scholarly journals Biochemical Characterization of Gyp6p, a Ypt/Rab-specific GTPase-activating Protein from Yeast

2001 ◽  
Vol 276 (15) ◽  
pp. 12135-12139 ◽  
Author(s):  
Elke Will ◽  
Dieter Gallwitz
Keyword(s):  
Biochemical Characterization ◽  
Three Dimensional ◽  
Significant Inhibition ◽  
Dimensional Structure ◽  
Physical Interaction ◽  
Mutant Proteins ◽  
Gtpase Activating Proteins ◽  
Two Hybrid ◽  
Intrinsic Gtpase Activity

Gyp6p from yeast belongs to the GYP family of Ypt/Rab-specific GTPase-activating proteins, and Ypt6p is its preferred substrate (Strom, M., Vollmer, P., Tan, T. J., and Gallwitz, D. (1993)Nature361, 736–739). We have investigated the kinetic parameters of Gyp6p/Ypt6p interactions and find that Gyp6p accelerates the intrinsic GTPase activity of Ypt6p (0.0002 min−1) by a factor of 5 × 106and that they have a very low affinity for its preferred substrate(Km= 592 μm). Substitution with alanine of several arginines, which Gyp6p shares with other GYP family members, resulted in significant inhibition of GAP activity. Replacement of arginine-155 with either alanine or lysine abolished its GAP activity, indicating a direct involvement of this strictly conserved arginine in catalysis. Physical interaction of the catalytically inactive Gyp6(R155A) mutant GAP with Ypt6 wild-type and Ypt6 mutant proteins could be demonstrated with the two-hybrid system. Short N-terminal and C-terminal truncations of Gyp6p resulted in a complete loss of GAP activity and Ypt6p binding, showing that in contrast to two other Gyp proteins studied previously, most of the 458 amino acid-long Gyp6p sequence is required to form a three-dimensional structure that allows substrate binding and catalysis.

scholarly journals High-resolution crystal structure and biochemical characterization of a GH11 endoxylanase from Nectria haematococca

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hina Andaleeb ◽  
Najeeb Ullah ◽  
Sven Falke ◽  
Markus Perbandt ◽  
Hévila Brognaro ◽  
...  
Keyword(s):  
Alternative Fuels ◽  
Biochemical Characterization ◽  
Three Dimensional ◽  
Detailed Comparison ◽  
Structural Features ◽  
Dimensional Structure ◽  
Nectria Haematococca ◽  
Chemical Reagents ◽  
Beechwood Xylan

Abstract Enzymatic degradation of vegetal biomass offers versatile procedures to improve the production of alternative fuels and other biomass-based products. Here we present the three-dimensional structure of a xylanase from Nectria haematococca (NhGH11) at 1.0 Å resolution and its functional properties. The atomic resolution structure provides details and insights about the complex hydrogen bonding network of the active site region and allowed a detailed comparison with homologous structures. Complementary biochemical studies showed that the xylanase can catalyze the hydrolysis of complex xylan into simple xylose aldopentose subunits of different lengths. NhGH11 can catalyze the efficient breakdown of beechwood xylan, xylan polysaccharide, and wheat arabinoxylan with turnover numbers of 1730.6 ± 318.1 min−1, 1648.2 ± 249.3 min−1 and 2410.8 ± 517.5 min−1 respectively. NhGH11 showed maximum catalytic activity at pH 6.0 and 45 °C. The mesophilic character of NhGH11 can be explained by distinct structural features in comparison to thermophilic GH11 enzymes, including the number of hydrogen bonds, side chain interactions and number of buried water molecules. The enzymatic activity of NhGH11 is not very sensitive to metal ions and chemical reagents that are typically present in associated industrial production processes. The data we present highlights the potential of NhGH11 to be applied in industrial biomass degradation processes.

scholarly journals Detailed structural and biochemical characterization of the nexin-dynein regulatory complex

