Thermodynamic analysis of the activation mechanism of the GCSF receptor induced by ligand binding

The granulocyte colony-stimulating factor receptor (GCSFR), containing the Ig-like domain (Ig) and cytokine receptor homologous region (CRH), was prepared as a preformed dimer (Ig-CRH-Fc)(2) after fusion to the mouse Fc region via an eight-residue linker (approximately 55 A). Monomer Ig-CRH was also prepared after the Fc region was removed from (Ig-CRH-Fc)(2). GCSF binding to Ig-CRH and (Ig-CRH-Fc)(2) was investigated using light scattering and isothermal titration calorimetry. The average molecular mass determined by light scattering showed that both Ig-CRH and (Ig-CRH-Fc)(2) formed a 2:2 dimer with GCSF. Moreover, isothermal titration calorimetry showed that the thermodynamic parameters upon binding of GCSF to Ig-CRH and (Ig-CRH-Fc)(2) were comparable, suggesting a similar binding stoichiometry and interface [including similar buried surface area (5700-6000 A(2))] despite the presence of the eight-residue linker. The buried surface area is much larger than that calculated from our previous report of the crystal structure of the GCSF-CRH complex [Aritomi, M., et al. (1999) Nature 401, 713-717], suggesting a substantial contribution of the Ig domain to GCSF binding. The data also indicate that the distance (55 A) between two CRH domains in the 2:2 complex is much shorter than in our previous model (approximately 90 A) predicted from the same crystal structure of the GCSF-CRH complex.     

Electrostatic interaction and complex formation between gum arabic and bovine serum albumin

The interaction of gum arabic (GA) and bovine serum albumin (BSA) has been investigated through turbidity and light scattering intensity measurements and by the use of dynamic light scattering, laser Doppler velocimetry, and isothermal titration calorimetry. It has been shown that GA and BSA can form soluble and insoluble complexes depending on the solution pH and the mixing ratio and is a function of the net charge on the complex. Soluble complexes were obtained when the electrophoretic mobility was greater than ±1. 5 μm s(-1) V(-1) cm(-1). Changes in the value of the isoelectric point of the complexes with mixing ratio and isothermal titration calorimetric data indicated that complexes formed at pHs 3 and 4 consisted of ～60 BSA molecules for every GA molecule, while at pH 5 there were ～10 BSA molecules per GA molecule. Calorimetric studies also indicated that the interaction occurred in two stages at both pH 3 and pH 4, but that the nature of the interaction at these two pH values was significantly different. This was attributed to differences in the relative magnitude of the positive and negative charges on the BSA and GA, respectively, and possibly due to changes in the BSA conformation. The fact that there is an interaction at pH 5, which is above the isoelectric point of the BSA, is due to the interaction of the carboxylate groups on the GA with positive patches on the BSA or to the charge regulation of the protein-polysaccharide system brought about by changes in dissociation equilibria. Complexation is reduced as the ionic strength of the solvent increases and is prevented at a NaCl concentration of 120 mM.     

Engineering a structure switching mechanism into a steroid-binding aptamer and hydrodynamic analysis of the ligand binding mechanism

The steroid binding mechanism of a DNA aptamer was studied using isothermal titration calorimetry (ITC), NMR spectroscopy, quasi-elastic light scattering (QELS), and small-angle X-ray spectroscopy (SAXS). Binding affinity determination of a series of steroid-binding aptamers derived from a parent cocaine-binding aptamer demonstrates that substituting a GA base pair with a GC base pair governs the switch in binding specificity from cocaine to the steroid deoxycholic acid (DCA). Binding of DCA to all aptamers is an enthalpically driven process with an unfavorable binding entropy. We engineered into the steroid-binding aptamer a ligand-induced folding mechanism by shortening the terminal stem by two base pairs. NMR methods were used to demonstrate that there is a transition from a state where base pairs are formed in one stem of the free aptamer, to where three stems are formed in the DCA-bound aptamer. The ability to generate a ligand-induced folding mechanism into a DNA aptamer architecture based on the three-way junction of the cocaine-binding aptamer opens the door to obtaining a series of aptamers all with ligand-induced folding mechanisms but triggered by different ligands. Hydrodynamic data from diffusion NMR spectroscopy, QELS, and SAXS show that for the aptamer with the full-length terminal stem there is a small amount of structure compaction with DCA binding. For ligand binding by the short terminal stem aptamer, we propose a binding mechanism where secondary structure forms upon DCA binding starting from a free structure where the aptamer exists in a compact form.     

