Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions
TL;DR: Flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which is demonstrated by addressing typically challenging types of binding events from various fields of life science.
About: This article is published in Methods.The article was published on 2013-03-01 and is currently open access. It has received 508 citations till now. The article focuses on the topics: Microscale thermophoresis & Thermophoresis.
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TL;DR: In this review, methods to adjust the polar solvation energy and to improve the performance of MM/PBSA and MM/GBSA calculations are reviewed and discussed and guidance is provided for practically applying these methods in drug design and related research fields.
Abstract: Molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) and molecular mechanics generalized Born surface area (MM/GBSA) are arguably very popular methods for binding free energy prediction since they are more accurate than most scoring functions of molecular docking and less computationally demanding than alchemical free energy methods. MM/PBSA and MM/GBSA have been widely used in biomolecular studies such as protein folding, protein-ligand binding, protein-protein interaction, etc. In this review, methods to adjust the polar solvation energy and to improve the performance of MM/PBSA and MM/GBSA calculations are reviewed and discussed. The latest applications of MM/GBSA and MM/PBSA in drug design are also presented. This review intends to provide readers with guidance for practically applying MM/PBSA and MM/GBSA in drug design and related research fields.
822 citations
TL;DR: This review presents recent progress and developments in MST technology and introduces alternative MST applications – such as determination of binding stoichiometries and binding modes, analysis of protein unfolding, thermodynamics and enzyme kinetics, as well as demonstrating the capability of MST to quantify high-affinity interactions with dissociation constants in the low picomolar (pM) range.
469 citations
TL;DR: This review attempted to combine a comprehensive list of various applications of nanodisc technology with systematic analysis of the most attractive features of this system and advantages provided by nanodISCs for structural and mechanistic studies of membrane proteins.
Abstract: Membrane proteins play a most important part in metabolism, signaling, cell motility, transport, development, and many other biochemical and biophysical processes which constitute fundamentals of life on the molecular level. Detailed understanding of these processes is necessary for the progress of life sciences and biomedical applications. Nanodiscs provide a new and powerful tool for a broad spectrum of biochemical and biophysical studies of membrane proteins and are commonly acknowledged as an optimal membrane mimetic system that provides control over size, composition, and specific functional modifications on the nanometer scale. In this review we attempted to combine a comprehensive list of various applications of nanodisc technology with systematic analysis of the most attractive features of this system and advantages provided by nanodiscs for structural and mechanistic studies of membrane proteins.
365 citations
TL;DR: The results illustrate that the MM/GBSA rescoring has better capability to distinguish the correct binding structures from the decoys than the ZDOCK scoring and may be a good choice for predicting the binding affinities and identifying correctbinding structures for protein-protein systems.
Abstract: Understanding protein–protein interactions (PPIs) is quite important to elucidate crucial biological processes and even design compounds that interfere with PPIs with pharmaceutical significance. Protein–protein docking can afford the atomic structural details of protein–protein complexes, but the accurate prediction of the three-dimensional structures for protein–protein systems is still notoriously difficult due in part to the lack of an ideal scoring function for protein–protein docking. Compared with most scoring functions used in protein–protein docking, the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) and Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) methodologies are more theoretically rigorous, but their overall performance for the predictions of binding affinities and binding poses for protein–protein systems has not been systematically evaluated. In this study, we first evaluated the performance of MM/PBSA and MM/GBSA to predict the binding affinities for 46 protein–protein complexes. On the whole, different force fields, solvation models, and interior dielectric constants have obvious impacts on the prediction accuracy of MM/GBSA and MM/PBSA. The MM/GBSA calculations based on the ff02 force field, the GB model developed by Onufriev et al. and a low interior dielectric constant (ein = 1) yield the best correlation between the predicted binding affinities and the experimental data (rp = −0.647), which is better than MM/PBSA (rp = −0.523) and a number of empirical scoring functions used in protein–protein docking (rp = −0.141 to −0.529). Then, we examined the capability of MM/GBSA to identify the possible near-native binding structures from the decoys generated by ZDOCK for 43 protein–protein systems. The results illustrate that the MM/GBSA rescoring has better capability to distinguish the correct binding structures from the decoys than the ZDOCK scoring. Besides, the optimal interior dielectric constant of MM/GBSA for re-ranking docking poses may be determined by analyzing the characteristics of protein–protein binding interfaces. Considering the relatively high prediction accuracy and low computational cost, MM/GBSA may be a good choice for predicting the binding affinities and identifying correct binding structures for protein–protein systems.
