scholarly journals Protein Phosphatase 2B (PP2B, Calcineurin) in Paramecium: Partial Characterization Reveals That Two Members of the Unusually Large Catalytic Subunit Family Have Distinct Roles in Calcium-Dependent Processes

2010 ◽  
Vol 9 (7) ◽  
pp. 1049-1063 ◽  
Author(s):  
D. Fraga ◽  
I. M. Sehring ◽  
R. Kissmehl ◽  
M. Reiss ◽  
R. Gaines ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Amino Acid Level ◽  
Three Dimensional ◽  
Large Family ◽  
Genome Project ◽  
Dimensional Structure ◽  
Sequence Information ◽  
Paramecium Tetraurelia ◽  
Content Type ◽  
Calcium Dependent

ABSTRACT We characterized the calcineurin (CaN) gene family, including the subunits CaNA and CaNB, based upon sequence information obtained from the Paramecium genome project. Paramecium tetraurelia has seven subfamilies of the catalytic CaNA subunit and one subfamily of the regulatory CaNB subunit, with each subfamily having two members of considerable identity on the amino acid level (≥55% between subfamilies, ≥94% within CaNA subfamilies, and full identity in the CaNB subfamily). Within CaNA subfamily members, the catalytic domain and the CaNB binding region are highly conserved and molecular modeling revealed a three-dimensional structure almost identical to a human ortholog. At 14 members, the size of the CaNA family is unprecedented, and we hypothesized that the different CaNA subfamily members were not strictly redundant and that at least some fulfill different roles in the cell. This was tested by selecting two phylogenetically distinct members of this large family for posttranscriptional silencing by RNA interference. The two targets resulted in differing effects in exocytosis, calcium dynamics, and backward swimming behavior that supported our hypothesis that the large, highly conserved CaNA family members are not strictly redundant and that at least two members have evolved diverse but overlapping functions. In sum, the occurrence of CaN in Paramecium spp., although disputed in the past, has been established on a molecular level. Its role in exocytosis and ciliary beat regulation in a protozoan, as well as in more complex organisms, suggests that these roles for CaN were acquired early in the evolution of this protein family.

scholarly journals Biochemical Characterization of the Amylase Activity from the New Haloarchaeal Strain Haloarcula sp. HS Isolated in the Odiel Marshlands

Biology ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 337
Author(s):  
Patricia Gómez-Villegas ◽  
Javier Vigara ◽  
Luis Romero ◽  
Cecilia Gotor ◽  
Sara Raposo ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Amylase Activity ◽  
Extreme Environments ◽  
Three Dimensional ◽  
Large Family ◽  
Industrial Applications ◽  
Dimensional Structure ◽  
Glycosyl Hydrolases ◽  
Calcium Dependent ◽  
Alpha Amylases

Alpha-amylases are a large family of α,1-4-endo-glycosyl hydrolases distributed in all kingdoms of life. The need for poly-extremotolerant amylases encouraged their search in extreme environments, where archaea become ideal candidates to provide new enzymes that are able to work in the harsh conditions demanded in many industrial applications. In this study, a collection of haloarchaea isolated from Odiel saltern ponds in the southwest of Spain was screened for their amylase activity. The strain that exhibited the highest activity was selected and identified as Haloarcula sp. HS. We demonstrated the existence in both, cellular and extracellular extracts of the new strain, of functional α-amylase activities, which showed to be moderately thermotolerant (optimum around 60 °C), extremely halotolerant (optimum over 25% NaCl), and calcium-dependent. The tryptic digestion followed by HPLC-MS/MS analysis of the partially purified cellular and extracellular extracts allowed to identify the sequence of three alpha-amylases, which despite sharing a low sequence identity, exhibited high three-dimensional structure homology, conserving the typical domains and most of the key consensus residues of α-amylases. Moreover, we proved the potential of the extracellular α-amylase from Haloarcula sp. HS to treat bakery wastes under high salinity conditions.

scholarly journals Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins

1993 ◽  
Vol 2 (3) ◽  
pp. 366-382 ◽  
Author(s):  
Miroslaw Cygler ◽  
Joseph D. Schrag ◽  
Joel L. Sussman ◽  
Michal Harel ◽  
Israel Silman ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Large Family ◽  
Sequence Conservation ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Related Proteins

The first crystal structure of the peptidase domain of the U32 peptidase family

2015 ◽  
Vol 71 (12) ◽  
pp. 2505-2512 ◽  
Author(s):  
Magdalena Schacherl ◽  
Angelika A. M. Montada ◽  
Elena Brunstein ◽  
Ulrich Baumann
Keyword(s):  
Crystal Structure ◽  
Catalytic Mechanism ◽  
Quaternary Structure ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Zinc Ion ◽  
Crystal Structure Analysis ◽  
Dimensional Structure ◽  
Sequence Motifs ◽  
Conserved Sequence

