Modern Biophysical Approaches to Study Protein–Ligand Interactions

2018 ◽  
Vol 13 (04) ◽  
pp. 133-155
Author(s):  
Priyanka Biswas
Keyword(s):  
Single Molecule ◽  
Analytical Ultracentrifugation ◽  
Correlation Spectroscopy ◽  
Titration Calorimetry ◽  
Resonance Fluorescence ◽  
Biological Interactions ◽  
Protein Ligand Interactions ◽  
Dual Polarization Interferometry ◽  
Ligand Interactions

Protein–ligand interactions act as a pivot to the understanding of most of the biological interactions. The study of interactions between proteins and cellular molecules has led to the establishment and identification of various important pathways that control biological systems. Investigators working in different fields of biological sciences have an intrinsic interest in this field and complement their findings by the application of different biophysical approaches and tools to quantify protein–ligand interactions that include protein–small molecules, protein–DNA, protein–RNA, protein–protein both in vitro and in vivo. In this paper, the various biophysical techniques that can be employed to study such interactions will be discussed. In addition to native gel electrophoresis and fluorescence-based methods, more details will be discussed, on the broad range of modern day biophysical tools such as Circular Dichroism, Fourier Transform Infrared (FTIR) Spectroscopy, Isothermal Titration Calorimetry, Analytical Ultracentrifugation, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy, Differential Scanning Fluorimetry, Nuclear Magnetic Resonance, Mass Spectroscopy, Single Molecule Spectroscopy, Dual Polarization Interferometry, Micro Scale Thermophoresis and Electro–switchable Biosensors that can be used to study the different aspects of protein–ligand interactions.

scholarly journals Protein–ligand interactions investigated by thermal shift assays (TSA) and dual polarization interferometry (DPI)

2015 ◽  
Vol 71 (1) ◽  
pp. 36-44 ◽  
Author(s):  
Morten K. Grøftehauge ◽  
Nelly R. Hajizadeh ◽  
Marcus J. Swann ◽  
Ehmke Pohl
Keyword(s):  
Spatial Information ◽  
Thermodynamic Description ◽  
Titration Calorimetry ◽  
Dual Polarization ◽  
Polarization Interferometry ◽  
Protein Ligand Interactions ◽  
Wide Range ◽  
Dual Polarization Interferometry ◽  
Ligand Interactions ◽  
Thermal Shift

Over the last decades, a wide range of biophysical techniques investigating protein–ligand interactions have become indispensable tools to complement high-resolution crystal structure determinations. Current approaches in solution range from high-throughput-capable methods such as thermal shift assays (TSA) to highly accurate techniques including microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) that can provide a full thermodynamic description of binding events. Surface-based methods such as surface plasmon resonance (SPR) and dual polarization interferometry (DPI) allow real-time measurements and can provide kinetic parameters as well as binding constants. DPI provides additional spatial information about the binding event. Here, an account is presented of new developments and recent applications of TSA and DPI connected to crystallography.

scholarly journals Arranging Small Molecules with Subnanometer Precision on DNA Origami Substrates for the Single‐Molecule Investigation of Protein–Ligand Interactions

2020 ◽  
Vol 1 (1) ◽  
pp. 2000038
Author(s):  
Jingyuan Huang ◽  
Antonio Suma ◽  
Meiying Cui ◽  
Guido Grundmeier ◽  
Vincenzo Carnevale ◽  
...  
Keyword(s):  
Small Molecules ◽  
Single Molecule ◽  
Dna Origami ◽  
Protein Ligand Interactions ◽  
Ligand Interactions

scholarly journals Avoiding accuracy-limiting pitfalls in the study of protein-ligand interactions with isothermal titration calorimetry

2015 ◽  
Author(s):  
Sarah E. Boyce ◽  
Joel Tellinghuisen ◽  
John D. Chodera
Keyword(s):  
Isothermal Titration Calorimetry ◽  
Protocol Design ◽  
Titration Calorimetry ◽  
Interlaboratory Comparisons ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Enthalpy And Entropy ◽  
Precision And Accuracy ◽  
Indispensable Tool ◽  
Limiting Errors

Isothermal titration calorimetry (ITC) can yield precise (±3%) estimates of the thermodynamic parameters describing biomolecular association (affinity, enthalpy, and entropy), making it an indispensable tool for biochemistry and drug discovery. Surprisingly, interlaboratory comparisons suggest that errors of ∼20% are common and widely underreported. Here, we show how to reduce precision- and accuracy-limiting errors while obtaining good estimates and minimizing material and time consumed by an experiment. We provide a simple spreadsheet that allows practitioners to identify precision-limiting operations during protocol design, track precision during the experiment, and propagate error to yield realistic final uncertainties.

scholarly journals A ClyA nanopore tweezer for analysis of functional states of protein-ligand interactions

2019 ◽  
Author(s):  
Xin Li ◽  
Kuohao Lee ◽  
Jianhan Chen ◽  
Min Chen
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Bound States ◽  
Conformational Dynamics ◽  
Electrical Current ◽  
Label Free ◽  
Protein Motions ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Molecular Tweezer

AbstractConformational changes of proteins are essential to their functions. Yet it remains challenging to measure the amplitudes and timescales of protein motions. Here we show that the ClyA nanopore can be used as a molecular tweezer to trap a single maltose-binding protein (MBP) within its lumen, which allows conformation changes to be monitored as electrical current fluctuations in real time. The current measurements revealed three distinct ligand-bound states for MBP in the presence of reducing saccharides. Our biochemical and kinetic analysis reveal that these three states represented MBP bound to different isomers of reducing sugars. These findings shed light on the mechanism of substrate recognition by MBP and illustrate that the nanopore tweezer is a powerful, label-free, single-molecule approach for studying protein conformational dynamics under functional conditions.

