◾ Isothermal Titration Calorimetry and Fluorescent Thermal and Pressure Shift Assays in Protein–Ligand Interactions

2016 ◽  
pp. 277-296
Keyword(s):  
Isothermal Titration Calorimetry ◽  
Titration Calorimetry ◽  
Pressure Shift ◽  
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.

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.

Structures of apo and GTP-bound molybdenum cofactor biosynthesis protein MoaC fromThermus thermophilusHB8

2010 ◽  
Vol 66 (7) ◽  
pp. 821-833 ◽  
Author(s):  
Shankar Prasad Kanaujia ◽  
Jeyaraman Jeyakanthan ◽  
Noriko Nakagawa ◽  
Sathyaramya Balasubramaniam ◽  
Akeo Shinkai ◽  
...  
Keyword(s):  
Thermus Thermophilus ◽  
Md Simulations ◽  
Molybdenum Cofactor ◽  
Titration Calorimetry ◽  
Ligand Complex ◽  
Protein Ligand Interactions ◽  
Enzyme Superfamily ◽  
Ligand Interactions ◽  
Cofactor Biosynthesis ◽  
Molybdenum Cofactor Biosynthesis

The first step in the molybdenum cofactor (Moco) biosynthesis pathway involves the conversion of guanosine triphosphate (GTP) to precursor Z by two proteins (MoaA and MoaC). MoaA belongs to theS-adenosylmethionine-dependent radical enzyme superfamily and is believed to generate protein and/or substrate radicals by reductive cleavage ofS-adenosylmethionine using an Fe–S cluster. MoaC has been suggested to catalyze the release of pyrophosphate and the formation of the cyclic phosphate of precursor Z. However, structural evidence showing the binding of a substrate-like molecule to MoaC is not available. Here, apo and GTP-bound crystal structures of MoaC fromThermus thermophilusHB8 are reported. Furthermore, isothermal titration calorimetry experiments have been carried out in order to obtain thermodynamic parameters for the protein–ligand interactions. In addition, molecular-dynamics (MD) simulations have been carried out on the protein–ligand complex of known structure and on models of relevant complexes for which X-ray structures are not available. The biophysical, structural and MD results reveal the residues that are involved in substrate binding and help in speculating upon a possible mechanism.

scholarly journals Structure-based screening of binding affinities via small-angle X-ray scattering

IUCrJ ◽  
2020 ◽  
Vol 7 (4) ◽  
pp. 644-655
Author(s):  
Po-chia Chen ◽  
Pawel Masiewicz ◽  
Kathryn Perez ◽  
Janosch Hennig
Keyword(s):  
Conformational Changes ◽  
Small Angle ◽  
Titration Calorimetry ◽  
X Ray ◽  
Protein Ligand Interactions ◽  
X Ray Scattering ◽  
Ligand Interactions ◽  
Titration Protocol ◽  
Screen Performance ◽  
Ray Scattering

Protein–protein and protein–ligand interactions often involve conformational changes or structural rearrangements that can be quantified by solution small-angle X-ray scattering (SAXS). These scattering intensity measurements reveal structural details of the bound complex, the number of species involved and, additionally, the strength of interactions if carried out as a titration. Although a core part of structural biology workflows, SAXS-based titrations are not commonly used in drug discovery contexts. This is because prior knowledge of expected sample requirements, throughput and prediction accuracy is needed to develop reliable ligand screens. This study presents the use of the histidine-binding protein (26 kDa) and other periplasmic binding proteins to benchmark ligand screen performance. Sample concentrations and exposure times were varied across multiple screening trials at four beamlines to investigate the accuracy and precision of affinity prediction. The volatility ratio between titrated scattering curves and a common apo reference is found to most reliably capture the extent of structural and population changes. This obviates the need to explicitly model scattering intensities of bound complexes, which can be strongly ligand-dependent. Where the dissociation constant is within 102 of the protein concentration and the total exposure times exceed 20 s, the titration protocol presented at 0.5 mg ml−1 yields affinities comparable to isothermal titration calorimetry measurements. Estimated throughput ranges between 20 and 100 ligand titrations per day at current synchrotron beamlines, with the limiting step imposed by sample handling and cleaning procedures.

scholarly journals The thermodynamic profile and molecular interactions of a C(9)-cytisine derivative-binding acetylcholine-binding protein from Aplysia californica

2020 ◽  
Vol 76 (2) ◽  
pp. 74-80
Author(s):  
Samuel Davis ◽  
Hugo Rego Campello ◽  
Timothy Gallagher ◽  
William N. Hunter
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Binding Protein ◽  
Aplysia Californica ◽  
Side Chain ◽  
Titration Calorimetry ◽  
Acetylcholine Binding Protein ◽  
Protein Ligand Interactions ◽  
Ligand Interactions

Cytisine, a natural product with high affinity for clinically relevant nicotinic acetylcholine receptors (nAChRs), is used as a smoking-cessation agent. The compound displays an excellent clinical profile and hence there is an interest in derivatives that may be further improved or find use in the treatment of other conditions. Here, the binding of a cytisine derivative modified by the addition of a 3-(hydroxypropyl) moiety (ligand 4) to Aplysia californica acetylcholine-binding protein (AcAChBP), a surrogate for nAChR orthosteric binding sites, was investigated. Isothermal titration calorimetry revealed that the favorable binding of cytisine and its derivative to AcAChBP is driven by the enthalpic contribution, which dominates an unfavorable entropic component. Although ligand 4 had a less unfavorable entropic contribution compared with cytisine, the affinity for AcAChBP was significantly diminished owing to the magnitude of the reduction in the enthalpic component. The high-resolution crystal structure of the AcAChBP–4 complex indicated close similarities in the protein–ligand interactions involving the parts of 4 common to cytisine. The point of difference, the 3-(hydroxypropyl) substituent, appears to influence the conformation of the Met133 side chain and helps to form an ordered solvent structure at the edge of the orthosteric binding site.

scholarly journals Designing ligands to bind proteins

2005 ◽  
Vol 38 (4) ◽  
pp. 385-395 ◽  
Author(s):  
George M. Whitesides ◽  
Vijay M. Krishnamurthy
Keyword(s):  
Tight Binding ◽  
Difficult Problem ◽  
Rational Drug Design ◽  
Titration Calorimetry ◽  
Protein Ligand Interactions ◽  
Rational Drug ◽  
Ligand Interactions ◽  
Model Protein ◽  
Technological Developments ◽  
Simple Systems

The ability to design drugs (so-called ‘rational drug design’) has been one of the long-term objectives of chemistry for 50 years. It is an exceptionally difficult problem, and many of its parts lie outside the expertise of chemistry. The much more limited problem – how to design tight-binding ligands (rational ligand design) – would seem to be one that chemistry could solve, but has also proved remarkably recalcitrant. The question is ‘Why is it so difficult?’ and the answer is ‘We still don't entirely know’. This perspective discusses some of the technical issues – potential functions, protein plasticity, enthalpy/entropy compensation, and others – that contribute, and suggests areas where fundamental understanding of protein–ligand interactions falls short of what is needed. It surveys recent technological developments (in particular, isothermal titration calorimetry) that will, hopefully, make now the time for serious progress in this area. It concludes with the calorimetric examination of the association of a series of systematically varied ligands with a model protein. The counterintuitive thermodynamic results observed serve to illustrate that, even in relatively simple systems, understanding protein–ligand association is challenging.

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.

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