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F1000Research 2018Voltage-gated calcium (Ca) channels are associated with β and αδ auxiliary subunits. This review will concentrate on the function of the αδ protein family, which... (Review)
Review
Voltage-gated calcium (Ca) channels are associated with β and αδ auxiliary subunits. This review will concentrate on the function of the αδ protein family, which has four members. The canonical role for αδ subunits is to convey a variety of properties on the Ca1 and Ca2 channels, increasing the density of these channels in the plasma membrane and also enhancing their function. More recently, a diverse spectrum of non-canonical interactions for αδ proteins has been proposed, some of which involve competition with calcium channels for αδ or increase αδ trafficking and others which mediate roles completely unrelated to their calcium channel function. The novel roles for αδ proteins which will be discussed here include association with low-density lipoprotein receptor-related protein 1 (LRP1), thrombospondins, α-neurexins, prion proteins, large conductance (big) potassium (BK) channels, and -methyl-d-aspartate (NMDA) receptors.
Topics: Animals; Calcium Channels; Calcium Channels, L-Type; Calcium Channels, N-Type; Humans; Protein Binding; Protein Subunits
PubMed: 30519455
DOI: 10.12688/f1000research.16104.1 -
Nucleic Acids Research Jul 2018CavityPlus is a web server that offers protein cavity detection and various functional analyses. Using protein three-dimensional structural information as the input,...
CavityPlus is a web server that offers protein cavity detection and various functional analyses. Using protein three-dimensional structural information as the input, CavityPlus applies CAVITY to detect potential binding sites on the surface of a given protein structure and rank them based on ligandability and druggability scores. These potential binding sites can be further analysed using three submodules, CavPharmer, CorrSite, and CovCys. CavPharmer uses a receptor-based pharmacophore modelling program, Pocket, to automatically extract pharmacophore features within cavities. CorrSite identifies potential allosteric ligand-binding sites based on motion correlation analyses between cavities. CovCys automatically detects druggable cysteine residues, which is especially useful to identify novel binding sites for designing covalent allosteric ligands. Overall, CavityPlus provides an integrated platform for analysing comprehensive properties of protein binding cavities. Such analyses are useful for many aspects of drug design and discovery, including target selection and identification, virtual screening, de novo drug design, and allosteric and covalent-binding drug design. The CavityPlus web server is freely available at http://repharma.pku.edu.cn/cavityplus or http://www.pkumdl.cn/cavityplus.
Topics: Allosteric Site; Binding Sites; Biophysical Phenomena; Internet; Ligands; Protein Binding; Protein Conformation; Proteins; Software
PubMed: 29750256
DOI: 10.1093/nar/gky380 -
Methods (San Diego, Calif.) Aug 2018Many biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these... (Review)
Review
Many biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these interactions is zonal elution affinity chromatography. This review discusses the theory and principles of zonal elution affinity chromatography, along with its general applications. Examples of applications that are examined include the use of this method to estimate the relative extent of solute-protein binding, to examine solute-solute competition and displacement from proteins, and to measure the strength of these interactions. It is also shown how zonal elution affinity chromatography can be used in solvent and temperature studies and to characterize the binding sites for solutes on proteins. In addition, several alternative applications of zonal elution affinity chromatography are discussed, which include the analysis of binding by a solute with a soluble binding agent and studies of allosteric effects. Other recent applications that are considered are the combined use of immunoextraction and zonal elution for drug-protein binding studies, and binding studies that are based on immobilized receptors or small targets.
Topics: Binding Sites; Chromatography, Affinity; Protein Binding; Thermodynamics
PubMed: 29409783
DOI: 10.1016/j.ymeth.2018.01.020 -
Biochemistry Mar 2010The NF-kappaB family of transcription factors responds to inflammatory cytokines with rapid transcriptional activation and subsequent signal repression. Much of the... (Review)
Review
The NF-kappaB family of transcription factors responds to inflammatory cytokines with rapid transcriptional activation and subsequent signal repression. Much of the system control depends on the unique characteristics of its major inhibitor, IkappaBalpha, which appears to have folding dynamics that underlie the biophysical properties of its activity. Theoretical folding studies followed by experiments have shown that a portion of the ankyrin repeat domain of IkappaBalpha folds on binding. In resting cells, IkappaBalpha is constantly being synthesized, but most of it is rapidly degraded, leaving only a very small pool of free IkappaBalpha. Nearly all of the NF-kappaB is bound to IkappaBalpha, resulting in near-complete inhibition of nuclear localization and transcriptional activation. Combined solution biophysical measurements and quantitative protein half-life measurements inside cells have allowed us to understand how the inhibition occurs, why IkappaBalpha can be degraded quickly in the free state but remain extremely stable in the bound state, and how signal activation and repression can be tuned by IkappaB folding dynamics. This review summarizes results of in vitro and in vivo experiments that converge demonstrating the effective interplay between biophysics and cell biology in understanding transcriptional control by the NF-kappaB signaling module.