2015 ◽  
Vol 26 (2) ◽  
pp. 294-304 ◽  
Author(s):  
Toshiyuki Oda ◽  
Haruaki Yanagisawa ◽  
Masahide Kikkawa
Keyword(s):  
Biochemical Characterization ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Dynein Motor ◽  
Cryo Electron Tomography ◽  
Regulatory Complex ◽  
And Function

The nexin-dynein regulatory complex (N-DRC) forms a cross-bridge between the outer doublet microtubules of the axoneme and regulates dynein motor activity in cilia/flagella. Although the molecular composition and the three-dimensional structure of N-DRC have been studied using mutant strains lacking N-DRC subunits, more accurate approaches are necessary to characterize the structure and function of N-DRC. In this study, we precisely localized DRC1, DRC2, and DRC4 using cryo–electron tomography and structural labeling. All three N-DRC subunits had elongated conformations and spanned the length of N-DRC. Furthermore, we purified N-DRC and characterized its microtubule-binding properties. Purified N-DRC bound to the microtubule and partially inhibited microtubule sliding driven by the outer dynein arms (ODAs). Of interest, microtubule sliding was observed even in the presence of fourfold molar excess of N-DRC relative to ODA. These results provide insights into the role of N-DRC in generating the beating motions of cilia/flagella.

scholarly journals Crystallization and biochemical characterization of an archaeal lectin fromMethanococcus voltaeA3

2017 ◽  
Vol 73 (5) ◽  
pp. 300-304 ◽  
Author(s):  
N. Sivaji ◽  
K. V. Abhinav ◽  
M. Vijayan
Keyword(s):  
Biochemical Characterization ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Asymmetric Unit ◽  
Sequence Comparisons ◽  
Space Group ◽  
Three Dimensional Structure ◽  
Methanococcus Voltae

A lectin fromMethanococcus voltaeA3 has been cloned, expressed, purified and characterized. The lectin appears to be specific for complex sugars. The protein crystallized in a tetragonal space group, with around 16 subunits in the asymmetric unit. Sequence comparisons indicate the lectin to have a β-prism I fold, with poor homology to lectins of known three-dimensional structure.

scholarly journals Purification and biochemical characterization of the D6 chemokine receptor

2004 ◽  
Vol 379 (2) ◽  
pp. 263-272 ◽  
Author(s):  
Paul E. BLACKBURN ◽  
Clare V. SIMPSON ◽  
Robert J. B. NIBBS ◽  
Maureen O'HARA ◽  
Rhona BOOTH ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Chemokine Receptor ◽  
Mammalian Cells ◽  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Affinity Column ◽  
Transfected Cells

There is much interest in chemokine receptors as therapeutic targets in diseases such as AIDS, autoimmune and inflammatory disorders, and cancer. Hampering such studies is the lack of accurate three-dimensional structural models of these molecules. The CC-chemokine receptor D6 is expressed at exceptionally high levels in heterologous transfectants. Here we report the purification and biochemical characterization of milligram quantities of D6 protein from relatively small cultures of transfected mammalian cells. Importantly, purified D6 retains full functional activity, shown by displaceable binding of 125I-labelled MIP-1β (macrophage inflammatory protein-1β) and by complete binding of the receptor to a MIP-1α affinity column. In addition, we show that D6 is decorated on the N-terminus by N-linked glycosylation. Mutational analysis reveals that this glycosylation is dispensable for ligand binding and high expression in transfected cells. Metabolic labelling has revealed the receptor to also be sulphated and phosphorylated. Phosphorylation is ligand independent and is not enhanced by ligand binding and internalization, suggesting similarities with the viral chemokine receptor homologue US28. Like US28, an analysis of the full cellular complement of D6 in transfected cells indicates that >80% is found associated with intracellular vesicular structures. This may account for the high quantities of D6 that can be synthesized in these cells. These unusual properties of D6, and the biochemical characterization described here, leads the way towards work aimed at generating the three-dimensional structure of this seven-transmembrane-spanning receptor.

