scholarly journals A protocol to automatically calculate homo-oligomeric protein structures through the integration of evolutionary constraints and ambiguous contacts derived from solid- or solution-state NMR

2019 ◽  
Author(s):  
Davide Sala ◽  
Linda Cerofolini ◽  
Marco Fragai ◽  
Andrea Giachetti ◽  
Claudio Luchinat ◽  
...  
Keyword(s):  
Protein Structures ◽  
3D Structure ◽  
Protein Assemblies ◽  
Coevolution Analysis ◽  
Oligomeric Protein ◽  
Intermolecular Contacts ◽  
Docking Calculations ◽  
Uniform Labeling ◽  
Time Required

ABSTRACTProtein assemblies are involved in many important biological processes. Solid-state NMR (SSNMR) spectroscopy is a technique suitable for the structural characterization of samples with high molecular weight and thus can be applied to such assemblies. A significant bottleneck in terms of both effort and time required is the manual identification of unambiguous intermolecular contacts. This is particularly challenging for homo-oligomeric complexes, where simple uniform labeling may not be effective. We tackled this challenge by exploiting coevolution analysis to extract information on homo-oligomeric interfaces from NMR-derived ambiguous contacts. After removing the evolutionary couplings (ECs) that are already satisfied by the 3D structure of the monomer, the predicted ECs are matched with the automatically generated list of experimental contacts. This approach provides a selection of potential interface residues that is used directly in monomer-monomer docking calculations. We validated the protocol on tetrameric L-asparaginase II and dimeric Sod1.

scholarly journals Comparative studies on O-acetylhomoserine sulfhydrylase: physiological role and characterization of the Aspergillus nidulans enzyme.

1993 ◽  
Vol 40 (3) ◽  
pp. 421-428 ◽  
Author(s):  
J Brzywczy ◽  
S Yamagata ◽  
A Paszewski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Substrate Specificity ◽  
Aspergillus Nidulans ◽  
Comparative Studies ◽  
Alternative Pathway ◽  
Physiological Role ◽  
Oligomeric Protein ◽  
Cysteine Synthesis ◽  
Sulfhydryl Compounds

O-acetylhomoserine sulfhydrylase (OAH SHLase) from Aspergillus nidulans is an oligomeric protein with a broad substrate specificity with regard to sulfhydryl compounds. As its Saccharomyces cerevisiae counterpart the enzyme also reacts with O-acetylserine and is inhibited by carbonyl reagents but not by antiserum raised against the yeast enzyme. In contrast to Saccharomyces cerevisiae the enzyme is not essential for Aspergillus nidulans as indicated by the completely prototrophic phenotype of OAH SHLase-negative mutants. Its major physiological role in Aspergillus nidulans seems to be recycling of the thiomethyl group of methylthio-adenosine but it is also a constituent of the alternative pathway of cysteine synthesis.

scholarly journals An operational implementation of the GHER model for the Black Sea, with SST and CTD data assimilation

2009 ◽  
Vol 6 (2) ◽  
pp. 1895-1911 ◽  
Author(s):  
L. Vandenbulcke ◽  
A. Capet ◽  
J. M. Beckers ◽  
M. Grégoire ◽  
S. Besiktepe
Keyword(s):  
Black Sea ◽  
Initial Conditions ◽  
Sea Surface ◽  
The Black Sea ◽  
Marmara Sea ◽  
Reduced Rank ◽  
Data Transfers ◽  
Time Required ◽  
Assimilation Scheme

Abstract. In this article, we describe the first operational implementation of the GHER hydrodynamic model. This happened onboard the research vessel "Alliance", in the context of the Turkish Straits System 2008 campaign, which aimed at the real-time characterization of the Marmara Sea and (south-western) Black Sea. The model performed badly at first, mainly because of poor initial conditions. Hence, as the model includes a reduced-rank extended Kalman filter assimilation scheme, after a hindcast where sea surface temperature and temperature and salinity profiles were assimilated, the model yielded realistic forecasts. Furthermore, the time required to run a one-day simulation (about 5 min of simulation, or 10 min with pre-processing and data transfers included) is very limited and thus operational use of the model is possible.

