scholarly journals New experimental approaches for investigating interactions betweenPyrococcus furiosuscarbamate kinase and carbamoyltransferases, enzymes involved in the channeling of thermolabile carbamoyl phosphate

Archaea ◽  
2005 ◽  
Vol 1 (6) ◽  
pp. 365-373 ◽  
Author(s):  
Jan Massant ◽  
Nicolas Glansdorff
Keyword(s):  
Protein Interactions ◽  
Thermal Protection ◽  
Pyrococcus Furiosus ◽  
Titration Calorimetry ◽  
Physical Interaction ◽  
Protein Protein Interactions ◽  
Carbamoyl Phosphate ◽  
Molecular Physiology ◽  
Metabolic Channeling ◽  
Experimental Approaches

A somewhat neglected but essential aspect of the molecular physiology of hyperthermophiles is the protection of thermolabile metabolites and coenzymes. An example is carbamoyl phosphate (CP), a precursor of pyrimidines and arginine, which is an extremely labile and potentially toxic intermediate. The first evidence for a biologically significant interaction between carbamate kinase (CK) and ornithine carbamoyltransferase (OTC) fromPyrococcus furiosuswas provided by affinity electrophoresis and co-immunoprecipitation in combination with cross-linking (Massant et al. 2002). Using the yeast two-hybrid system, Hummel-Dreyer chromatography and isothermal titration calorimetry, we obtained additional concrete evidence for an interaction between CK and OTC, the first evidence for an interaction between CK and aspartate carbamoyltransferase (ATC) and an estimate of the binding constant between CK and ATC. The physical interaction between CK and OTC or ATC may prevent thermodenaturation of CP in the aqueous cytoplasmic environment. Here we emphasize the importance of developing experimental approaches to investigate the mechanism of thermal protection of metabolic intermediates by metabolic channeling and the molecular basis of transient protein–protein interactions in the physiology of hyperthermophiles.

Metabolic channelling of carbamoyl phosphate in the hyperthermophilic archaeon Pyrococcus furiosus: dynamic enzyme–enzyme interactions involved in the formation of the channelling complex

2004 ◽  
Vol 32 (2) ◽  
pp. 306-309 ◽  
Author(s):  
J. Massant ◽  
N. Glansdorff
Keyword(s):  
Protein Interactions ◽  
Pyrococcus Furiosus ◽  
Hyperthermophilic Archaea ◽  
Physical Interaction ◽  
Protein Protein Interactions ◽  
Carbamoyl Phosphate ◽  
Molecular Physiology ◽  
Metabolic Channelling ◽  
Aspartate Carbamoyltransferase ◽  
Carbamate Kinase

Protection of thermolabile metabolites and coenzymes is a somewhat neglected but essential aspect of the molecular physiology of hyperthermophiles. Detailed information about the mechanisms used by thermophiles to protect these thermolabile metabolites and coenzymes is still scarce. A case in point is CP (carbamoyl phosphate), a precursor of pyrimidines and arginine, which is an extremely labile and potentially toxic intermediate. Recently we obtained the first evidence for a physical interaction between two hyperthermophilic enzymes for which kinetic evidence had suggested that these enzymes channel a highly thermolabile and potentially toxic intermediate. By physically interacting with each other, CKase (carbamate kinase) and OTCase (ornithine carbamoyltransferase) prevent thermodenaturation of CP in the aqueous cytoplasmic environment. The CP channelling complex involving CKase and OTCase or ATCase (aspartate carbamoyltransferase), identified in hyperthermophilic archaea, provides a good model system to investigate the mechanism of metabolic channelling and the molecular basis of protein–protein interactions in the physiology of extreme thermophiles.

scholarly journals UniProt-Related Documents (UniReD): assisting wet lab biologists in their quest on finding novel counterparts in a protein network

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Theodosios Theodosiou ◽  
Nikolaos Papanikolaou ◽  
Maria Savvaki ◽  
Giulia Bonetto ◽  
Stella Maxouri ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Computational Prediction ◽  
Biomedical Literature ◽  
Protein Network ◽  
Protein Protein Interactions ◽  
High Coverage ◽  
Depth Study ◽  
Experimental Approaches ◽  
Wet Lab ◽  
User Friendly

