Steady-state Fluorescence Titration Research Articles

Successful Identification of Cardiac Troponin Calcium Sensitizers Using a Combination of Virtual Screening and ROC Analysis of Known Troponin C Binders

Calcium dependent cardiac muscle contraction is regulated by the protein complex troponin. Calcium binds to the N-terminal domain of troponin C (cNTnC) which initiates the process of contraction. Heart failure is a consequence of a disruption of this process. With the prevalence of this condition, a strong need exists to find novel compounds to increase the calcium sensitivity of cNTnC. Desirable are small chemical molecules that bind to the interface between cTnC and the cTnI switch peptide and exhibit calcium sensitizing properties by possibly stabilizing cTnC in an open conformation. To identify novel drug candidates, we employed a structure-based drug discovery protocol that incorporated the use of a molecular dynamics simuations. In preparation for the virtual screening, cNTnC conformations were identified based on their ability to correctly predict known cNTnC binders using a receiver operating characteristics analysis. Following a virtual screen of the National Cancer Institute’s Developmental Therapeutic Program database, a small number of molecules were experimentally tested using stopped-flow kinetics and steady-state fluorescence titrations. We identified two novel compounds that show increased calcium sensitivity of cTnC in the presence of the regulatory domain of cTnI. The effects of those compounds on the calcium dissociation rate was stronger than that of the known calcium sensitizer bepridil. Additionally, we identified a 3-phenylchromane group as a possible key pharmacophore in the sensitization of cardiac muscle contraction. Finally, we are also developing new computational techniques to quantitatively assess the impact of cardiomyopathy mutations and small molecules on troponin function.

Determination of Effector Binding Affinities Using Photoacoustic Calorimetry

Determination of binding affinity and thermodynamic parameters of interactions between two molecules is a common procedure in biochemical contexts. Isothermal titration calorimetry and steady-state fluorescence titration are commonly used to determine the binding affinity of small effector compounds and ligands to large biomolecules such as proteins. These methods however have several disadvantages, such large quantity of protein in case of ITC and the presence of a fluorescence probe in case of fluorescence. Photoacoustic calorimetry (PAC) is often used to monitor volume and enthalpy change associated with a photo-triggered reaction. Here we have tested the application of PAC to measure equilibrium binding constant and thermodynamic parameters for small organic molecules binding to hemoglobin. This technique does not require a fluorescent probe to be present and instead takes advantage of the fact that the association of small organic molecules to hemoglobin modulates the amplitude of PAC signal for O2 photo-dissociation from this protein. From the temperature dependence of the equilibrium constants, the enthalpy changes were determined using Van’t Hoff plots. So far, we have determined the dissociation constant (KD) and enthalpy change (ΔH) for binding of inositol hexakisphosphate (IHP) (KD = 73.5 μM at 20 °C, ΔH = −9.8 kJ mol−1) and 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine) (KD = 46.7 µM at 20 °C, ΔH = 13.2 kJ mol−1) to fully-oxygenated adult human hemoglobin; and the determined parameters are in excellent agreement with those published previously. Being able to determine the binding affinity and thermodynamic parameters for ligand binding to proteins is extremely useful for understanding the environment of the ligand binding site, potentially allowing us to create new and more effective drugs. The use of PAC may provide a new approach of evaluating ligand protein interactions quickly and with smaller quantities of protein.

Successful Identification of Cardiac Troponin Calcium Sensitizers Using a Combination of Virtual Screening and ROC Analysis of Known Troponin C Binders.

Calcium-dependent cardiac muscle contraction is regulated by the protein complex troponin. Calcium binds to the N-terminal domain of troponin C (cNTnC) which initiates the process of contraction. Heart failure is a consequence of a disruption of this process. With the prevalence of this condition, a strong need exists to find novel compounds to increase the calcium sensitivity of cNTnC. Desirable are small chemical molecules that bind to the interface between cTnC and the cTnI switch peptide and exhibit calcium sensitizing properties by possibly stabilizing cTnC in an open conformation. To identify novel drug candidates, we employed a structure-based drug discovery protocol that incorporated the use of a relaxed complex scheme (RCS). In preparation for the virtual screening, cNTnC conformations were identified based on their ability to correctly predict known cNTnC binders using a receiver operating characteristics analysis. Following a virtual screen of the National Cancer Institute's Developmental Therapeutic Program database, a small number of molecules were experimentally tested using stopped-flow kinetics and steady-state fluorescence titrations. We identified two novel compounds, 3-(4-methoxyphenyl)-6,7-chromanediol (NSC600285) and 3-(4-methylphenyl)-7,8-chromanediol (NSC611817), that show increased calcium sensitivity of cTnC in the presence of the regulatory domain of cTnI. The effects of NSC600285 and NSC611817 on the calcium dissociation rate was stronger than that of the known calcium sensitizer bepridil. Thus, we identified a 3-phenylchromane group as a possible key pharmacophore in the sensitization of cardiac muscle contraction. Building on this finding is of interest to researchers working on development of drugs for calcium sensitization.