The U32 family is a collection of over 2500 annotated peptidases in the MEROPS database with unknown catalytic mechanism. They mainly occur in bacteria and archaea, but a few representatives have also been identified in eukarya. Many of the U32 members have been linked to pathogenicity, such as proteins fromHelicobacterandSalmonella. The first crystal structure analysis of a U32 catalytic domain fromMethanopyrus kandleri(genemk0906) reveals a modified (βα)8TIM-barrel fold with some unique features. The connecting segment between strands β7 and β8 is extended and helix α7 is located on top of the C-terminal end of the barrel body. The protein exhibits a dimeric quaternary structure in which a zinc ion is symmetrically bound by histidine and cysteine side chains from both monomers. These residues reside in conserved sequence motifs. No typical proteolytic motifs are discernible in the three-dimensional structure, and biochemical assays failed to demonstrate proteolytic activity. A tunnel in which an acetate ion is bound is located in the C-terminal part of the β-barrel. Two hydrophobic grooves lead to a tunnel at the C-terminal end of the barrel in which an acetate ion is bound. One of the grooves binds to aStrep-Tag II of another dimer in the crystal lattice. Thus, these grooves may be binding sites for hydrophobic peptides or other ligands.

scholarly journals An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Sabino Pacheco ◽  
Isabel Gómez ◽  
Jorge Sánchez ◽  
Blanca-Ines García-Gómez ◽  
Mario Soberón ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Double Point ◽  
Single Point ◽  
Three Dimensional ◽  
Point Mutations ◽  
Salt Bridge ◽  
Dimensional Structure ◽  
Cry Toxins ◽  
Content Type ◽  
Domain I

ABSTRACT Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity.

Prediction of Structural and Functional Aspects of Protein

2014 ◽  
pp. 317-333
Author(s):  
Arun G. Ingale
Keyword(s):  
Protein Structure ◽  
Protein Structure Prediction ◽  
Structure Prediction ◽  
Tertiary Structure ◽  
Protein Structures ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Sequence Information ◽  
Predict Protein Structure ◽  
Basic Ideas

To predict the structure of protein from a primary amino acid sequence is computationally difficult. An investigation of the methods and algorithms used to predict protein structure and a thorough knowledge of the function and structure of proteins are critical for the advancement of biology and the life sciences as well as the development of better drugs, higher-yield crops, and even synthetic bio-fuels. To that end, this chapter sheds light on the methods used for protein structure prediction. This chapter covers the applications of modeled protein structures and unravels the relationship between pure sequence information and three-dimensional structure, which continues to be one of the greatest challenges in molecular biology. With this resource, it presents an all-encompassing examination of the problems, methods, tools, servers, databases, and applications of protein structure prediction, giving unique insight into the future applications of the modeled protein structures. In this chapter, current protein structure prediction methods are reviewed for a milieu on structure prediction, the prediction of structural fundamentals, tertiary structure prediction, and functional imminent. The basic ideas and advances of these directions are discussed in detail.

scholarly journals TransformerCPI: improving compound–protein interaction prediction by sequence-based deep learning with self-attention mechanism and label reversal experiments

2020 ◽  
Vol 36 (16) ◽  
pp. 4406-4414 ◽  
Author(s):  
Lifan Chen ◽  
Xiaoqin Tan ◽  
Dingyan Wang ◽  
Feisheng Zhong ◽  
Xiaohong Liu ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Three Dimensional ◽  
Prediction Performance ◽  
Dimensional Structure ◽  
Sequence Information ◽  
Biological Targets ◽  
Interaction Prediction ◽  
Protein Interaction Prediction ◽  
Improved Performance ◽  
Ligand Bias

Abstract Motivation Identifying compound–protein interaction (CPI) is a crucial task in drug discovery and chemogenomics studies, and proteins without three-dimensional structure account for a large part of potential biological targets, which requires developing methods using only protein sequence information to predict CPI. However, sequence-based CPI models may face some specific pitfalls, including using inappropriate datasets, hidden ligand bias and splitting datasets inappropriately, resulting in overestimation of their prediction performance. Results To address these issues, we here constructed new datasets specific for CPI prediction, proposed a novel transformer neural network named TransformerCPI, and introduced a more rigorous label reversal experiment to test whether a model learns true interaction features. TransformerCPI achieved much improved performance on the new experiments, and it can be deconvolved to highlight important interacting regions of protein sequences and compound atoms, which may contribute chemical biology studies with useful guidance for further ligand structural optimization. Availability and implementation https://github.com/lifanchen-simm/transformerCPI.

scholarly journals DUF3380 Domain from a Salmonella Phage Endolysin Shows PotentN-Acetylmuramidase Activity

2016 ◽  
Vol 82 (16) ◽  
pp. 4975-4981 ◽  
Author(s):  
Lorena Rodríguez-Rubio ◽  
Hans Gerstmans ◽  
Simon Thorpe ◽  
Stéphane Mesnage ◽  
Rob Lavigne ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Thermal Resistance ◽  
High Performance ◽  
Antimicrobial Agents ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Gram Negative ◽  
Content Type

ABSTRACTBacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novelSalmonellabacteriophage endolysin, Gp110, which comprises an uncharacterizeddomain ofunknownfunction (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/μM) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7- to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 hasN-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond betweenN-acetylmuramic acid andN-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents.IMPORTANCEWe report the functional and biochemical characterization of theSalmonellaphage endolysin Gp110. This endolysin has a modular structure with an enzymatically active domain and a cell wall binding domain. The enzymatic activity of this endolysin exceeds that of all other endolysins previously characterized using the same methods. A domain of unknown function (DUF3380) is responsible for this high enzymatic activity. We report that DUF3380 hasN-acetylmuramidase activity against directly cross-linked peptidoglycans with various peptide stem compositions. This experimentally verified activity allows better classification and understanding of the enzymatic activities of endolysins, which mostly are inferred by sequence similarities. Three-dimensional structure predictions for Gp110 suggest a fold that is completely different from that of known structures of enzymes with the same peptidoglycan cleavage specificity, making this endolysin quite unique. All of these features, combined with increased thermal resistance, make Gp110 an attractive candidate for engineering novel endolysin-based antibacterials.

Structure of the Calcium Pump from Sarcoplasmic Reticulum at 8 Å Resolution: Architecture of the Transmembrane Helices and Localization of the Binding Site for Thapsigargin

1998 ◽  
Vol 4 (S2) ◽  
pp. 462-463
Author(s):  
P. Zhang ◽  
C. Toyoshima ◽  
K. Yonekura ◽  
G. Inesi ◽  
M. Green ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Transmembrane Domain ◽  
Three Dimensional ◽  
Specific Inhibitor ◽  
Large Family ◽  
Calcium Pump ◽  
Dimensional Structure ◽  
Transmembrane Helices ◽  
Site Mutagenesis ◽  
P Type

The calcium pump (Ca2+-ATPase) from sarcoplasmic reticulum (SR) is a prominent member of the large family of ATP-dependent cation pumps, which include Na+ /K+-ATPase, H+/K+-ATPase from the stomach, H+-ATPase from yeast and Neurospora, and various detoxifying pumps for Cd+, Cu+ and other metals. In muscle, calcium is stored inside the SR and contraction is initiated by regulated release through specific calcium channels; Ca2+ -ATPase is responsible for relaxation by pumping calcium back into the SR lumen. Many techniques (chemical modification, site mutagenesis, reaction kinetics) have been used to correlate Ca2+-ATPase sequence with function, but no high resolution three-dimensional structure of Ca2+-ATPase, or any P-type pump, has yet been determined. In the current work, we have determined the structure from helical crystals at 8 A resolution and thus revealed the alpha-helical architecture of the transmembrane domain. In addition, a specific inhibitor of Ca2+-ATPase, thapsigargin, was used to promote crystallization and we have characterized the structural consequences of its inhibition.

Hospitality situations, consumer expertise, and perceptions of wine attributes: three empirical studies

2019 ◽  
Vol 31 (1) ◽  
pp. 68-88 ◽  
Author(s):  
Dale F. Duhan ◽  
Shannon B. Rinaldo ◽  
Natalia Velikova ◽  
Tim Dodd ◽  
Brent Trela
Keyword(s):  
Empirical Studies ◽  
Three Dimensional ◽  
Total Sample ◽  
Dimensional Structure ◽  
Product Knowledge ◽  
Product Attributes ◽  
Content Type ◽  
Wine Consumption ◽  
Consumer Expertise ◽  
Academic Researchers

PurposeWine choices are not always fully understood by academic researchers or the industry. This paper aims to outline and test a theoretical model proposing that wine consumption may be dependent on differences in consumer expertise, the hospitality situation, characteristics of the wine itself and an interaction of these variables.Design/methodology/approachThree empirical studies (total sample size = 356) tested these theoretical propositions. Consumers with varying levels of wine knowledge were presented with experimental vignettes showing videos of wine opening and pouring and were asked to pair wines with hospitality situations.FindingsStudy 1 found that consumers with low product knowledge were more sensitive to hospitality situations and extrinsic product attributes (closures) than were the experts. Study 2 found that wine hospitality situations fall into three predicted categories, namely, food, friends and formality, although contrary to prediction, the presence of food was the weakest predictors. Study 3 demonstrated the robustness of the three-dimensional structure of wine hospitality situations.Practical implicationsThese studies provided important practical information because targeting various market segments requires the industry to know what product attributes are favored by different groups of consumers different situations.Originality/valuePrevious researchers have discussed the difficulty of measuring consumption situations. By limiting these studies to wine consumption within hospitality situations, the authors learned much about how consumers’ characteristics, product attributes and the situations interact to influence not only product assessments but also choices.

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