Abstract The in-depth study of protein–protein interactions (PPIs) is of key importance for understanding how cells operate. Therefore, in the past few years, many experimental as well as computational approaches have been developed for the identification and discovery of such interactions. Here, we present UniReD, a user-friendly, computational prediction tool which analyses biomedical literature in order to extract known protein associations and suggest undocumented ones. As a proof of concept, we demonstrate its usefulness by experimentally validating six predicted interactions and by benchmarking it against public databases of experimentally validated PPIs succeeding a high coverage. We believe that UniReD can become an important and intuitive resource for experimental biologists in their quest for finding novel associations within a protein network and a useful tool to complement experimental approaches (e.g. mass spectrometry) by producing sorted lists of candidate proteins for further experimental validation. UniReD is available at http://bioinformatics.med.uoc.gr/unired/

scholarly journals Assembly of the Complex between Archaeal RNase P Proteins RPP30 and Pop5

Archaea ◽  
2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Brandon L. Crowe ◽  
Christopher J. Bohlen ◽  
Ross C. Wilson ◽  
Venkat Gopalan ◽  
Mark P. Foster
Keyword(s):  
Protein Interactions ◽  
Pyrococcus Furiosus ◽  
Resonance Assignments ◽  
Rnase P ◽  
Size Exclusion ◽  
Titration Calorimetry ◽  
Protein Protein Interaction ◽  
Model Complex ◽  
Exclusion Chromatography ◽  
P Proteins

RNase P is a highly conserved ribonucleoprotein enzyme that represents a model complex for understanding macromolecular RNA-protein interactions. Archaeal RNase P consists of one RNA and up to five proteins (Pop5, RPP30, RPP21, RPP29, and RPP38/L7Ae). Four of these proteins function in pairs (Pop5-RPP30 and RPP21–RPP29). We have used nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to characterize the interaction between Pop5 and RPP30 from the hyperthermophilic archaeonPyrococcus furiosus(Pfu). NMR backbone resonance assignments of free RPP30 (25 kDa) indicate that the protein is well structured in solution, with a secondary structure matching that observed in a closely related crystal structure. Chemical shift perturbations upon the addition of Pop5 (14 kDa) reveal its binding surface on RPP30. ITC experiments confirm a net 1 : 1 stoichiometry for this tight protein-protein interaction and exhibit complex isotherms, indicative of higher-order binding. Indeed, light scattering and size exclusion chromatography data reveal the complex to exist as a 78 kDa heterotetramer with two copies each of Pop5 and RPP30. These results will inform future efforts to elucidate the functional role of the Pop5-RPP30 complex in RNase P assembly and catalysis.

scholarly journals Computational and experimental studies of plasmodium falciparum protein PfAMA1 domain-II loop dynamics: implications in PfAMA1-PfRON2 binding event

2021 ◽  
Author(s):  
Suman Sinha ◽  
Anamika Biswas ◽  
Jagannath Mondal ◽  
Kalyaneswar Mandal
Keyword(s):  
Drug Discovery ◽  
Protein Interactions ◽  
Experimental Studies ◽  
Alpha Helix ◽  
Dynamics Simulation ◽  
Malarial Parasite ◽  
Titration Calorimetry ◽  
Protein Protein Interactions ◽  
Human Red Blood Cells ◽  
Developed Nations

