Measurement of Nanomolar Dissociation Constants by Titration Calorimetry and Thermal Shift Assay – Radicicol Binding to Hsp90 and Ethoxzolamide Binding to CAII

The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90αN) and the binding of ethoxzolamide to human carbonic anhydrase (hCAII) were too strong to be measured accurately by direct ITC titration and therefore were measured by displacement ITC and by observing the temperature-denaturation transitions of ligand-free and ligand-bound protein. Stabilization of both proteins by their ligands was profound, increasing the melting temperature by more than 10 ºC, depending on ligand concentration. Analysis of the melting temperature dependence on the protein and ligand concentrations yielded dissociation constants equal to 1 nM and 2 nM for Hsp90αN-radicicol and hCAII-ethoxzolamide, respectively. The ligand-free and ligand-bound protein fractions melt separately, and two melting transitions are observed. This phenomenon is especially pronounced when the ligand concentration is equal to about half the protein concentration. The analysis compares ITC and TSA data, accounts for two transitions and yields the ligand binding constant and the parameters of protein stability, including the Gibbs free energy and the enthalpy of unfolding

Thermodynamics of Aryl-Dihydroxyphenyl-Thiadiazole Binding to Human Hsp90

The design of specific inhibitors against the Hsp90 chaperone and other enzyme relies on the detailed and correct understanding of both the thermodynamics of inhibitor binding and the structural features of the protein-inhibitor complex. Here we present a detailed thermodynamic study of binding of aryl-dihydroxyphenyl-thiadiazole inhibitor series to recombinant human Hsp90 alpha isozyme. The inhibitors are highly potent, with the intrinsic Kd approximately equal to 1 nM as determined by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). Dissection of protonation contributions yielded the intrinsic thermodynamic parameters of binding, such as enthalpy, entropy, Gibbs free energy, and the heat capacity. The differences in binding thermodynamic parameters between the series of inhibitors revealed contributions of the functional groups, thus providing insight into molecular reasons for improved or diminished binding efficiency. The inhibitor binding to Hsp90 alpha primarily depended on a large favorable enthalpic contribution combined with the smaller favorable entropic contribution, thus suggesting that their binding was both enthalpically and entropically optimized. The enthalpy-entropy compensation phenomenon was highly evident when comparing the inhibitor binding enthalpies and entropies. This study illustrates how detailed thermodynamic analysis helps to understand energetic reasons for the binding efficiency and develop more potent inhibitors that could be applied for therapeutic use as Hsp90 inhibitors

Type II restriction-modification system from Gardnerella vaginalis ATCC 14018

Intensive horizontal gene transfer may generate diversity and heterogeneity within the genus Gardnerella. Restriction-modification (R-M) systems and CRISPR-Cas are the principal defense tools against foreign DNA in bacteria. Nearly half of the tested Gardnerella spp. isolates harbored the CRISPR-Cas system. Several putative R-M systems of Gardnerella spp. strains were identified in the REBASE database. However, there was no experimental evidence for restriction endonuclease (REase) activity in the isolates. We showed that G. vaginalis strain ATCC 14018 contains the REase R.Gva14018I, which recognizes GGCC and most probably generates blunt ends on cleavage. Bioinformatics evidence and the activity of recombinant methyltransferase M.Gva14018I in vivo indicate that ATCC 14018 possesses a HaeIII-like R-M system. The truncated R.Gva14018I-4 lacking the C-terminal region was expressed in Escherichia coli and displayed wild-type REase specificity. Polyclonal antibodies against R.Gva14018I-4 detected the wild-type REase in the cell lysate of ATCC 14018. The cofactor requirements for activity and bioinformatics analysis indicated that R.Gva14018I belongs to the PD-(D/E)XK family of REases. The REase-like activity was observed in 5 of 31 tested Gardnerella spp. strains, although none of these matched the DNA digestion pattern of R.Gva14018I

Thiazide and other Cl-benzenesulfonamide-bearing clinical drug affinities for human carbonic anhydrases