scholarly journals Phytoconstituents from Markhamia Tomentosa Bind To HPV Oncoprotein with Apoptogenic Potential: A Molecular Modeling Approach

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Mutiat B. Ibrahim ◽  
Adeola T. Kola-Mustapha ◽  
Niyi S. Adelakun ◽  
Neil A. Koorbanally
Keyword(s):  
Drug Targets ◽  
Active Sites ◽  
Caspase 3 ◽  
Cancer Cell Line ◽  
Hpv 16 ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Potent Growth ◽  
The Stability

Abstract Markhamia tomentosa crude extract and fractions exhibited potent growth inhibitory effects capable to induce apoptosis in cervical (HeLa) cancer cell line via in vitro model. Presently, interaction of M. tomentosa phytoconstituents with molecular drug targets to exert its anticancer property is evaluated via in silico study. Identified phytoconstituents from M. tomentosa were retrieved from PubChem database and docked in active sites of HPV 16 E6, caspase -3 and caspase -8 targets using AutoDockVina from PyRx software. Screening for druglikeness; and absorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions was carried out with the use of SwissADME and pkCSM web servers. Standard melphalan and co-crystallized ligands of caspases -3 and -8 enzymes were used to validate protein-ligand interactions. Molecular dynamic simulation was used to validate the stability of the hit molecules complexed with caspases -3 and -8. All identified phytoconstituents from M. tomentosa showed binding affinity for HPV with docking scores range of - 5.4 to -2.6 kcal/mol. Ajugol, carnosol, luteolin and phytol showed good docking energy range of -6.8 to -3.6 kcal/mol; and -4.8 to -1.9 kcal/mol for the active sites of caspases -3 and -8 targets respectively. Based on docking scores; drug-likeliness; and ADMET predictions; luteolin and carnosol were selected as hit compounds. These molecules were found to be stable within the binding site of caspase -3 target throughout the 40ns simulation time. These findings identified hit ligands from M. tomentosa phytoconstituents that inhibit HPV 16 E6 oncogene expression with stimulation of caspases -3 and -8 targets.

scholarly journals Variants of Escherichia coli Subtilase Cytotoxin Subunits Show Differences in Complex Formation In Vitro

Toxins ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 703 ◽  
Author(s):  
Maike Krause ◽  
Katharina Sessler ◽  
Anna Kaziales ◽  
Richard Grahl ◽  
Sabrina Noettger ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hela Cells ◽  
Analytical Ultracentrifugation ◽  
Cd Spectroscopy ◽  
Size Exclusion ◽  
Titration Calorimetry ◽  
Target Cells ◽  
Binding Behavior ◽  
Subtilase Cytotoxin

The subtilase cytotoxin (SubAB) of Shiga toxin-producing Escherichia coli (STEC) is a member of the AB5 toxin family. In the current study, we analyzed the formation of active homo- and hetero-complexes of SubAB variants in vitro to characterize the mode of assembly of the subunits. Recombinant SubA1-His, SubB1-His, SubA2-2-His, and SubB2-2-His subunits, and His-tag-free SubA2-2 were separately expressed, purified, and biochemically characterized by circular dichroism (CD) spectroscopy, size-exclusion chromatography (SEC), and analytical ultracentrifugation (aUC). To confirm their biological activity, cytotoxicity assays were performed with HeLa cells. The formation of AB5 complexes was investigated with aUC and isothermal titration calorimetry (ITC). Binding of SubAB2-2-His to HeLa cells was characterized with flow cytometry (FACS). Cytotoxicity experiments revealed that the analyzed recombinant subtilase subunits were biochemically functional and capable of intoxicating HeLa cells. Inhibition of cytotoxicity by Brefeldin A demonstrated that the cleavage is specific. All His-tagged subunits, as well as the non-tagged SubA2-2 subunit, showed the expected secondary structural compositions and oligomerization. Whereas SubAB1-His complexes could be reconstituted in solution, and revealed a Kd value of 3.9 ± 0.8 μmol/L in the lower micromolar range, only transient interactions were observed for the subunits of SubAB2-2-His in solution, which did not result in any binding constant when analyzed with ITC. Additional studies on the binding characteristics of SubAB2-2-His on HeLa cells revealed that the formation of transient complexes improved binding to the target cells. Conclusively, we hypothesize that SubAB variants exhibit different characteristics in their binding behavior to their target cells.

scholarly journals Footprinting of Inhibitor Interactions ofIn SilicoIdentified Inhibitors of Trypanothione Reductase ofLeishmaniaParasite

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Santhosh K. Venkatesan ◽  
Vikash Kumar Dubey
Keyword(s):  
Virtual Screening ◽  
Leishmania Infantum ◽  
Trypanothione Reductase ◽  
Inhibition Kinetics ◽  
Screening Process ◽  
Protein Ligand Interactions ◽  
Enzymatic Reduction ◽  
Ligand Interactions ◽  
First Time

Structure-based virtual screening of NCI Diversity set II compounds was performed to indentify novel inhibitor scaffolds of trypanothione reductase (TR) fromLeishmania infantum. The top 50 ranked hits were clustered using the AuPoSOM tool. Majority of the top-ranked compounds were Tricyclic. Clustering of hits yielded four major clusters each comprising varying number of subclusters differing in their mode of binding and orientation in the active site. Moreover, for the first time, we report selected alkaloids and dibenzothiazepines as inhibitors ofLeishmania infantumTR. The mode of binding observed among the clusters also potentiates the probablein vitroinhibition kinetics and aids in defining key interaction which might contribute to the inhibition of enzymatic reduction of T[S] 2. The method provides scope for automation and integration into the virtual screening process employing docking softwares, for clustering the small molecule inhibitors based upon protein-ligand interactions.

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