Protein-protein interactions are interesting targets for various drug discovery campaigns. One such promising and therapeutically pertinent protein-protein complex is PfAMA1-PfRON2, which is involved in malarial parasite invasion into human red blood cells. A thorough understanding of the interactions between these macromolecular binding partners is absolutely necessary to design better therapeutics to fight against the age-old disease affecting mostly under-developed nations. Although crystal structures of several PfAMA1-PfRON2 complexes have been solved to understand the molecular interactions between these two proteins, the mechanistic aspects of the domain II loop-PfRON2 association is far from clear. The current work investigates a crucial part of the recognition event; i.e., how the domain II loop of PfAMA1 exerts its effect on the alpha helix of the PfRON2, thus influencing the overall kinetics of this intricate recognition phenomenon. To this end, we have conducted thorough computational investigation of the dynamics and free energetics of domain II loop closing processes using molecular dynamics simulation. The computational results are validated by systematic alanine substitutions of the PfRON2 peptide helix. The subsequent evaluation of the binding affinity of Ala-substituted PfRON2 peptide ligands by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) provides a rank of the relative importance of the residues in context. Our combined (computational and experimental) investigation has revealed that the domain II loop of PfAMA1 is in fact responsible for arresting the PfRON2 molecule from egress, K2027 and D2028 of PfRON2 being the determinant residues for the capturing event. Our study provides a comprehensive understanding of the molecular recognition event between PfAMA1 and PfRON2, specifically in the post binding stage, which potentially can be utilized for drug discovery against malaria.

scholarly journals UHM–ULM interactions in the RBM39–U2AF65 splicing-factor complex

2016 ◽  
Vol 72 (4) ◽  
pp. 497-511 ◽  
Author(s):  
Galina A. Stepanyuk ◽  
Pedro Serrano ◽  
Eigen Peralta ◽  
Carol L. Farr ◽  
Herbert L. Axelrod ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Binding ◽  
Splicing Factor ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Protein Protein Interactions ◽  
Rrm Domain ◽  
Nmr Structures ◽  
Binding Interface ◽  
Cell Extracts

RNA-binding protein 39 (RBM39) is a splicing factor and a transcriptional co-activator of estrogen receptors and Jun/AP-1, and its function has been associated with malignant progression in a number of cancers. The C-terminal RRM domain of RBM39 belongs to the U2AF homology motif family (UHM), which mediate protein–protein interactions through a short tryptophan-containing peptide known as the UHM-ligand motif (ULM). Here, crystal and solution NMR structures of the RBM39-UHM domain, and the crystal structure of its complex with U2AF65-ULM, are reported. The RBM39–U2AF65 interaction was confirmed by co-immunoprecipitation from human cell extracts, by isothermal titration calorimetry and by NMR chemical shift perturbation experiments with the purified proteins. When compared with related complexes, such as U2AF35–U2AF65 and RBM39–SF3b155, the RBM39-UHM–U2AF65-ULM complex reveals both common and discriminating recognition elements in the UHM–ULM binding interface, providing a rationale for the known specificity of UHM–ULM interactions. This study therefore establishes a structural basis for specific UHM–ULM interactions by splicing factors such as U2AF35, U2AF65, RBM39 and SF3b155, and a platform for continued studies of intermolecular interactions governing disease-related alternative splicing in eukaryotic cells.

Isothermal Titration Calorimetry of Protein–Protein Interactions

Methods ◽  
1999 ◽  
Vol 19 (2) ◽  
pp. 213-221 ◽  
Author(s):  
Michael M. Pierce ◽  
C.S. Raman ◽  
Barry T. Nall
Keyword(s):  
Isothermal Titration Calorimetry ◽  
Protein Interactions ◽  
Titration Calorimetry ◽  
Protein Protein Interactions

scholarly journals Self-association of the erythroid transcription factor GATA-1 mediated by its zinc finger domains.

1995 ◽  
Vol 15 (5) ◽  
pp. 2448-2456 ◽  
Author(s):  
M Crossley ◽  
M Merika ◽  
S H Orkin
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Zinc Finger ◽  
Protein Interactions ◽  
Physical Interaction ◽  
Erythroid Cells ◽  
Protein Protein Interactions ◽  
Self Association ◽  
Zinc Finger Region ◽  
In Erythroid Cells