3D Luminescent Amide-Functionalized Cadmium Tetrazolate Framework for Selective Detection of 2,4,6-Trinitrophenol

A new 3D fluorescent amide-functionalized Cd(II)-based metal–organic framework (MOF) with molecular formula [Cd5Cl6(L)(HL)2]·7H2O (1) was synthesized under solvothermal conditions using CdCl2·H2O and 4-(1H-tetrazol-5-yl)-N-[4-(1H-tetrazol-5-yl)phenyl]benzamide (H2L) in DMF/methanol in the presence of conc. HCl. Single-crystal X-diffraction analysis reveals that the 3D framework structure of 1 is constructed from octahedrally coordinated Cd2+ ions interconncted by chloride anions and ditopic tetrazolate-based ligand molecules. The phase-purity of the compound was confirmed by X-ray powder diffraction (XRPD) analysis, infrared spectroscopy, and elemental analysis. Thermogravimetric analysis suggests that 1 is thermally stable up to 300 °C. Steady-state fluorescence titration experiments reveal that activated 1′ can selectively detect 2,4,6-trinitrophenol (TNP) in the presence of other competing nitroaromatic compounds with a detection limit of 42.84 ppb. Recyclability experiments reveal that 1′ retains its ...

A thiadiazole-functionalized Zr(iv)-based metal–organic framework as a highly fluorescent probe for the selective detection of picric acid

A new, strongly luminescent Zr(IV)-based metal–organic framework (MOF) material (1) having a UiO-68 (UiO = University of Oslo) framework topology and incorporating the π-conjugated, thiadiazole-functionalized H2BTDB {H2BTDB = 4,4′-(benzo[c][1,2,5]thiadiazole-4,7-diyl)dibenzoic acid} ligand was synthesized under solvothermal conditions (150 °C, 24 h) using ZrCl4 and the H2BTDB ligand in DMF (DMF = N,N-dimethylformamide). The phase-purity of as-synthesized 1 was ascertained by X-ray powder diffraction (XRPD) analysis, Fourier transform infrared (FT-IR) spectroscopy and elemental analysis. Based on the thermogravimetric analyses, 1 is thermally stable up to 400 °C. XRPD experiments verify that activated 1′ retains its crystallinity when exposed to water, acetic acid and 1 M HCl solutions. As revealed by the steady-state fluorescence titration experiments, the thermally activated form of the compound (1′) showed a selective sensing behaviour towards 2,4,6-trinitrophenol (TNP, commonly known as picric acid), even in the presence of other potentially competing nitroaromatic explosive compounds. The estimated detection limit of 1′ for sensing TNP in methanol suspension was found to be 1.63 × 10−6 M. The highest fluorescence quenching ability of TNP can be attributed to both energy and electron transfer processes as well as electrostatic interactions between the hydroxyl group of TNP and the Lewis basic N-donor sites of the BTDB ligand. Endowed with its excellent detection efficiency, 1′ is a promising luminescent sensor material for the long-term, practical sensing of TNP.

