2 resulted in both increased resistance to SeMet as well as reduced levels of Cys3p. A methionine auxotrophic strain lacking CYS3 was able to grow in media with SeMet as the only source of Met, achieving essentially 100% occupancy in total proteins. The CYS3 deletion strain provides advantages for an easy and cost-effective method to prepare SeMet-substituted protein in yeast and perhaps other eukaryotic systems.

scholarly journals Cloning, expression, purification, crystallization and preliminary crystallographic analysis of 5-aminolaevulinic acid dehydratase fromBacillus subtilis

2010 ◽  
Vol 66 (9) ◽  
pp. 1053-1055 ◽  
Author(s):  
Qianda Lu ◽  
Jinming Ma ◽  
Hui Rong ◽  
Jun Fan ◽  
Ye Yuan ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Gene Encoding ◽  
Data Set ◽  
X Ray ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Strain Bl21 ◽  
Aminolaevulinic Acid Dehydratase

5-Aminolaevulinic acid dehydratase (ALAD), a crucial enzyme in the biosynthesis of tetrapyrrole, catalyses the condensation of two 5-aminolaevulinic acid (ALA) molecules to form porphobilinogen (PBG). The gene encoding ALAD was amplified from genomic DNA ofBacillus subtilisand the protein was overexpressed inEscherichia colistrain BL21 (DE3). The protein was purified and crystallized with an additional MGSSHHHHHHSSGLVPRGSH– tag at the N-terminus of the target protein. Diffraction-quality single crystals were obtained by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected at a resolution of 2.7 Å.

scholarly journals O2 and Reactive Oxygen Species Detoxification Complex, Composed of O2-Responsive NADH:Rubredoxin Oxidoreductase-Flavoprotein A2-Desulfoferrodoxin Operon Enzymes, Rubperoxin, and Rubredoxin, in Clostridium acetobutylicum

2009 ◽  
Vol 75 (4) ◽  
pp. 1021-1029 ◽  
Author(s):  
Shinji Kawasaki ◽  
Yu Sakai ◽  
Tohru Takahashi ◽  
Ippei Suzuki ◽  
Youichi Niimura
Keyword(s):  
Clostridium Acetobutylicum ◽  
Nadh Oxidase ◽  
Culture Conditions ◽  
Superoxide Reductase ◽  
Obligate Anaerobe ◽  
Gene Encoding ◽  
High Affinity ◽  
Nadh Peroxidase ◽  
Genes Encoding ◽  
Nadh Oxidase Activity

ABSTRACT Clostridium acetobutylicum, an obligate anaerobe, grows normally under continuous-O2-flow culture conditions, where the cells consume O2 proficiently. An O2-responsive NADH:rubredoxin oxidoreductase operon composed of three genes (nror, fprA2, and dsr), encoding NROR, functionally uncharacterized flavoprotein A2 (FprA2), and the predicted superoxide reductase desulfoferrodoxin (Dsr), has been proposed to participate in defense against O2 stress. To functionally characterize these proteins, native NROR from C. acetobutylicum, recombinant NROR (rNROR), FprA2, Dsr, and rubredoxin (Rd) expressed in Escherichia coli were purified. Purified native NROR and rNROR both exhibited weak H2O2-forming NADH oxidase activity that was slightly activated by Rd. A mixture of NROR, Rd, and FprA2 functions as an efficient H2O-forming NADH oxidase with a high affinity for O2 (the Km for O2 is 2.9 � 0.4 μM). A mixture of NROR, Rd, and Dsr functions as an NADH-dependent O2 − reductase. A mixture of NROR, Rd, and rubperoxin (Rpr, a rubrerythrin homologue) functions as an inefficient H2O-forming NADH oxidase but an efficient NADH peroxidase with a low affinity for O2 and a high affinity for H2O2 (the Km s for O2 and H2O2 are 303 � 39 μM and ≤1 μM, respectively). A gene encoding Rd is dicistronically transcribed with a gene encoding a glutaredoxin (Gd) homologue, and the expression levels of the genes encoding Gd and Rd were highly upregulated upon exposure to O2. Therefore, nror operon enzymes, together with Rpr, efficiently function to scavenge O2, O2 −, and H2O2 by using an O2-responsive rubredoxin as a common electron carrier protein.