Topics: Animals; Humans; I-kappa B Proteins; Models, Biological; NF-KappaB Inhibitor alpha; NF-kappa B; Protein Binding; Protein Folding; Signal Transduction
PubMed: 20055496
DOI: 10.1021/bi901948j -
Postepy Higieny I Medycyny... Jul 2010Poly(ADP-ribose)polymerase-1 (PARP-1) catalyzes the polymerization of ADP-ribose units from NAD+ modules on target proteins, resulting in the attachment of linear or... (Review)
Review
Poly(ADP-ribose)polymerase-1 (PARP-1) catalyzes the polymerization of ADP-ribose units from NAD+ modules on target proteins, resulting in the attachment of linear or branched polymers. PARP-1 and its product poly(ADP-ribose)--PAR have recently received considerable attention because of their involvement in a wide range of cellular processes including chromatin modification, metabolism of nucleic acids, transcription regulation, and cell death. This review summarizes recent work on modular structure of six functional domains (A-F) of PARP-1 molecule in the context of three classic domains, i.e., DNA binding (DBD), automodification (AD) and catalytic (CD) released by proteolytic enzymes. A special attention is paid to subcellular localization and molecular mechanisms of PARP-1 posttranslational modifications, such as: poly(ADP-ribosylation), phosphorylation, acetylation and sumolyation. In addition, main functions of PARP-1 are discussed, focusing on the activity of this enzyme in DNA damage detection and repair, genome stability, and cell death.
Topics: Animals; Humans; Poly(ADP-ribose) Polymerases; Protein Binding
PubMed: 20679690
DOI: No ID Found -
Methods in Molecular Biology (Clifton,... 2021The confirmation of a small molecule binding to a protein target can be challenging when switching from biochemical assays to physiologically relevant cellular models....
The confirmation of a small molecule binding to a protein target can be challenging when switching from biochemical assays to physiologically relevant cellular models. The cellular thermal shift assay (CETSA) is an approach to validate ligand-protein binding in a cellular environment by examining a protein's melting profile which can shift to a higher or lower temperature when bound by a small molecule. Traditional CETSA uses SDS-PAGE and Western blotting to quantify protein levels, a process that is both time consuming and low-throughput when screening multiple compounds and concentrations. Herein, we outline the reagents and methods to implement split Nano Luciferase (SplitLuc) CETSA, which is a reporter-based target engagement assay designed for high-throughput screening in 384- or 1536-well plate formats.
Topics: Biological Assay; High-Throughput Screening Assays; Ligands; Luciferases; Protein Binding
PubMed: 34432237
DOI: 10.1007/978-1-0716-1665-9_2 -
PLoS Computational Biology Mar 2018Cell division, endocytosis, and viral budding would not function without the localization and assembly of protein complexes on membranes. What is poorly appreciated,...
Cell division, endocytosis, and viral budding would not function without the localization and assembly of protein complexes on membranes. What is poorly appreciated, however, is that by localizing to membranes, proteins search in a reduced space that effectively drives up concentration. Here we derive an accurate and practical analytical theory to quantify the significance of this dimensionality reduction in regulating protein assembly on membranes. We define a simple metric, an effective equilibrium constant, that allows for quantitative comparison of protein-protein interactions with and without membrane present. To test the importance of membrane localization for driving protein assembly, we collected the protein-protein and protein-lipid affinities, protein and lipid concentrations, and volume-to-surface-area ratios for 46 interactions between 37 membrane-targeting proteins in human and yeast cells. We find that many of the protein-protein interactions between pairs of proteins involved in clathrin-mediated endocytosis in human and yeast cells can experience enormous increases in effective protein-protein affinity (10-1000 fold) due to membrane localization. Localization of binding partners thus triggers robust protein complexation, suggesting that it can play an important role in controlling the timing of endocytic protein coat formation. Our analysis shows that several other proteins involved in membrane remodeling at various organelles have similar potential to exploit localization. The theory highlights the master role of phosphoinositide lipid concentration, the volume-to-surface-area ratio, and the ratio of 3D to 2D equilibrium constants in triggering (or preventing) constitutive assembly on membranes. Our simple model provides a novel quantitative framework for interpreting or designing in vitro experiments of protein complexation influenced by membrane binding.
