Salt bridge exchange binding mechanism between streptavidin and its DNA aptamer – thermodynamics and spectroscopic evidences

Protein‐nucleic acids binding driven by electrostatic interactions typically are characterized by the release of counter ions, and the salt‐inhibited binding association constant (K_a) and the magnitude of exothermic binding enthalpy (ΔH). Here, we report a non‐classical thermodynamics of streptavidin (SA)–aptamer binding in NaCl (140–350 mM) solutions near room temperatures (23–27 °C). By using isothermal titration calorimetry (ITC) and circular dichroism (CD)/fluorescence spectroscopy, we found that the binding was enthalpy driven with a large entropy cost (ΔH ?20.58 kcal mol^?1, TΔS ?10.99 kcal mol^?1, and K_a 1.08 × 10⁷ M^?1 at 140 mM NaCl 25 °C). With the raise of salt concentrations, the ΔH became more exothermic, yet the K_a was almost unchanged (ΔH ?26.29 kcal mol^?1 and K_a 1.50 × 10⁷ M^?1 at 350 mM NaCl 25 °C). The data suggest that no counter Na⁺ was released in the binding. Spectroscopy data suggest that the binding, with a stoichiometry of 2, was accompanied with substantial conformational changes on SA, and the changes were insensitive to the variation of salt concentrations. To account for the non‐classical results, we propose a salt bridge exchange model. The intramolecular binding‐site salt bridge(s) of the free SA and the charged phosphate group of aptamers re‐organize to form the binding complex by forming a new intermolecular salt bridge(s). The salt bridge exchange binding process requires minimum amount of counter ions releasing but dehydration of the contacting surface of SA and the aptamer. The energy required for dehydration is reduced in the case of binding solution with higher salt concentration and account for the higher binding exothermic mainly. Copyright © 2013 John Wiley & Sons, Ltd.     

Heparin binding domain peptides of antithrombin III: analysis by isothermal titration calorimetry and circular dichroism spectroscopy.

The serine proteinase inhibitor antithrombin III (ATIII) is a key regulatory protein of intrinsic blood coagulation. ATIII attains its full biological activity only upon binding polysulfated oligosaccharides, such as heparin. A series of synthetic peptides have been prepared based on the proposed heparin binding regions of ATIII and their ability to bind heparin has been assessed by CD spectrometry, by isothermal titration calorimetry, and by the ability of the peptides to compete with ATIII for binding heparin in a factor Xa procoagulant enzyme assay. Peptide F123-G148, which encompasses both the purported high-affinity pentasaccharide binding region and an adjacent, C-terminally directed segment of ATIII, was found to bind heparin with good affinity, but amino-terminal truncations of this sequence, including L130-G148 and K136-G148 displayed attenuated heparin binding activities. In fact, K136-G148 appears to encompass only a low-affinity heparin binding site. In contrast, peptides based solely on the high-affinity binding site (K121-A134) displayed much higher affinities for heparin. By CD spectrometry, these high-affinity peptides are chiefly random coil in nature, but low microM concentrations of heparin induce significant alpha-helix conformation. K121-A134 also effectively competes with ATIII for binding heparin. Thus, through the use of synthetic peptides that encompass part, if not all, of the heparin binding site(s) within ATIII, we have further elucidated the structure-function relations of heparin-ATIII interactions.     

Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain

Riboswitches are cis-acting genetic regulatory elements found commonly in bacterial mRNAs that consist of a metabolite-responsive aptamer domain coupled to a regulatory switch. Purine riboswitches respond to intracellular concentrations of either adenine or guanine/hypoxanthine to control gene expression. The aptamer domain of the purine riboswitch contains a pyrimidine residue (Y74) that forms a Watson-Crick base-pairing interaction with the bound purine nucleobase ligand that discriminates between adenine and guanine. We sought to understand the structural basis of this specificity and the mechanism of ligand recognition by the purine riboswitch. Here, we present the 2,6-diaminopurine-bound structure of a C74U mutant of the xpt-pbuX guanine riboswitch, along with a detailed thermodynamic and kinetic analysis of nucleobase recognition by both the native and mutant riboswitches. These studies demonstrate clearly that the pyrimidine at position 74 is the sole determinant of purine riboswitch specificity. In addition, the mutant riboswitch binds adenine and adenine derivatives well compared with the guanine-responsive riboswitch. Under our experimental conditions, 2,6-diaminopurine binds the RNA with DeltaH=-40.3 kcal mol(-1), DeltaS=-97.6 cal mol(-1)K(-1), and DeltaG=-10.73 kcal mol(-1). A kinetic determination of the slow rate (0.15 x 10(5)M(-1)s(-1) and 2.1 x 10(5)mM(-1)s(-1) for 2-aminopurine binding the adenine-responsive mutant riboswitch and 7-deazaguanine-binding guanine riboswitch, respectively) of association under varying experimental conditions allowed us to propose a mechanism for ligand recognition by the purine riboswitch. A conformationally dynamic unliganded state for the binding pocket is stabilized first by the Watson-Crick base pairing between the ligand and Y74, and by the subsequent ordering of the J2/3 loop, enclosing the ligand within the three-way junction.     

Interaction of epitope-related and -unrelated peptides with anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab fragment

The binding of four epitope-related peptides and three library-derived, epitope-unrelated peptides of different lengths (10-14 amino acids) and sequence by anti-p24 (HIV-1) monoclonal antibody CB4-1 and its Fab fragment was studied by isothermal titration calorimetry. The binding constants K(A) at 25 degrees C vary between 5.1 x 10(7) M (-1) for the strongest and 1.4 x 10(5) M (-1) for the weakest binder. For each of the peptides complex formation is enthalpically driven and connected with unfavorable entropic contributions; however, the ratio of enthalpy and entropy contributions to deltaG(0) differs markedly for the individual peptides. A plot of -deltaH(0) vs -TdeltaS(0) shows a linear correlation of the data for a wide variety of experimental conditions as expected for a process with deltaC(p) much larger than deltaS(0). The dissimilarity of deltaC(p) and deltaS(0) also explains why deltaH(0) and TdeltaS(0) show similar temperature dependences resulting in relatively small changes of deltaG(0) with temperature. The heat capacity changes deltaC(p) upon antibody-peptide complex formation determined for three selected peptides vary only in a small range, indicating basic thermodynamic similarity despite different key residues interacting in the complexes. Furthermore, the comparison of van't Hoff and calorimetric enthalpies point to a non-two-state binding mechanism. Protonation effects were excluded by measurements in buffers of different ionization enthalpies. Differences in the solution conformation of the peptides as demonstrated by circular dichroic measurements do not explain different binding affinities of the peptides; specifically a high helix content in solution is not essential for high binding affinity despite the helical epitope conformation in the crystal structure of p24.     

Studies of lysozyme binding to histamine as a ligand for hydrophobic charge induction chromatography

Histamine was immobilized on Sepharose CL‐6B (Sepharose) for use as a ligand of hydrophobic charge induction chromatography (HCIC) of proteins. Lysozyme adsorption onto Histamine‐Sepharose (HA‐S) was studied by adsorption equilibrium and calorimetry to uncover the thermodynamic mechanism of the protein binding. In both the experiments, the influence of salt (ammonium sulfate and sodium sulfate) was examined. Adsorption isotherms showed that HA‐S exhibited a high salt tolerance in lysozyme adsorption. This property was well explained by the combined contributions of hydrophobic interaction and aromatic stacking. The isotherms were well fitted to the Langmuir equation, and the equilibrium parameters for lysozyme adsorption were obtained. In addition, thermodynamic parameters (ΔH_ads, ΔS_ads, and ΔG_ads) for the adsorption were obtained by isothermal titration calorimetry by titrating lysozyme solutions into the adsorbent suspension. Furthermore, free histamine was titrated into lysozyme solution in the same salt‐buffers. Compared with the binding of lysozyme to free histamine, lysozyme adsorption onto HA‐S was characterized by a less favorable ΔG_ads and an unfavorable ΔS_ads because histamine was covalently attached to Sepharose via a three‐carbon‐chain spacer. Consequently, the immobilized histamine could only associate with the residues on the protein surface rather than those in the hydrophobic pocket, causing a less favorable orientation between histamine and lysozyme. Further comparison of thermodynamic parameters indicated that the unfavorable ΔS_ads was offset by a favorable ΔH_ads, thus exhibiting typical enthalpy‐entropy compensation. Moreover, thermodynamic analyses indicated the importance of the dehydration of lysozyme molecule and HA‐S during the adsorption and a substantial conformational change of the protein during adsorption. The results have provided clear insights into the adsorption mechanisms of lysozyme onto the new HCIC material. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Kinetics and energetics of ligand binding determined by microcalorimetry: insights into active site mobility in a psychrophilic alpha-amylase