GATA-1, the founding member of a distinctive family of transcription factors, is expressed predominantly in erythroid cells and participates in the expression of numerous erythroid cell-expressed genes. GATA-binding sites are found in the promoters and enhancers of globin and nonglobin erythroid genes as well as in the alpha- and beta-globin locus control regions. To elucidate how GATA-1 may function in a variety of regulatory contexts, we have examined its protein-protein interactions. Here we show that GATA-1 self-associates in solution and in whole-cell extracts and that the zinc finger region of the molecule is sufficient to mediate this interaction. This physical interaction can influence transcription, as GATA-1 self-association is able to recruit a transcriptionally active but DNA-binding-defective derivative of GATA-1 to promoter-bound GATA-1 and result in superactivation. Through in vitro studies with bacterially expressed glutathione S-transferase fusion proteins, we have localized the minimal domain required for GATA-1 self-association to 40 amino acid residues within the C-terminal zinc finger region. Finally, we have detected physical interaction of GATA-1 with other GATA family members (GATA-2 and GATA-3) also mediated through the zinc finger domain. These findings have broad implications for the involvement of GATA factors in transcriptional control. In particular, the interaction of GATA-1 with itself and with other transcription factors may facilitate its function at diverse promoters in erythroid cells and also serve to bring together, or stabilize, loops between distant regulatory elements, such as the globin locus control regions and downstream globin promoters. We suggest that the zinc finger region of GATA-1, and related proteins, is multifunctional and mediates not only DNA binding but also important protein-protein interactions.

scholarly journals Brief Guide: Experimental Strategies for High-Quality Hit Selection from Small-Molecule Screening Campaigns

2021 ◽  
pp. 247255522110088
Author(s):  
Ina Rothenaigner ◽  
Kamyar Hadian
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Protein Protein Interactions ◽  
High Quality ◽  
Biological Targets ◽  
Small Molecule Screening ◽  
Powerful Approach ◽  
Reporter Assays ◽  
Experimental Approaches

Small-molecule screening is a powerful approach to identify modulators of either specific biological targets or cellular pathways with phenotypic endpoints. A myriad of assay technologies are available to assess the activity of enzymes, monitor protein–protein interactions, measure transcription factor activity in reporter assays, or detect cellular features and activities using high-content imaging. A common challenge during small-molecule screening is, however, the presence of hit compounds generating assay interference, thereby producing false-positive hits. Thus, efforts are needed to uncover such interferences to prioritize high-quality hits for further analysis. This process encompasses (1) computational approaches to flag undesirable compounds, and (2) the use of experimental approaches like counter, orthogonal, and cellular fitness screens to identify and eliminate artifacts. In this brief guide, we provide an overview for first-time users, highlighting experimental screening strategies to prioritize high-quality bioactive hits from high-throughput screening/high-content screening (HTS/HCS) campaigns.

Computational Screening and Design for Compounds that Disrupt Protein-protein Interactions

2017 ◽  
Vol 17 (23) ◽  
pp. 2703-2714 ◽  
Author(s):  
David K. Johnson ◽  
John Karanicolas
Keyword(s):  
Small Molecule ◽  
Protein Interactions ◽  
Target Space ◽  
Computational Techniques ◽  
Protein Protein Interactions ◽  
Protein Surfaces ◽  
Design And Optimization ◽  
Computational Screening ◽  
Experimental Approaches ◽  
High Throughput Screens

Protein-protein interactions play key roles in all biological processes, motivating numerous campaigns to seek small-molecule disruptors of therapeutically relevant interactions. Two decades ago, the prospect of developing small-molecule inhibitors was thought to be perhaps impossible due to the potentially undruggable nature of the protein surfaces involved; this viewpoint was reinforced by the limited successes provided from traditional high-throughput screens. To date, however, refinement of new experimental approaches has led to a multitude of inhibitors against many different targets. Having thus established the feasibility of attaining success in this valuable and diverse target space, attention now turns to incorporating computational techniques that might assist during various stages of drug design and optimization. Here we review cases in which computational approaches – virtual screening, docking, and ligand optimization – have contributed to discovery of new inhibitors of protein-protein interactions. We conclude by providing an outlook into the upcoming challenges and recent advances likely to shape this field moving forward.

Sign in / Sign up

Export Citation Format

Share Document