scholarly journals Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control

2014 ◽  
Vol 207 (4) ◽  
pp. 463-480 ◽  
Author(s):  
Jonathan Bizarro ◽  
Christophe Charron ◽  
Séverine Boulon ◽  
Belinda Westman ◽  
Bérengère Pradet-Balade ◽  
...  
Keyword(s):  
Isotope Labeling ◽  
Core Protein ◽  
3D Structure ◽  
Assembly Mechanism ◽  
Core Proteins ◽  
Assembly Factors ◽  
Assembly Factor

In vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the HSP90–R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu13p/15.5K bound to a fragment of the assembly factor Rsa1p/NUFIP. This revealed several unexpected features: (a) the existence of a protein-only pre-snoRNP complex containing five assembly factors and two core proteins, 15.5K and Nop58; (b) the characterization of ZNHIT3, which is present in the protein-only complex but gets released upon binding to C/D snoRNAs; (c) the dynamics of the R2TP complex, which appears to load/unload RuvBL AAA+ adenosine triphosphatase from pre-snoRNPs; and (d) a potential mechanism for preventing premature activation of snoRNP catalytic activity. These data provide a framework for understanding the assembly of box C/D snoRNPs.

New insights on the Golgi complex ofTritrichomonas foetus

Parasitology ◽  
2013 ◽  
Vol 141 (2) ◽  
pp. 241-253 ◽  
Author(s):  
IVONE De ANDRADE ROSA ◽  
MARJOLLY BRIGIDO CARUSO ◽  
SILAS PESSINI RODRIGUES ◽  
REINALDO BARROS GERALDO ◽  
LUIZA WILGES KIST ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Trichomonas Vaginalis ◽  
Adenosine Triphosphatase ◽  
Protein Structures ◽  
3D Structure ◽  
Tritrichomonas Foetus ◽  
Beta Tubulin ◽  
Human Parasite ◽  
New Information ◽  
Fluorescence And Electron Microscopy

SUMMARYTritrichomonas foetusis a protist that causes bovine trichomoniasis and presents a well-developed Golgi. There are very few studies concerning the Golgi in trichomonads. In this work, monoclonal antibodies were raised against Golgi ofT. foetusand used as a tool on morphologic and biochemical studies of this organelle. Among the antibodies produced, one was named mAb anti-Golgi 20.3, which recognized specifically the Golgi complex by fluorescence and electron microscopy. By immunoblotting this antibody recognized two proteins with 60 and 66 kDa that were identified as putative beta-tubulin and adenosine triphosphatase, respectively. The mAb 20.3 also recognized the Golgi complex of theTrichomonas vaginalis, a human parasite. In addition, the nucleotide coding sequences of these proteins were identified and included in theT. foetusdatabase, and the 3D structure of the proteins was predicted. In conclusion, this study indicated: (1) adenosine triphosphatase is present in the Golgi, (2) ATPase is conserved betweenT. foetusandT. vaginalis, (3) there is new information concerning the nucleic acid sequences and protein structures of adenosine triphosphatase and beta-tubulin fromT. foetusand (4) the mAb anti-Golgi 20.3 is a good Golgi marker and can be used in future studies.

scholarly journals Characterization of a porin channel in the endosymbiont of the trypanosomatid protozoan Crithidia deanei

Microbiology ◽  
2011 ◽  
Vol 157 (10) ◽  
pp. 2818-2830 ◽  
Author(s):  
Iamara da Silva Andrade ◽  
João Lídio Vianez-Júnior ◽  
Carolina Lage Goulart ◽  
Fabrice Homblé ◽  
Jean-Marie Ruysschaert ◽  
...  
Keyword(s):  
3D Structure ◽  
Symbiotic Bacterium ◽  
Gram Negative Bacteria ◽  
Genome Database ◽  
Gram Negative ◽  
Secondary Structure Analysis ◽  
Electrophysiological Measurements ◽  
Mutualistic Relationship ◽  
Obligate Intracellular Bacteria