Twelve carbonic anhydrase (CA) isoforms catalyze carbon dioxide hydration to bicarbonate and acid protons and are responsible for many biological functions in human body. Despite their vital functions, they are also responsible for, or implicated in, numerous ailments and diseases such as glaucoma, high altitude sickness, and cancer. Because CA isoforms are highly homologous, clinical drugs designed to inhibit enzymatic activity of a particular isoform, can also bind to others with similar affinity causing toxic side effects. In this study, the affinities of twelve CA isoforms have been determined for nineteen clinically used drugs used to treat hypertension related diseases, i.e. thiazides, indapamide, and metolazone. Their affinities were determined using a fluorescent thermal shift assay. Stopped flow assay and isothermal titration calorimetry were also employed on a subset of compounds and proteins to confirm inhibition of CA enzymatic activity and verify the quantitative agreement between different assays. The findings of this study showed that pharmaceuticals could bind to human CA isoforms with variable affinities and inhibit their catalytic activity, even though the drug was intended to interact with a different (non-CA) protein target. Relatively minor structural changes of the compounds may cause significant changes in affinity and selectivity for a particular CA isoform

Protein–Ligand Binding Volume Determined from a Single 2D NMR Spectrum with Increasing Pressure

[Image: see text] Proteins undergo changes in their partial volumes in numerous biological processes such as enzymatic catalysis, unfolding–refolding, and ligand binding. The change in the protein volume upon ligand binding—a parameter termed the protein–ligand binding volume—can be extensively studied by high-pressure NMR spectroscopy. In this study, we developed a method to determine the protein–ligand binding volume from a single two-dimensional (2D) (1)H–(15)N heteronuclear single quantum coherence (HSQC) spectrum at different pressures, if the exchange between ligand-free and ligand-bound states of a protein is slow in the NMR time-scale. This approach required a significantly lower amount of protein and NMR time to determine the protein–ligand binding volume of two carbonic anhydrase isozymes upon binding their ligands. The proposed method can be used in other protein–ligand systems and expand the knowledge about protein volume changes upon small-molecule binding

Representative ITC data for the binding of ICPD compounds to recombinant human Hsp90 protein constructs as a function of pH, temperature, and buffer ionization enthalpy.

<p>Repeated experiments at identical conditions in different series are shown to illustrate the level of experimental error involved in these results.</p>a<p>Pi is sodium phosphate buffer. The standard deviations were about 3 kJ×mol⁻¹ for the enthalpy and up to 1.6 fold for the <i>K_d</i>, especially when the binding was too tight to be measured accurately using ITC.</p

Bar chart comparing relative contributions of intrinsic enthalpies and intrinsic entropies to the Gibbs free energies of binding of ICPD compound to Hsp90 at 37°C.

<p>Note, that with the partial exception of ICPD62, the compounds bound with major exothermic enthalpy contribution and minor favorable entropy contribution. Favorable contributions of both components make the compounds such potent binders.</p

Enthalpic contributions of the binding-linked reactions are shown, including, the protonation of the compound (ICPD47 ) hydroxy group (bold and red), buffer ionization, and the intrinsic enthalpy of binding into the experimentally observed values.

<p>Enthalpic contributions of the binding-linked reactions are shown, including, the protonation of the compound (ICPD47 ) hydroxy group (bold and red), buffer ionization, and the intrinsic enthalpy of binding into the experimentally observed values.</p

The observed binding constant (<i>K_{b_obs}</i>) dependence on pH.

<p>The observed binding constants of the interaction of ICPD47 with Hsp90αN obtained using three experimental approaches: ▵ – ITC in phosphate buffer, □ – ITC in Tris buffer, and • – TSA, all at 37°C. There is a clear decrease in the binding affinity at higher pH. The line is fitted according to Eq. (1) for a linked protonation event using p<i>K_a^f</i>  = 6.72. Note, that the ITC data does not provide an accurate measure of the binding constant at pH values below 8.0 because the binding is too tight, as shown by the dashed line drawn for the ITC <i>c</i> factor equal to 500.</p