A new microcalorimetric method for recording the kinetic parameters k(cat), K(m) and K(i) of alpha-amylases using polysaccharides and oligosaccharides as substrates is described. This method is based on the heat released by glycosidic bond hydrolysis. The method has been developed to study the active site properties of the cold-active alpha-amylase produced by an Antarctic psychrophilic bacterium in comparison with its closest structural homolog from pig pancreas. It is shown that the psychrophilic alpha-amylase is more active on large macromolecular substrates and that the higher rate constants k(cat) are gained at the expense of a lower affinity for the substrate. The active site is able to accommodate larger inhibitory complexes, resulting in a mixed-type inhibition of starch hydrolysis by maltose. A method for recording the binding enthalpies by isothermal titration calorimetry in a low-affinity system has been developed, allowing analysis of the energetics of weak ligand binding using the allosteric activator chloride. It is shown that the low affinity of the psychrophilic alpha-amylase for chloride is entropically driven. The high enthalpic and entropic contributions of activator binding suggest large structural fluctuations between the free and the bound states of the cold-active enzyme. The kinetic and thermodynamic data for the psychrophilic alpha-amylase indicate that the strictly conserved side-chains involved in substrate binding and catalysis possess an improved mobility, responsible for activity in the cold, and resulting from the disappearance of stabilizing interactions far from the active site.     

Structural destabilization of tropomyosin induced by the cardiomyopathy‐linked mutation R21H

The missense mutation R21H in striated muscle tropomyosin is associated with hypertrophic cardiomyopathy, a genetic cardiac disease and a leading cause of sudden cardiac death in young people. Tropomyosin adopts conformation of a coiled coil which is critical for regulation of muscle contraction. In this study, we investigated the effects of the R21H mutation on the coiled‐coil structure of tropomyosin and its interactions with its binding partners, tropomodulin and leiomodin. Using circular dichroism and isothermal titration calorimetry, we found that the mutation profoundly destabilized the structural integrity of αTM1a_1‐28Zip, a chimeric peptide containing the first 28 residues of tropomyosin. The mutated αTM1a_1‐28Zip was still able to interact with tropomodulin and leiomodin. However, the mutation resulted in a ～30‐fold decrease of αTM1a_1‐28Zip's binding affinity to leiomodin. We used a crystal structure of αTM1a_1‐28Zip that we solved at 1.5 Å resolution to study the mutation's effect in silico by means of molecular dynamics simulation. The simulation data indicated that while the mutation disrupted αTM1a_1‐28Zip's coiled‐coil structure, most notably from residue Ala18 to residue His31, it may not affect the N‐terminal end of tropomyosin. The drastic decrease of αTM1a_1‐28Zip's affinity to leiomodin caused by the mutation may lead to changes in the dynamics at the pointed end of thin filaments. Therefore, the R21H mutation is likely interfering with the regulation of the normal thin filament length essential for proper muscle contraction.     