scholarly journals High-Affinity IgE Recognition of a Conformational Epitope of the Major Respiratory Allergen Phl p 2 As Revealed by X-Ray Crystallography

2009 ◽  
Vol 182 (4) ◽  
pp. 2141-2151 ◽  
Author(s):  
Sivaraman Padavattan ◽  
Sabine Flicker ◽  
Tilman Schirmer ◽  
Christoph Madritsch ◽  
Stefanie Randow ◽  
...  
Keyword(s):  
Conformational Epitope ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Affinity

scholarly journals MBP-binding DARPins facilitate the crystallization of an MBP fusion protein

2018 ◽  
Vol 74 (9) ◽  
pp. 549-557 ◽  
Author(s):  
Rajesh Gumpena ◽  
George T. Lountos ◽  
David S. Waugh
Keyword(s):  
Fusion Protein ◽  
Catalytic Domain ◽  
Fusion Partner ◽  
High Quality ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Affinity ◽  
Repeat Proteins ◽  
Dual Specificity ◽  
Ankyrin Repeat Proteins

The production of high-quality crystals is the main bottleneck in determining the structures of proteins using X-ray crystallography. In addition to being recognized as a very effective solubility-enhancing fusion partner,Escherichia colimaltose-binding protein (MBP) has also been successfully employed as a `fixed-arm' crystallization chaperone in more than 100 cases. Here, it is reported that designed ankyrin-repeat proteins (DARPins) that bind with high affinity to MBP can promote the crystallization of an MBP fusion protein when the fusion protein alone fails to produce diffraction-quality crystals. As a proof of principle, three different co-crystal structures of MBP fused to the catalytic domain of human dual-specificity phosphatase 1 in complex with DARPins are reported.

A New, Simple, High-Affinity Glycosidase Inhibitor:  Analysis of Binding through X-ray Crystallography, Mutagenesis, and Kinetic Analysis

2000 ◽  
Vol 122 (17) ◽  
pp. 4229-4230 ◽  
Author(s):  
Spencer J. Williams ◽  
Valerie Notenboom ◽  
Jacqueline Wicki ◽  
David R. Rose ◽  
Stephen G. Withers
Keyword(s):  
Kinetic Analysis ◽  
X Ray ◽  
X Ray Crystallography ◽  
High Affinity ◽  
Inhibitor Analysis ◽  
Glycosidase Inhibitor

scholarly journals Synthesis, Characterization, and Bioactivity of Schiff Bases and TheirCd2+,Zn2+,Cu2+, andNi2+Complexes Derived from Chloroacetophenone Isomers with S-Benzyldithiocarbazate and the X-Ray Crystal Structure of S-Benzyl-β-N-(4-chlorophenyl)methylenedithiocarbazate

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Mohammed Khaled bin Break ◽  
M. Ibrahim M. Tahir ◽  
Karen A. Crouse ◽  
Teng-Jin Khoo
Keyword(s):  
Crystal Structure ◽  
Phenyl Ring ◽  
Crystallographic Analysis ◽  
Gram Negative Bacteria ◽  
Schiff Base Ligands ◽  
Gram Negative ◽  
X Ray ◽  
X Ray Crystallography ◽  
Gram Positive Bacteria ◽  
Antimicrobial Assay