Crithidia deanei is a trypanosomatid protozoan that harbours a symbiotic bacterium. The partners maintain a mutualistic relationship, thus constituting an excellent model for studying metabolic exchanges between the host and the symbiont, the origin of organelles and cellular evolution. According to molecular analysis, symbionts of different trypanosomatid species share high identity and descend from a common ancestor, a β-proteobacterium of the genus Bordetella. The endosymbiont is surrounded by two membranes, like Gram-negative bacteria, but its envelope presents special features, since phosphatidylcholine is a major membrane component and the peptidoglycan layer is highly reduced, as described in other obligate intracellular bacteria. Like the process that generated mitochondria and plastids, the endosymbiosis in trypanosomatids depends on pathways that facilitate the intensive metabolic exchanges between the bacterium and the host protozoan. A search of the annotated symbiont genome database identified one sequence with identity to porin-encoding genes of the genus Bordetella. Considering that the symbiont outer membrane has a great accessibility to cytoplasm host factors, it was important to characterize this single porin-like protein using biochemical, molecular, computational and ultrastructural approaches. Antiserum against the recombinant porin-like molecule revealed that it is mainly located in the symbiont envelope. Secondary structure analysis and comparative modelling predicted the protein 3D structure as an 18-domain β-barrel, which is consistent with porin channels. Electrophysiological measurements showed that the porin displays a slight preference for cations over anions. Taken together, the data presented herein suggest that the C. deanei endosymbiont porin is phylogenetically and structurally similar to those described in Gram-negative bacteria, representing a diffusion channel that might contribute to the exchange of nutrients and metabolic precursors between the symbiont and its host cell.

Primary structure of a human mitochondrial protein homologous to the bacterial and plant chaperonins and to the 65-kilodalton mycobacterial antigen

1989 ◽  
Vol 9 (5) ◽  
pp. 2279-2283
Author(s):  
S Jindal ◽  
A K Dudani ◽  
B Singh ◽  
C B Harley ◽  
R S Gupta
Keyword(s):  
Mammalian Cells ◽  
Mitochondrial Protein ◽  
Protein Structures ◽  
Mycobacterial Antigen ◽  
Related Protein ◽  
Bisphosphate Carboxylase ◽  
Oligomeric Protein ◽  
Strong Homology ◽  
Major Antigen ◽  
High Degree

The complete cDNA for a human mitochondrial protein designated P1, which was previously identified as a microtubule-related protein, has been cloned and sequenced. The deduced amino acid sequence of P1 shows strong homology (40 to 50% identical residues and an additional 20% conservative replacements) to the 65-kilodalton major antigen of mycobacteria, to the GroEL protein of Escherichia coli, and to the ribulose 1,5-bisphosphate carboxylase-oxygenase (rubisco) subunit binding protein of plant chloroplasts. Similar to the case with the latter two proteins, which have been shown to act as chaperonins in the posttranslational assembly of oligomeric protein structures, it is suggested that P1 may play a similar role in mammalian cells. The observed high degree of homology between human P1 and mycobacterial antigen also suggests the possible involvement of this protein in certain autoimmune diseases.

scholarly journals Identification and Characterization of a Novel Thermostable and Salt-Tolerant β-1,3 Xylanase from Flammeovirga pacifica Strain WPAGA1

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1287
Author(s):  
Zhiwei Yi ◽  
Zhengwen Cai ◽  
Bo Zeng ◽  
Runying Zeng ◽  
Guangya Zhang
Keyword(s):  
Thermal Stability ◽  
Salt Tolerance ◽  
3D Structure ◽  
Catalytic Efficiency ◽  
Dynamics Simulation ◽  
Carbohydrate Binding ◽  
Salt Tolerant ◽  
Excellent Thermal Stability ◽  
Moderately Thermophilic

β-1,3 xylanase is an important enzyme in the biorefinery process for some algae. The discovery and characterization of new β-1,3 xylanase is a hot research topic. In this paper, a novel β-1,3 xylanase (Xyl88) is revealed from the annotated genome of Flammeovirga pacifica strain WPAGA1. Bioinformatic analysis shows that Xyl88 belongs to the glycoside hydrolase 26 (GH26) with a suspected CBM (carbohydrate-binding module) sequence. The activity of rXyl88 is 75% of the highest enzyme activity (1.5 mol/L NaCl) in 3 mol/L NaCl buffer, which suggests good salt tolerance of rXy188. The optimum reaction temperature in the buffer without NaCl and with 1.5 mol/L NaCl is 45 °C and 55 °C, respectively. Notably, the catalytic efficiency of rXyl88 (kcat/Km) is approximately 20 higher than that of the thermophilic β-1,3 xylanase that has the highest catalytic efficiency. Xyl88 in this study becomes the most efficient enzyme ever found, and it is also the first reported moderately thermophilic and salt-tolerant β-1,3 xylanase. Results of molecular dynamics simulation further prove the excellent thermal stability of Xyl88. Moreover, according to the predicted 3D structure of the Xyl88, the surface of the enzyme is distributed with more negative charges, which is related to its salt tolerance, and significantly more hydrogen bonds and Van der Waals force between the intramolecular residues, which is related to its thermal stability.