Structural and binding studies of the three-metal center in two mycobacterial PPM Ser/Thr protein phosphatases

Phospho-Ser/Thr protein phosphatases (PPs) are dinuclear metalloenzymes classed into two large families, PPP and PPM, on the basis of sequence similarity and metal ion dependence. The archetype of the PPM family is the α isoform of human PP2C (PP2Cα), which folds into an α/β domain similar to those of PPP enzymes. The recent structural studies of three bacterial PPM phosphatases, Mycobacterium tuberculosis MtPstP, Mycobacterium smegmatis MspP, and Streptococcus agalactiae STP, confirmed the conservation of the overall fold and dinuclear metal center in the family, but surprisingly revealed the presence of a third conserved metal-binding site in the active site. To gain insight into the roles of the three-metal center in bacterial enzymes, we report structural and metal-binding studies of MtPstP and MspP. The structure of MtPstP in a new trigonal crystal form revealed a fully active enzyme with the canonical dinuclear metal center but without the third metal ion bound to the catalytic site. The absence of metal correlates with a partially unstructured flap segment, indicating that the third manganese ion contributes to reposition the flap, but is dispensable for catalysis. Studies of metal binding to MspP using isothermal titration calorimetry revealed that the three Mn²⁺-binding sites display distinct affinities, with dissociation constants in the nano- and micromolar range for the two catalytic metal ions and a significantly lower affinity for the third metal-binding site. In agreement, the structure of inactive MspP at acidic pH was determined at atomic resolution and shown to lack the third metal ion in the active site. Structural comparisons of all bacterial phosphatases revealed positional variations in the third metal-binding site that are correlated with the presence of bound substrate and the conformation of the flap segment, supporting a role of this metal ion in assisting enzyme-substrate interactions.     

Thermodynamics of DNA binding and distortion by the hyperthermophile chromatin protein Sac7d

Sac7d is a hyperthermophile chromatin protein which binds non-specifically to the minor groove of duplex DNA and induces a sharp kink of 66 degrees with intercalation of valine and methionine side-chains. We have utilized the thermal stability of Sac7d and the lack of sequence specificity to define the thermodynamics of DNA binding over a wide temperature range. The binding affinity for poly(dGdC) was moderate at 25 degrees C (Ka = 3.5(+/-1.6) x 10(6) M(-1)) and increased by nearly an order of magnitude from 10 degrees C to 80 degrees C. The enthalpy of binding was unfavorable at 25 degrees C, and decreased linearly from 5 degrees C to 60 degrees C. A positive binding heat at 25 degrees C is attributed in part to the energy of distorting DNA, and ensures that the temperature of maximal binding affinity (75.1+/-5.6 degrees C) is near the growth temperature of Sulfolobus acidocaldarius. Truncation of the two intercalating residues to alanine led to a decreased ability to bend and unwind DNA at 25 degrees C with a small decrease in binding affinity. The energy gained from intercalation is slightly greater than the free energy penalty of bending duplex DNA. Surprisingly, reduced distortion from the double alanine substitution did not lead to a significant decrease in the heat of binding at 25 degrees C. In addition, an anomalous positive DeltaCp of binding was observed for the double alanine mutant protein which could not be explained by the change in polar and apolar accessible surface areas. Both the larger than expected binding enthalpy and the positive heat capacity can be explained by a temperature dependent structural transition in the protein-DNA complex with a Tm of 15-20 degrees C and a DeltaH of 15 kcal/mol. Data are discussed which indicate that the endothermic transition in the complex is consistent with DNA distortion.     

Calorimetric investigation of phosphorylated and non-phosphorylated peptide ligand binding to the human Grb7-SH2 domain

Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20-30% of breast cancers. In general, growth factor receptor bound (Grb) proteins bind to activated membrane-bound receptor tyrosine kinases (RTKs; e.g., the epidermal growth factor receptor, EGFR) through their Src homology 2 (SH2) domains. In particular, Grb7 binds to erbB2 (a.k.a. EGFR2) and may be involved in cell signaling pathways that promote the formation of metastases and inflammatory responses. In previous studies, we reported the solution structure and the backbone relaxation behavior of the Grb7-SH2/erbB2 peptide complex. In this study, isothermal titration calorimetry studies have been completed by measuring the thermodynamic binding parameters of several phosphorylated and non-phosphorylated peptides representative of natural Grb7 receptor ligands as well as ligands developed through combinatorial peptide screening methods. The entirety of these calorimetric studies is interpreted in an effort to describe the specific ligand binding characteristics of the Grb7 protein.     