Observation and characterization of the interaction between a single immunoglobulin binding domain of protein L and two equivalents of human kappa light chains

Detailed stopped-flow studies in combination with site-directed mutagenesis, isothermal titration calorimetry data and x-ray crystallographic knowledge have revealed that the biphasic pre-equilibrium fluorescence changes reported for a single Ig-binding domain of protein L from Peptostreptococcus magnus binding to kappa light chain are due to the binding of the kappa light chain at two separate sites on the protein L molecule. Elimination of binding site 2 through the mutation A66W has allowed the K(d) for kappa light chain binding at site 1 to be measured by stopped-flow fluorescence and isothermal titration calorimetry techniques, giving values of 48.0 +/- 8.0 nM and 37.5 +/- 7.3 nM respectively. Conversely, a double mutation Y53F/L57H eliminates binding at site 1 and has allowed the K(d) for binding at site 2 to be determined. Stopped-flow fluorimetry suggests this to be 3.4 +/- 0.8 microM in good agreement with the value of 4.6 +/- 0.8 microM determined by isothermal titration calorimetry. The mutation Y53F reduces the affinity of site 1 to approximately that of site 2.     

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L(-1)) than in its absence (93 micromol L(-1)). Apparent binding constant values were 2160 and 10,000 L mol(-1), respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide.     

Multithermal titration calorimetry: a rapid method to determine binding heat capacities

Herein a new method that allows binding DeltaCp to be determined with a single experiment is presented. Multithermal titration calorimetry (MTC) is a simple extension of isothermal titration calorimetry (ITC) that explicitly takes into account the thermal dependences of DeltaH and the binding constant. Experimentally, this is accomplished by performing a single stepwise titration with ITC equipment, allowing temperature re-adjustments of the system at intermediate states of the titration process. Thus, from the resulting multitherm, DeltaCp can also be determined. The experimental feasibility of MTC was tested by using the well-characterized lysozyme-chitotriose complex as a model system.     

Entropic Enhancement of Protein-DNA Affinity by Oxygen-to-Sulfur Substitution in DNA Phosphate

Dithioation of DNA phosphate is known to enhance binding affinities, at least for some proteins. We mechanistically characterized this phenomenon for the Antennapedia homeodomain-DNA complex by integrated use of fluorescence, isothermal titration calorimetry, NMR spectroscopy, and x-ray crystallography. By fluorescence and isothermal titration calorimetry, we found that this affinity enhancement is entropy driven. By NMR, we investigated the ionic hydrogen bonds and internal motions of lysine side-chain NH₃⁺ groups involved in ion pairs with DNA. By x-ray crystallography, we compared the structures of the complexes with and without dithioation of the phosphate. Our NMR and x-ray data show that the lysine side chain in contact with the DNA phosphate becomes more dynamic upon dithioation. Our thermodynamic, structural, and dynamic investigations collectively suggest that the affinity enhancement by the oxygen-to-sulfur substitution in DNA phosphate is largely due to an entropic gain arising from mobilization of the intermolecular ion pair at the protein-DNA interface.     

Molecular level interaction of inositol hexaphosphate with the C2B domain of human synaptotagmin I

Synaptotagmin I is a synaptic vesicle membrane protein that serves as a multifunctional regulator during the exocytosis of neurotransmitter release. It contains C2A and C2B domains. The binding of Ca(2+) to the C2A domain activates the exocytosis of secretory vesicles, while the binding of inositol polyphosphates (IP4-IP6) to the C2B domain inhibits this process. To understand the IP6-induced inhibition of exocytosis of secretory vesicles, we determined the three-dimensional structure of the C2B-IP6 complex by nuclear magnetic resonance (NMR). In this study, we have determined the binding constant by isothermal titration calorimetry. The circular dichroism measurements demonstrated that IP6 can stabilize the C2B molecule. We identified the binding site using (1)H-(15)N heteronuclear single-quantum coherence spectroscopy titration data and determined the structure of the C2B-IP6 complex using multidimensional NMR studies. This information will aid in the design of better pharmacological treatments for neurological disorders.     