scholarly journals Preparation and Characterization of Perforated SERS Active Array for Particle Trapping and Sensitive Molecular Analysis

Biosensors ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 93 ◽  
Author(s):  
István Rigó ◽  
Miklós Veres ◽  
Tamás Váczi ◽  
Eszter Holczer ◽  
Orsolya Hakkel ◽  
...  
Keyword(s):  
Molecular Analysis ◽  
Silicon Surface ◽  
3D Structure ◽  
Raman Spectroscopic ◽  
Cell Trapping ◽  
Highly Sensitive ◽  
Surface Enhanced ◽  
Surface Enhanced Raman ◽  
Flow Through

A gold-coated array of flow-through inverse pyramids applicable as substrate for entrapment and immobilization of micro-objects and for surface enhanced Raman spectroscopic measurements was fabricated using bulk micromachining techniques from silicon. Surface morphology, optical reflectance, immobilization properties, and surface enhanced Raman amplification of the array were modelled and characterized. It was found that the special perforated periodic 3D structure can be used for parallel particle and cell trapping and highly sensitive molecular analysis of the immobilized objects.

Molecular and Biomechanical Characterization of Mineralized Tissue by Dental Pulp Cells on Titanium

2005 ◽  
Vol 84 (6) ◽  
pp. 515-520 ◽  
Author(s):  
H. Nakamura ◽  
L. Saruwatari ◽  
H. Aita ◽  
K. Takeuchi ◽  
T. Ogawa
Keyword(s):  
Dental Pulp ◽  
Titanium Surface ◽  
Healing Time ◽  
Dental Pulp Cells ◽  
Mineralized Tissue ◽  
Osteoblastic Phenotype ◽  
Pulp Cells ◽  
Implant Therapy ◽  
Time Required

The application of implant therapy is still limited, because of various risk factors and the long healing time required for bone-titanium integration. This study explores the potential for osseointegration engineering with dental pulp cells (DPCs) by testing a hypothesis that DPCs generate mineralized tissue on titanium. DPCs extracted from rat incisors positive for CD44, alkaline phosphatase activity, and mineralizing capability were cultured on polystyrene and on machined and dual-acid-etched (DAE) titanium. Tissue cultured on titanium with a Ca/P ratio of 1.4 exhibited plate-like morphology, while that on the polystyrene exhibited fibrous and punctate structures. Tissues cultured on titanium were harder than those on polystyrene, 1.5 times on the machined and 3 times on the DAE. Collagen I, osteopontin, and osteocalcin genes were up-regulated on titanium, especially the DAE surface. In conclusion, DPCs showing some characteristics of the previously identified dental pulp stem cells can generate mineralized tissue on titanium via the osteoblastic phenotype, which can be enhanced by titanium surface roughness.

Solving the 3D structure of metal nanoparticles

2007 ◽  
Vol 222 (11/2007) ◽  
Author(s):  
Anatoly Frenkel
Keyword(s):  
Metal Nanoparticles ◽  
Short Range ◽  
Short Range Order ◽  
3D Structure ◽  
Three Dimensional ◽  
X Ray ◽  
Particle Distributions ◽  
Structure Of Metal ◽  
X Ray Absorption

We discuss methods of Extended X-ray Absorption Fine-Structure (EXAFS) analysis that provide three-dimensional structural characterization of metal nanoparticles, both mono- and bi-metallic. For the bimetallic alloys, we use short range order measurements to discriminate between random and non-random inter-particle distributions of atoms. We also discuss the application of EXAFS to heterogeneous nanoparticle systems.

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