Characterization of Monoclonal Anti-human Factor VII Antibody (B101/B1) that Recognized Three-dimensional Structures near Arg at Position 79 in the First EGF-like Domain

1998 ◽  
Vol 79 (01) ◽  
pp. 104-109 ◽  
Author(s):  
Osamu Takamiya
Keyword(s):  
Monoclonal Antibodies ◽  
Tissue Factor ◽  
Human Factor ◽  
Solid Phase ◽  
Three Dimensional ◽  
Coagulation Factors ◽  
Factor Vii ◽  
Dimensional Structure ◽  
Epidermal Growth

SummaryMurine monoclonal antibodies (designated hVII-B101/B1, hVIIDC2/D4 and hVII-DC6/3D8) directed against human factor VII (FVII) were prepared and characterized, with more extensive characterization of hVII-B101/B1 that did not bind reduced FVIIa. The immunoglobulin of the three monoclonal antibodies consisted of IgG1. These antibodies did not inhibit procoagulant activities of other vitamin K-dependent coagulation factors except FVII and did not cross-react with proteins in the immunoblotting test. hVII-DC2/D4 recognized the light chain after reduction of FVIIa with 2-mercaptoethanol, and hVIIDC6/3D8 the heavy chain. hVII-B101/B1 bound FVII without Ca2+, and possessed stronger affinity for FVII in the presence of Ca2+. The Kd for hVII-B101/B1 to FVII was 1.75 x 10–10 M in the presence of 5 mM CaCl2. The antibody inhibited the binding of FVII to tissue factor in the presence of Ca2+. hVII-B101/B1 also inhibited the activation of FX by the complex of FVIIa and tissue factor in the presence of Ca2+. Furthermore, immunoblotting revealed that hVII-B101/B1 reacted with non-reduced γ-carboxyglutaminic acid (Gla)-domainless-FVII and/or FVIIa. hVII-B101/B1 showed a similar pattern to that of non-reduced proteolytic fragments of FVII by trypsin with hVII-DC2/D4 on immunoblotting test. hVII-B101/B1 reacted differently with the FVII from the dysfunctional FVII variant, FVII Shinjo, which has a substitution of Gln for Arg at residue 79 in the first epidermal growth factor (1st EGF)-like domain (Takamiya O, et al. Haemosta 25, 89-97,1995) compared with normal FVII, when used as a solid phase-antibody for ELISA by the sandwich method. hVII-B101/B1 did not react with a series of short peptide sequences near position 79 in the first EGF-like domain on the solid-phase support for epitope scanning. These results suggested that the specific epitope of the antibody, hVII-B101/B1, was located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII.

Ribosomal protein S17: Characterization of the three-dimensional structure by proton and nitrogen-15 NMR

Biochemistry ◽  
1993 ◽  
Vol 32 (47) ◽  
pp. 12812-12820 ◽  
Author(s):  
Barbara L. Golden ◽  
David W. Hoffman ◽  
V. Ramakrishnan ◽  
Stephen W. White
Keyword(s):  
Ribosomal Protein ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Three Dimensional Structure

scholarly journals Structural characterization of quaternary selenites of tungsten(VI), A 2W3SeO12 (A = NH4, Cs, Rb, K or Tl)

2021 ◽  
Vol 77 (4) ◽  
pp. 406-411
Author(s):  
Vineela Balisetty ◽  
Kanamaluru Vidyasagar
Keyword(s):  
Three Dimensional ◽  
Spectroscopic Studies ◽  
Solid State Reactions ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
Compound K ◽  
Three Dimensional Structure ◽  
Thermal And Spectroscopic Studies ◽  
Anionic Framework

The quaternary A 2W3SeO12 (A = NH4, Cs, Rb, K or Tl) selenites have been prepared in the form of single crystals by hydrothermal and novel solid-state reactions. They were characterized by X-ray diffraction, thermal and spectroscopic studies. All of them have a hexagonal tungsten oxide (HTO) related [W3SeO12]2− anionic framework with pyramidally coordinated Se4+ ions. The known A 2W3SeO12 (A = NH4, Cs or Rb) compounds are isostructural with the Cs2W3TeO12 compound and have a non-centrosymmetric layered structure containing intra-layer Se—O bonds. The new compound K2W3SeO12(α) is isostructural with the K2W3TeO12 compound and has a centrosymmetric three-dimensional structure containing interlayer Se—O bonds. It is inferred that the new Tl2W3SeO12 compound has the same three-dimensional structure as K2W3SeO12(α).