Electrostatic selectivity in protein-nanoparticle interactions

The binding of bovine serum albumin (BSA) and β-lactoglobulin (BLG) to TTMA (a cationic gold nanoparticle coupled to 3,6,9,12-tetraoxatricosan-1-aminium, 23-mercapto-N,N,N-trimethyl) was studied by high-resolution turbidimetry (to observe a critical pH for binding), dynamic light scattering (to monitor particle growth), and isothermal titration calorimetry (to measure binding energetics), all as a function of pH and ionic strength. Distinctively higher affinities observed for BLG versus BSA, despite the lower pI of the latter, were explained in terms of their different charge anisotropies, namely, the negative charge patch of BLG. To confirm this effect, we studied two isoforms of BLG that differ in only two amino acids. Significantly stronger binding to BLGA could be attributed to the presence of the additional aspartates in the negative charge domain for the BLG dimer, best portrayed in DelPhi. This selectivity decreases at low ionic strength, at which both isoforms bind well below pI. Selectivity increases with ionic strength for BLG versus BSA, which binds above pI. This result points to the diminished role of long-range repulsions for binding above pI. Dynamic light scattering reveals a tendency for higher-order aggregation for TTMA-BSA at pH above the pI of BSA, due to its ability to bridge nanoparticles. In contrast, soluble BLG-TTMA complexes were stable over a range of pH because the charge anisotropy of this protein at makes it unable to bridge nanoparticles. Finally, isothermal titration calorimetry shows endoenthalpic binding for all proteins: the higher affinity of TTMA for BLGA versus BLGB comes from a difference in the dominant entropy term.     

Lectins as probes for assessing the accessibility of N‐linked glycans in relation to the conformational changes of fibronectin

Fibronectin, a ≈450‐kDa protein with 4–9% (w/w) glycosylation, is a key component of extracellular matrices and has a high conformational lability regarding its functions. However, the accessibility and the role of glycosylated moieties associated with the conformational changes of fibronectin are poorly understood. Using lectins as probes, we developed an approach comprising dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry to assess the accessibility of glycosylated moieties of fibronectin undergoing thermal‐induced conformational changes. Among a set of 14 lectins, fibronectin mainly reacted with mannose‐binding lectins, specifically concanavalin A. When temperature was raised from 25 to 50 °C, fibronectin underwent progressive unfolding, but the conformation of concanavalin A was unaffected. Dynamic light scattering, turbidimetry measurements, and isothermal titration calorimetry showed increased concanavalin A binding to fibronectin during progressive thermal‐induced unfolding of the protein core. Such data suggest that mannosylated residues are progressively exposed as fibronectin unfolds. Because oligosaccharide moieties can be differently exposed to cells, and the cell's responses could be modified physiologically or pathologically, modulation of fibronectin sugar chains could be relevant to its biological functions. Thus, lectins might be useful tools to probe the glycosylation accessibility accompanying changes in protein core folding, for which a better understanding would be of value for biological and biomedical research. Copyright © 2015 John Wiley & Sons, Ltd.     

Involvement of water molecules in the association of monoclonal antibody HyHEL-5 with bobwhite quail lysozyme.

Fluorescence polarization spectroscopy and isothermal titration calorimetry were used to study the influence of osmolytes on the association of the anti-hen egg lysozyme (HEL) monoclonal antibody HyHEL-5 with bobwhite quail lysozyme (BWQL). BWQL is an avian species variant with an Arg-->Lys mutation in the HyHEL-5 epitope, as well as three other mutations outside the HyHEL-5 structural epitope. This mutation decreases the equilibrium association constant of HyHEL-5 for BWQL by over 1000-fold as compared to HEL. The three-dimensional structure of this complex has been obtained recently. Fluorescein-labeled BWQL, obtained by labeling at pH 7.5 and purified by hydrophobic interaction chromatograpy, bound HyHEL-5 with an equilibrium association constant close to that determined for unlabeled BWQL by isothermal titration calorimetry. Fluorescence titration, stopped-flow kinetics, and isothermal titration calorimetry experiments using various concentrations of the osmolytes glycerol, ethylene glycol, and betaine to perturb binding gave a lower limit of the uptake of approximately 6-12 water molecules upon formation of the HyHEL-5/BWQL complex.     