Two bidentate Schiff base ligands having nitrogen sulphur donor sequence were derived from the condensation of S-benzyldithiocarbazate (SBDTC) with 2-chloroacetophenone and 4-chloroacetophenone to give S-benzyl-β-N-(2-chlorophenyl)methylenedithiocarbazate (NS2) and S-benzyl-β-N-(4-chlorophenyl)methylenedithiocarbazate (NS4) isomers. Each of the ligands was then chelated with Cd2+, Zn2+, Cu2+, and Ni2+. The compounds were characterized via IR spectroscopy and melting point while the structure of NS4 was revealed via X-ray crystallography. Finally, the compounds were screened for antimicrobial activity to investigate the effect that is brought by the introduction of the chlorine atom to the benzene ring. X-ray crystallographic analysis showed that the structure of NS4 is planar with a phenyl ring that is nearly perpendicular to the rest of the molecules. The qualitative antimicrobial assay results showed that NS4 and its complexes lacked antifungal activity while Gram-positive bacteria were generally inhibited more strongly than Gram-negative bacteria. Furthermore, NS4 metal complexes were inhibited more strongly than the ligand while the opposite was seen with NS2 ligand and its complexes due to the partial solubility in dimethyl sulfoxide (DMSO). It was concluded that generally NS2 derivatives have higher bioactivity than that of NS4 derivatives and that the Cd complexes of both ligands have pronounced activity specifically onK. rhizophila.

scholarly journals Expression, crystallization and preliminary X-ray crystallographic analysis of DNA-directed RNA polymerase subunit L fromThermococcus onnurineusNA1

2014 ◽  
Vol 70 (5) ◽  
pp. 639-642
Author(s):  
Thien-Hoang Ho ◽  
Myoung-Ki Hong ◽  
Ho-Phuong-Thuy Ngo ◽  
Lin-Woo Kang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Multiprotein Complex ◽  
Hanging Drop ◽  
Gene Encoding ◽  
X Ray ◽  
Cell Parameters ◽  
And Function

RNA polymerase (RNAP) plays a crucial role in gene expression in all organisms. It is a multiprotein complex that produces primary transcript RNA. Generally, the basal transcription apparatus in archaea is simpler than the eukaryotic RNA polymerase II counterpart. To understand the structure and function of archaeal RNAP, theTON-0309gene encoding DNA-directed RNA polymerase subunit L (ToRNAP_L) fromThermococcus onnurineusNA1 was cloned and the protein was overexpressed inEscherichia coli, purified and crystallized. The purified protein was crystallized using the hanging-drop vapour-diffusion method and the crystal diffracted to 2.10 Å resolution. The crystal belonged to the hexagonal space groupP6122, with unit-cell parametersa=b= 42.3,c= 211.2 Å. One molecule was present in the asymmetric unit, with a correspondingVMof 2.5 Å3 Da−1and a solvent content of 50.0%.

Condensation of cis-9-methyl-2-decalone with cyclic β-amino esters: synthesis and X-ray crystallography of perhydroazacyclopentaanthracenes and perhydroazabenzanthracenes

1976 ◽  
Vol 54 (10) ◽  
pp. 1512-1520 ◽  
Author(s):  
Ronald B. Kelly ◽  
Barry A. Beckett ◽  
Ijaz Mumtaz ◽  
Eric Stanley ◽  
Peter S. White
Keyword(s):  
Carbon Atom ◽  
Crystallographic Analysis ◽  
Amino Ketone ◽  
Structural Isomers ◽  
X Ray ◽  
X Ray Crystallography ◽  
Amino Esters ◽  
Condensation Products ◽  
Vinylogous Amides ◽  
Pure Isomer

Condensation of cis-9-methyl-2-decalone, 9, with ethyl 2-pyrrolidineacetate, 10, afforded a mixture of two isomeric vinylogous amides, 11. Oxidation of either one pure isomer, 11a, or the mixture 11 to the same pyridone, 12, revealed that the two condensation products were stereoisomeric at a single carbon atom and not structural isomers. Reduction of 11a afforded the amino ketone 13. The structures assigned to 11, 12, and 13 are based on X-ray crystallographic analysis of 13.Similarly, condensation of 9 with ethyl 2-piperidineacetate, 14, afforded the isomers 15a and 15b both of which were oxidized to the same pyridone, 16. Reduction of 15a afforded 17. The structures assigned to 15, 16, and 17 are based on X-ray crystallographic analysis of 17.

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