scholarly journals Structural characterization of a Thorarchaeota profilin indicates eukaryotic-like features but with an extended N-terminus

2021 ◽  
Author(s):  
Celestine N Chi ◽  
Ravi Teja Inturi ◽  
Sandra Martinez Lara ◽  
Mahmoud Darweesh
Keyword(s):  
Common Ancestor ◽  
Three Dimensional ◽  
Eukaryotic Cell ◽  
Dimensional Structure ◽  
Resonance Spectroscopy ◽  
Last Eukaryotic Common Ancestor ◽  
Rigid Core ◽  
N Terminus ◽  
Metagenomics Analysis

The emergence of the first eukaryotic cell was preceded by evolutionary events which are still highly debatable. Recently, comprehensive metagenomics analysis has uncovered that the Asgard super-phylum is the closest yet known archaea host of eukaryotes. However, it remains to be established if a large number of eukaryotic signature proteins predicated to be encoded by the Asgard super-phylum are functional at least, in the context of a eukaryotic cell. Here, we determined the three-dimensional structure of profilin from Thorarchaeota by nuclear magnetic resonance spectroscopy and show that this profilin has a rigid core with a flexible N-terminus which was previously implicated in polyproline binding. In addition, we also show that thorProfilin co-localizes with eukaryotic actin in cultured HeLa cells. This finding reaffirm the notion that Asgardean encoded proteins possess eukaryotic-like characteristics and strengthen likely existence of a complex cytoskeleton already in a last eukaryotic common ancestor

scholarly journals Structural Features of a Bacteroidetes-Affiliated Cellulase Linked with a Polysaccharide Utilization Locus

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
A.E. Naas ◽  
A.K. MacKenzie ◽  
B. Dalhus ◽  
V.G.H. Eijsink ◽  
P.B. Pope
Keyword(s):  
Active Site ◽  
Three Dimensional ◽  
Structural Features ◽  
Structure And Function ◽  
Dimensional Structure ◽  
Active Site Cleft ◽  
Polysaccharide Utilization Locus ◽  
And Function ◽  
Rumen Metagenome

Abstract Previous gene-centric analysis of a cow rumen metagenome revealed the first potentially cellulolytic polysaccharide utilization locus, of which the main catalytic enzyme (AC2aCel5A) was identified as a glycoside hydrolase (GH) family 5 endo-cellulase. Here we present the 1.8 Å three-dimensional structure of AC2aCel5A and characterization of its enzymatic activities. The enzyme possesses the archetypical (β/α)8-barrel found throughout the GH5 family and contains the two strictly conserved catalytic glutamates located at the C-terminal ends of β-strands 4 and 7. The enzyme is active on insoluble cellulose and acts exclusively on linear β-(1,4)-linked glucans. Co-crystallization of a catalytically inactive mutant with substrate yielded a 2.4 Å structure showing cellotriose bound in the −3 to −1 subsites. Additional electron density was observed between Trp178 and Trp254, two residues that form a hydrophobic “clamp”, potentially interacting with sugars at the +1 and +2 subsites. The enzyme’s active-site cleft was narrower compared to the closest structural relatives, which in contrast to AC2aCel5A, are also active on xylans, mannans and/or xyloglucans. Interestingly, the structure and function of this enzyme seem adapted to less-substituted substrates such as cellulose, presumably due to the insufficient space to accommodate the side-chains of branched glucans in the active-site cleft.

scholarly journals In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

2003 ◽  
Vol 77 (6) ◽  
pp. 3669-3679 ◽  
Author(s):  
Caterina Trozzi ◽  
Linda Bartholomew ◽  
Alessandra Ceccacci ◽  
Gabriella Biasiol ◽  
Laura Pacini ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Serine Protease ◽  
In Vitro Selection ◽  
Three Dimensional ◽  
Host Cells ◽  
Dimensional Structure ◽  
In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

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