Topics: Cell Membrane; Computer Simulation; Cytoplasm; Cytosol; Diffusion; Endocytosis; Membrane Proteins; Models, Biological; Multiprotein Complexes; Protein Binding
PubMed: 29505559
DOI: 10.1371/journal.pcbi.1006031 -
Proceedings of the National Academy of... Aug 2008Allostery, the coupling between ligand binding and protein conformational change, is the heart of biological network and it has often been explained by two...
Allostery, the coupling between ligand binding and protein conformational change, is the heart of biological network and it has often been explained by two representative models, the induced-fit and the population-shift models. Here, we clarified for what systems one model fits better than the other by performing molecular simulations of coupled binding and conformational change. Based on the dynamic energy landscape view, we developed an implicit ligand-binding model combined with the double-basin Hamiltonian that describes conformational change. From model simulations performed for a broad range of parameters, we uncovered that each of the two models has its own range of applicability, stronger and longer-ranged interaction between ligand and protein favors the induced-fit model, and weaker and shorter-ranged interaction leads to the population-shift model. We further postulate that the protein binding to small ligand tends to proceed via the population-shift model, whereas the protein docking to macromolecules such as DNA tends to fit the induced-fit model.
Topics: Antibodies, Monoclonal; DNA; Ligands; Models, Molecular; Protein Binding; Protein Structure, Quaternary
PubMed: 18678900
DOI: 10.1073/pnas.0802524105 -
Molecular Therapy : the Journal of the... Apr 2016Protein-protein interactions (PPIs) underlie most biological processes. An increasing interest to investigate the unexplored potential of PPIs in drug discovery is... (Review)
Review
Protein-protein interactions (PPIs) underlie most biological processes. An increasing interest to investigate the unexplored potential of PPIs in drug discovery is driven by the need to find novel therapeutic targets for a whole range of diseases with a high unmet medical need. To date, PPI inhibition with small molecules is the mechanism that has most often been explored, resulting in significant progress towards drug development. However, also PPI stabilization is gradually gaining ground. In this review, we provide a focused overview of a number of PPIs that control critical regulatory pathways and constitute targets for the design of novel therapeutics. We discuss PPI-modulating small molecules that are already pursued in clinical trials. In addition, we review a number of PPIs that are still under preclinical investigation but for which preliminary data support their use as therapeutic targets.
Topics: Animals; Drug Discovery; Drug Evaluation, Preclinical; Gene Regulatory Networks; Humans; Protein Binding; Protein Interaction Mapping; Proteins
PubMed: 26675501
DOI: 10.1038/mt.2015.214 -
Methods (San Diego, Calif.) Sep 2018Among the tools of structural biology, NMR spectroscopy is unique in that it not only derives a static three-dimensional structure, but also provides an atomic-level... (Review)
Review
Among the tools of structural biology, NMR spectroscopy is unique in that it not only derives a static three-dimensional structure, but also provides an atomic-level description of the local fluctuations and global dynamics around this static structure. A battery of NMR experiments is now available to probe the motions of proteins and nucleic acids over the whole biologically relevant timescale from picoseconds to hours. Here we focus on one of these methods, relaxation dispersion, which resolves dynamics on the micro- to millisecond timescale. Key biological processes that occur on this timescale include enzymatic catalysis, ligand binding, and local folding. In other words, relaxation-dispersion-resolved dynamics are often closely related to the function of the molecule and therefore highly interesting to the structural biochemist. With an astounding sensitivity of ∼0.5%, the method detects low-population excited states that are invisible to any other biophysical method. The kinetics of the exchange between the ground state and excited states are quantified in the form of the underlying exchange rate, while structural information about the invisible excited state is obtained in the form of its chemical shift. Lastly, the population of the excited state can be derived. This diversity in the information that can be obtained makes relaxation dispersion an excellent method to study the detailed mechanisms of conformational transitions and molecular interactions. Here we describe the two branches of relaxation dispersion, R and R, discussing their applicability, similarities, and differences, as well as recent developments in pulse sequence design and data processing.
Topics: Ligands; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Protein Conformation; Proteins
PubMed: 29704666
DOI: 10.1016/j.ymeth.2018.04.026