Bacterial expression,purification and biophysical characterization of wheat germ agglutinin and its four hevein-like domains

Wheat germ agglutinin (WGA), a chitin binding lectin, has attracted increasing interest because of its unique characteristics such as conformational stability, binding specificity and transcytosis capacity. To pave the way for the study of the molecular basis of WGA's structural stability and binding capacity, as well as to facilitate its use in biomedical and biotechnological developments, we produced recombinant WGA and its 4 isolated hevein-like domains in a bacterial system. All the proteins were expressed as fusion constructs linked to a thioredoxin domain, which was enzymatically or chemically released. The structural and ligand-binding properties of recombinant WGA were similar to the wild lectin. The 4 isolated domains folded and were ligand-binding competent, indicating that each domain constitutes an independent folding unity. The biophysical characterization of the recombinant domains sheds new light on the intricate folding and binding behavior of this emblematic lectin.     

Probing the sensory rhodopsin II binding domain of its cognate transducer by calorimetry and electrophysiology

Sensory rhodopsin II, a repellent phototaxis receptor from Natronobacterium pharaonis (NpSRII) forms a tight complex with its cognate transducer (NpHtrII). Light excitation of the receptor triggers conformational changes in both proteins, thereby activating the cellular two-component signalling cascade. In membranes, the two proteins form a 2:2 complex, which dissociates to a 1:1 heterodimer in micelles. Complexed to the transducer sensory rhodopsin II is no longer capable of light-driven proton pumping. In order to elucidate the dimerisation and the size of the receptor-binding domain of the transducer, isothermal titration calorimetry and electrophysiological experiments have been carried out. It is shown, that an N-terminal sequence of 114 amino acid residues is sufficient for tight binding (K(d)=240nM; DeltaH=-17.6kJmol(-1)) and for inhibiting the proton transfer. These data and results obtained from selected site-directed mutants indicate a synergistic interplay of transducer transmembrane domain (1-82) and cytoplasmic peptide (83-114) leading to an optimal and specific interaction between receptor and transducer.     

Linear correlation between thermal stability and folding kinetics of lysozyme

We have studied the refolding and thermal denaturation of hen egg white lysozyme in a wide range of pH values (from 1.5 to 9.4) using stopped-flow circular dichroism (CD) and differential scanning calorimetry (DSC). A linear correlation was found between the thermal denaturation temperature (T(m)) and the logarithm of the refolding rate of the slow folding phase of hen egg white lysozyme (lnk(2)).     

In-vitro assessment of the binding mechanism of oxyfluorfen (herbicide) with garlic phytocystatin: multi-spectroscopic and isothermal titration calorimetric study

Abstract

Oxyfluorfen (2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene) is a nitrophenyl ether herbicide. Phytocystatins are crucial plant proteins which regulate various physiological processes and are also responsible for maintaining protease–antiprotease balance within plants. Thus, the present article deciphers the interaction of oxyfluorfen with garlic phytocystatin (GPC) through various spectroscopic and calorimetric techniques. The cysteine proteinase inhibitory assay was done to assess the inhibitory action of GPC in the presence of oxyfluorfen. The GPC loses its inhibitory activity in the presence of oxyfluorfen. The complex formation of GPC-oxyfluorfen was shown by UV absorption spectroscopy. The intrinsic fluorescence experiment affirmed the quenching of GPC in the presence of oxyfluorfen. The Stern–Volmer quenching constant and binding constant was obtained as 6.89?×?10³ M^?1 and 9.72?×?10³ M^?1, respectively. Synchronous fluorescence showed the alteration in the microenvironment around tyrosine residues. 3D fluorescence suggested the perturbation in the polarity around aromatic residues. The isothermal titration experiment suggests that the interaction of oxyfluorfen with GPC is a thermodynamically favorable reaction. Secondary structure alteration of GPC in the presence of oxyfluorfen was studied by circular dichroism (CD). The CD result showed a reduction in the α-helical content of GPC on interaction with oxyfluorfen. Consequently, all these outcomes affirmed the formation of GPC–oxyfluorfen complex along with the structural and conformational alteration. This study identifies and signifies that the exposure of oxyfluorfen induces stress within the plant system.