Nucleotides sequestered at different subsite loci within DNA-binding pockets of two OB-fold single-stranded DNA-binding proteins are unstacked to different extents

The gene 5 protein (g5p) encoded by the Ff strains of Escherichia coli bacteriophages is a dimeric single-stranded DNA-binding protein (SSB) that consists of two identical OB-fold (oligonucleotide/oligosaccharide-binding) motifs. Ultrafast time-resolved fluorescence measurements were carried out to investigate the effect of g5p binding on the conformation of 2-aminopurine (2AP) labels positioned between adenines or cytosines in the 16-nucleotide antiparallel tails of DNA hairpins. The measurements revealed significant changes in the conformational heterogeneity of the 2AP labels caused by g5p binding. The extent of the changes was dependent on sub-binding-site location, but generally resulted in base unstacking. When bound by g5p, the unstacked 2AP population increased from ∼ 22% to 59-67% in C-2AP-C segments and from 39% to 77% in an A-2AP-A segment. The OB-fold RPA70A domain of the human replication protein A also caused a significant amount of base unstacking at various locations within the DNA binding site as evidenced by steady-state fluorescence titration measurements using 2AP-labeled 5-mer DNAs. These solution studies support the concept that base unstacking at most of a protein's multiple sub-binding-site loci may be a feature that allows non-sequence specific OB-fold proteins to bind to single-stranded DNAs (ssDNAs) with minimal preference for particular sequences.

Synthesis, characterization, fluorescence and computational studies of new Cu2+, Ni2+ and Hg2+ complexes with emissive thienylbenzoxazolyl-alanine ligands

The interaction of four fluorescent compounds containing thiophene and benzoxazole moieties combined with an alanine residue with alkaline, alkaline-earth, transition and post-transition metal ions was explored. The highly fluorescent heterocyclic alanine derivatives are strongly quenched in the solid state after complexation with the paramagnetic metal ions Cu2+ and Ni2+, and with the diamagnetic Hg2+. Absorption and steady-state fluorescence titrations reveal a selective interaction with Cu2+, Ni2+ and Hg2+. In all cases the formation of mononuclear or dinuclear metal complexes in solid state and in solution are postulated. DFT calculations on the mercury(II) complexes confirm the formation of dinuclear species. Our results suggest that one metal ion is coordinated by the chelate group formed by the amine and the protonated carboxylic groups present in the amino acid residue while a second metal ion is directly linked to the chromophore. As parent compound, L4 shows no interaction with Cu2+ and Ni2+ salts. However, the interaction with Hg2+ induces a strong quenching and a red shift of the fluorescence emission.

Energy Transfer Followed by Electron Transfer in a Porphyrin Macrocycle and Central Acceptor Ligand: A Model for a Photosynthetic Composite of the Light‐Harvesting Complex and Reaction Center

A system that models a photosynthetic composite of the light-harvesting complex and reaction center is reported in which light energy collected by cyclic antenna porphyrins is transferred to a central energy-acceptor porphyrin, followed by photoinduced electron transfer to a fullerene positioned above the ring plane. Pyridyl tripodal ligands appended with bis(phenylethynyl)porphyrinatozinc(II) (ZnP-Tripod) and additional fulleropyrrolidine moieties (C(60)-ZnP-Tripod) were synthesized as the reaction center units. The tripodal ligand was strongly accommodated by the light-harvesting porphyrin macrocycle N-(1-Zn)(3) (1-Zn = trisporphyrinatozinc(II)) by using three-point coordination of pyridyl to uncoordinated porphyrinatozinc sites to afford a stable 1:1 composite. The binding constants for ZnP-Tripod and C(60)-ZnP-Tripod in benzonitrile were estimated from steady-state fluorescence titrations to be 1.4x10(7) and 1.6x10(7) M(-1), respectively. The steady-state fluorescence titration, fluorescence lifetime, and transient absorption studies revealed that in both composites the excitation energy collected by the nine porphyrins of N-(1-Zn)(3) was efficiently transferred to a ZnP moiety by means of a through-space mechanism with a quantum yield of approximately 90%. Furthermore, in the composite with C(60)-ZnP-Tripod, the converged energy at the ZnP moiety induced electron transfer to the C(60) moiety, which afforded the stable charge-separated state (Phi(CS)>90%).

A New Generation of Peptide-based Inhibitors Targeting HIV-1 Reverse Transcriptase Conformational Flexibility

The biologically active form of human immunodeficiency virus (HIV) type 1 reverse transcriptase (RT) is a heterodimer. The formation of RT is a two-step mechanism, including a rapid protein-protein interaction "the dimerization step," followed by conformational changes "the maturation step," yielding the biologically active form of the enzyme. We have previously proposed that the heterodimeric organization of RT constitutes an interesting target for the design of new inhibitors. Here, we propose a new class of RT inhibitors that targets protein-protein interactions and conformational changes involved in the maturation of heterodimeric reverse transcriptase. Based on a screen of peptides derived from the thumb domain of this enzyme, we have identified a short peptide P(AW) that inhibits the maturation step and blocks viral replication at subnanomolar concentrations. P(AW) only binds dimeric RT and stabilizes it in an inactive/non-processive conformation. From a mechanistic point of view, P(AW) prevents proper binding of primer/template by affecting the structural dynamics of the thumb/fingers of p66 subunit. Taken together, these results demonstrate that HIV-1 RT maturation constitutes an attractive target for AIDS chemotherapeutics.