325 citations
TL;DR: It is indicated that quercetin showing inhibitory activity in the early stage of influenza infection provides a future therapeutic option to develop effective, safe and affordable natural products for the treatment and prophylaxis of IAV infections.
Abstract: Influenza A viruses (IAVs) cause seasonal pandemics and epidemics with high morbidity and mortality, which calls for effective anti-IAV agents. The glycoprotein hemagglutinin of influenza virus plays a crucial role in the initial stage of virus infection, making it a potential target for anti-influenza therapeutics development. Here we found that quercetin inhibited influenza infection with a wide spectrum of strains, including A/Puerto Rico/8/34 (H1N1), A/FM-1/47/1 (H1N1), and A/Aichi/2/68 (H3N2) with half maximal inhibitory concentration (IC50) of 7.756 ± 1.097, 6.225 ± 0.467, and 2.738 ± 1.931 μg/mL, respectively. Mechanism studies identified that quercetin showed interaction with the HA2 subunit. Moreover, quercetin could inhibit the entry of the H5N1 virus using the pseudovirus-based drug screening system. This study indicates that quercetin showing inhibitory activity in the early stage of influenza infection provides a future therapeutic option to develop effective, safe and affordable natural products for the treatment and prophylaxis of IAV infections.
286 citations
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References
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3,724 citations
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TL;DR: It is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed, and this gel method is applied to the study of the E. coli lactose operon regulatory system.
Abstract: The use of gel electrophoresis for quantitative studies of DNA-protein interactions is described. This rapid and simple technique involves separation of free DNA from DNA-protein complexes based on differences in their electrophoretic mobilities in polyacrylamide gels. Under favorable conditions both unbound DNA and DNA associated with protein can be quantified. This gel method is applied to the study of the E. coli lactose operon regulatory system. At ionic strengths in the physiological range, the catabolite activator protein (CAP) is shown to form a long-lived complex with the wild type lac promotor, but not with a CAP-insensitive mutant. Formation of a stable "open" or "melted-in" complex of RNA polymerase with the wild type promoter requires the participation of CAP and cyclic AMP. Further, it is demonstrated that even when pre-formed in the presence of CAP-cAMP, the polymerase-promoter open complex becomes unstable if CAP is then selectively removed.
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TL;DR: The most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions are identified and commonly used variants are discussed.
Abstract: The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this protocol, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.
948 citations
TL;DR: The results indicate that the Grbl/hSos1 complex couples activated EGF receptor to Ras signalling, and a synthetic 10-amino-acid peptide containing the sequence PPVPPR specifically blocks the interaction.
Abstract: Many of the actions of receptor tyrosine kinases are mediated by the protein Ras, including the activation of various downstream serine/threonine kinases and the stimulation of growth and differentiation. The human protein Grb2 binds to ligand-activated growth factor receptors and downstream effector proteins through its Src-homology (SH) domains SH2 and SH3, respectively, and like its homologue from Caenorhabditis elegans, Sem-5, apparently forms part of a highly conserved pathway by which these receptors can control Ras activity. Here we show that the SH3 domains of Grb2 bind to the carboxy-terminal part of hSos1, the human homologue of the Drosophila guanine-nucleotide-releasing factor for Ras, which is essential for control of Ras activity by epidermal growth factor receptor and sevenless. Moreover, a synthetic 10-amino-acid peptide containing the sequence PPVPPR specifically blocks the interaction. These results indicate that the Grb2/hSos1 complex couples activated EGF receptor to Ras signalling.
944 citations