Communicated by Ramaswamy H. Sarma     

Site-directed mutagenesis indicates an important role of cysteines 76 and 181 in the catalysis of hydantoin racemase from Sinorhizobium meliloti

Hydantoin racemase enzyme plays a crucial role in the reaction cascade known as "hydantoinase process." In conjunction with a stereoselective hydantoinase and a stereospecific carbamoylase, it allows the total conversion from D,L-5-monosubstituted hydantoins, with a low rate of racemization, to optically pure D- or L-amino acids. Residues Cys76 and Cys181 belonging to hydantoin racemase from Sinorhizobium meliloti (SmeHyuA) have been proved to be involved in catalysis. Here, we report biophysical data of SmeHyuA Cys76 and Cys181 to alanine mutants, which point toward a two-base mechanism for the racemization of 5-monosubstituted hydantoins. The secondary and the tertiary structure of the mutants were not significantly affected, as shown by circular dichroism. Calorimetric and fluorescence experiments have shown that Cys76 is responsible for recognition and proton retrieval of D-isomers, while Cys181 is responsible for L-isomer recognition and racemization. This recognition process is further supported by measurements of protein stability followed by chemical denaturation in the presence of the corresponding compound.     

The third 20 amino acid repeat is the tightest binding site of APC for beta-catenin

Adenomatous polyposis coli (APC) plays a critical role in the Wnt signaling pathway by tightly regulating beta-catenin turnover and localization. The central region of APC is responsible for APC-beta-catenin interactions through its seven 20 amino acid (20aa) repeats and three 15 amino acid (15aa) repeats. Using isothermal titration calorimetry, we have determined the binding affinities of beta-catenin with an APC 15aa repeat fragment and each of the seven 20aa repeats in both phosphorylated and unphosphorylated states. Despite sequence homology, different beta-catenin binding repeats of APC have dramatically different binding affinities with beta-catenin and thus may play different biological roles. The third 20aa repeat is by far the tightest binding site for beta-catenin among all the repeats. The fact that most APC mutations associated with colon cancers have lost the third 20aa repeat underlines the importance of APC-beta-catenin interaction in Wnt signaling and human diseases. For every 20aa repeat, phosphorylation dramatically increases its binding affinity for beta-catenin, suggesting phosphorylation has a critical regulatory role in APC function. In addition, our CD and NMR studies demonstrate that the central region of APC is unstructured in the absence of beta-catenin and Axin, and suggest that beta-catenin may interact with each of the APC 15aa and 20aa repeats independently.     

Thermodynamics of zinc binding to hepatitis C virus NS3 protease: A folding by binding event

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease is responsible for the processing of the non‐structural region of the viral precursor polyprotein in infected hepatic cells. HCV NS3 is a zinc‐dependent serine protease. The zinc ion, which is bound far away from the active site and considered to have a structural role, is essential for the structural integrity of the protein; furthermore, the ion is required for the hydrolytic activity. Consequently, the NS3 zinc binding site has been considered for a long time as a possible target for drug discovery. As a first step towards this goal, the energetics of the NS3‐zinc interaction and its effect on the NS3 conformation must be established and discussed. The thermodynamic characterization of zinc binding to NS3 protease by isothermal titration calorimetry and spectroscopy is presented here. Spectroscopic and calorimetric results suggest that a considerable conformational change in the protein is coupled to zinc binding. The energetics of the conformational change is comparable to that of the folding of a protein of similar size. Therefore, zinc binding to NS3 protease can be considered as a “folding by binding” event. Proteins 2009. © 2009 Wiley‐Liss, Inc.