Structure of heat-induced beta-lactoglobulin aggregates and their complexes with sodium-dodecyl sulfate

We report on the conformation of heat-induced bovine beta-lactoglobulin (betalg) aggregates prepared at different pH conditions, and their complexes with model anionic surfactants such as sodium dodecyl sulfate (SDS). The investigation was carried out by combining a wide range of techniques such as ultra small angle light scattering, static and dynamic light scattering, small angle neutron scattering, small-angle X-ray scattering, electrophoretic mobility, isothermal titration calorimetry (ITC) and transmission electron microscopy. Three types of aggregates were generated upon heating betalg aqueous dispersions at increasing pH from 2.0 to 5.8 to 7.0: rod-like aggregates, spherical aggregates, and worm-like primary aggregates, respectively. These aggregates were shown not only to differ for their sizes and morphologies, but also for their internal structures and fractal dimensions. The main differences between aggregates are discussed in terms of the ionic charge and conformational changes arising for betalg at different pHs. The formation of complexes between SDS and the various protein aggregates at pH 3.0 was shown to occur by two main mechanisms: at low concentration of SDS, the complex formation occurs essentially by ionic binding between the positive residues of the protein and the negative sulfate heads of the surfactant. At complete neutralization of charges, precipitation of the complexes is observed. Upon further increase in SDS concentration, complex formation of SDS and the protein aggregates occurs primarily by hydrophobic interactions, leading to (i) the formation of an SDS double layer around the protein aggregates, (ii) the inversion of the total ionic charge of each individual protein aggregate, and (iii) the complete redispersion of the protein aggregate-SDS complexes in water. Remarkably, the SDS double layer around the protein aggregates provides an efficient protective shield, preventing precipitation of the aggregates at any possible pH values, including those values corresponding to the isoelectric pH of the aggregates.     

Solution NMR structure of the barrier-to-autointegration factor-Emerin complex

The barrier-to-autointegration factor BAF binds to the LEM domain (Em(LEM)) of the nuclear envelope protein emerin and plays an essential role in the nuclear architecture of metazoan cells. In addition, the BAF(2) dimer bridges and compacts double-stranded DNA nonspecifically via two symmetry-related DNA binding sites. In this article we present biophysical and structural studies on a complex of BAF(2) and Em(LEM). Light scattering, analytical ultracentrifugation, and NMR indicate a stoichiometry of one molecule of Em(LEM) bound per BAF(2) dimer. The equilibrium dissociation constant (K(d)) for the interaction of the BAF(2) dimer and Em(LEM), determined by isothermal titration calorimetry, is 0.59 +/- 0.03 microm. Z-exchange spectroscopy between corresponding cross-peaks of the magnetically non-equivalent subunits of the BAF(2) dimer in the complex yields a dissociation rate constant of 78 +/- 2s(-1). The solution NMR structure of the BAF(2)-Em(LEM) complex reveals that the LEM and DNA binding sites on BAF(2) are non-overlapping and that both subunits of the BAF(2) dimer contribute approximately equally to the Em(LEM) binding site. The relevance of the implications of the structural and biophysical data on the complex in the context of the interaction between the BAF(2) dimer and Em(LEM) at the nuclear envelope is discussed.     

New insight into the stereoselective interactions of quinine and quinidine,with bovine serum albumin

Quinine (QN) and quinidine (QD), the chief quinoline alkaloids of various species of cinchona bark, are stereoisomers to each other. In this study, a series of appropriate and efficient methods have been applied to compare the binding modes of QN and QD with bovine serum albumin (BSA). The isothermal titration calorimetry and room temperature phosphorescence results show that both QN and QD can interact with BSA at one binding site to form drug–protein complexes, mainly through enthalpic driving force with the binding affinity order: QN > QD. The fluorescence resonance energy transfer and time‐resolved fluorescence spectroscopy exhibits that QN has a larger energy transfer and more intensified binding capacity for BSA than QD. Data of dynamic light scattering reveal that the aggregate state of BSA is changed during this binding process, and the particle size distribution of QN‐BSA bioconjugate is larger than that of QD. Nuclear magnetic resonance analysis indicates that aromatic protons make more contribution during ligand‐protein complexation than that of aliphatic protons. The circular dichroism spectra exhibit different degrees of changes in BSA secondary structures in the presence of QN and QD, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Chymotrypsin — Eudragit® complex formation

Eudragit® L100 (EuL) and Eudragit® S100 (EuS) are synthetic polyanions differing on their electric charge density. They interact with chymotrypsin (ChTRP), a basic protein forming soluble and non-soluble complexes. The complex formation was studied by dynamic light scattering, isothermal titration calorimetry, native fluorescence emission, circular dichroism and thermodynamical thermal stability of the enzyme. EuS was able to bind 33 ChTRP molecules while EuL, 60. The binding of ChTRP to both Eu was slightly endothermic and the entropic factor was responsible for the soluble complexes formation. The ChTRP-Eu size increases with pH and the binding of ChTRP to Eu modifies the Eu hydrodynamic radium. The interaction of ChTRP with Eu did not modify its secondary or tertiary structure. The thermal stability of ChTRP was increased when it interacted with both Eu.     

The MDMX Acidic Domain Uses Allovalency to Bind Both p53 and MDMX

Autoinhibition of p53 binding to MDMX requires two short-linear motifs (SLiMs) containing adjacent tryptophan (WW) and tryptophan-phenylalanine (WF) residues. NMR spectroscopy was used to show the WW and WF motifs directly compete for the p53 binding site on MDMX and circular dichroism spectroscopy was used to show the WW motif becomes helical when it is bound to the p53 binding domain (p53BD) of MDMX. Binding studies using isothermal titration calorimetry showed the WW motif is a stronger inhibitor of p53 binding than the WF motif when they are both tethered to p53BD by the natural disordered linker. We also investigated how the WW and WF motifs interact with the DNA binding domain (DBD) of p53. Both motifs bind independently to similar sites on DBD that overlap the DNA binding site. Taken together our work defines a model for complex formation between MDMX and p53 where a pair of disordered SLiMs bind overlapping sites on both proteins.     

Molecular interactions of yeast frequenin (Frq1) with the phosphatidylinositol 4-kinase isoform,Pik1

Frq1, a 190-residue N-myristoylated calcium-binding protein, associates tightly with the N terminus of Pik1, a 1066-residue phosphatidylinositol 4-kinase. Deletion analysis of an Frq1-binding fragment, Pik1-(10-192), showed that residues within 80-192 are necessary and sufficient for Frq1 association in vitro. A synthetic peptide (residues 151-199) competed for binding of [(35)S]Pik1-(10-192) to bead-immobilized Frq1, whereas shorter peptides (164-199 and 174-199) did not. Correspondingly, a deletion mutant, Pik1(delta152-191), did not co-immunoprecipitate efficiently with Frq1 and did not support growth at elevated temperature. Site-directed mutagenesis of Pik1-(10-192) suggested that recognition determinants lie over an extended region. Titration calorimetry demonstrated that binding of an 83-residue fragment, Pik1-(110-192), or the 151-199 peptide to Frq1 shows high affinity (K(d) approximately 100 nm) and is largely entropic, consistent with hydrophobic interaction. Stoichiometry of Pik1-(110-192) binding to Frq1 was 1:1, as judged by titration calorimetry, by changes in NMR spectrum and intrinsic tryptophan fluorescence, and by light scattering. In cell extracts, Pik1 and Frq1 exist mainly in a heterodimeric complex, as shown by size exclusion chromatography. Cys-15 in Frq1 is not S-palmitoylated, as assessed by mass spectrometry; a Frq1(C15A) mutant and even a non-myristoylated Frq1(G2A,C15A) double mutant rescued the inviability of frq1Delta cells. This study defines the segment of Pik1 required for high affinity binding of Frq1.     

How does dextran sulfate prevent heat induced aggregation of protein? The mechanism and its limitation as aggregation inhibitor

The effect of dextran sulfate on protein aggregation was investigated to provide the clues of its biochemical mechanism. The interaction between dextran sulfate and BSA varied with the pH values of the solution, which led to the different extent of aggregation prevention by dextran sulfate. Light scattering data with thermal scan showed that dextran sulfate suppressed BSA aggregation at pH 5.1 and pH 6.2, while it had no effect at pH 7.5. Isothermal titration calorimetric analysis suggested that the pH dependency of the role of dextran sulfate on BSA aggregation would be related to the difference in the mode of BSA-dextran sulfate complex formation. Isothermal titration calorimetric analysis at pH 6.2 indicated that dextran sulfate did not bind to native BSA at this pH, but interacted with partially unfolded BSA. While stabilizing native form of protein by the complex formation has been suggested as the suitable mechanism of preventing aggregation, our observation of conformational changes by circular dichroism spectroscopy showed that strong electrostatic interaction between dextran sulfate and BSA rather facilitated the denaturation of BSA. Combining the data from isothermal titration calorimetry, circular dichroism, and dynamic light scattering, we found that the complex formation of the intermediate state of denatured BSA with dextran sulfate is a prerequisite to suppress the aggregation by preventing further oligomerization/aggregation process of denatured protein.