Role of Chloride Ions in Modulation of the Interaction between von Willebrand Factor and ADAMTS-13

The degradation of von Willebrand factor (VWF) depends on the activity of a zinc protease (referred to as ADAMTS-13), which cleaves VWF at the Tyr(1605)-Met(1606) peptide bond. Little information is available on the physiological mechanisms involved in regulation of AD-AMTS-13 activity. In this study, the role of ions on the ADAMTS-13/VWF interaction was investigated. In the presence of 1.5 m urea, the protease cleaved multimeric VWF in the absence of NaCl at pH 8.00 and 37 degrees C, with an apparent k(cat)/K(m) congruent with 3.4 x 10(4) M(-1) s(-1), but this value decreased by approximately 10-fold in the presence of 0.15 M NaCl. Using several monovalent salts, the inhibitory effect was attributed mostly to anions, whose potency was inversely related to the corresponding Jones-Dole viscosity B coefficients (ClO(4)(-) > Cl(-) > F(-)). The specific inhibitory effect of anions was due to their binding to VWF, which caused a conformational change responsible for quenching the intrinsic fluorescence of the protein and reducing tyrosine exposition to bulk solvent. Ristocetin binding to VWF could reduce the apparent affinity and reverse the inhibitory effect of chloride. We hypothesize that, after secretion into the extracellular compartment, VWF is bound by chloride ions abundantly present in this milieu, becoming unavailable to proteolysis by AD-AMTS-13. Shear forces, which facilitate GpIbalpha binding (this effect being artificially obtained by ristocetin), can reverse the inhibitory effect of chloride, whose concentration gradient across the cell membrane may represent a simple but efficient strategy to regulate the enzymatic activity of ADAMTS-13.

Thermodynamic Dissection of the Polymerizing and Editing Modes of a DNA Polymerase

DNA polymerases with intrinsic proofreading activity interact with DNA primer/templates in two distinct modes, corresponding to the complexes formed during the 5′–3′ polymerization or 3′–5′ editing of a nascent DNA chain. Thermodynamic measurements designed to quantify the energetic contributions of individual DNA–protein contacts in either the polymerizing or editing complexes are complicated by the fact that both species exist in solution and are not resolved in conventional DNA–protein binding assays. To overcome this problem, we have developed a new binding analysis that combines information from steady-state and time-resolved fluorescence experiments and uses the Klenow fragment of Escherichia coli DNA polymerase I (KF) and fluorescently labeled primer/template oligonucleotides as a model polymerase–DNA system. Steady-state fluorescence titrations are used to evaluate the overall affinity of KF for the primer/template, while time-resolved fluorescence anisotropy is used to quantify the equilibrium fractions of the primer/template bound in the polymerizing and editing modes. From a combined analysis of both data, the equilibrium constant and hence standard free energy change associated with each binding mode can be obtained unequivocally. This method is initially used to determine the equilibrium constants describing binding of a correctly base-paired primer/template to the 5′–3′ polymerase and 3′–5′ exonuclease sites of KF. It is then extended to quantify the extent to which these parameters are affected by the introduction of mismatches into the primer/template, and by rearrangement of specific side-chains in the exonuclease domain of the protein. While these perturbants were originally designed to demonstrate the utility of our new approach, they are also relevant in their own right since they have helped identify some hitherto unknown determinants of polymerase fidelity.

Examination of the differences in structure–function of human and rat 3α-hydroxysteroid dehydrogenase

Human 3α-hydroxysteroid dehydrogenases (HSDs) are potential drug targets since they regulate the occupancy and trans-activation of steroid hormone receptors by interconverting potent hormones with their cognate inactive metabolites. The human isoforms (AKR1C1-4) are all members of the aldo–keto reductase superfamily and display distinctive differences in steroid specificity and catalytic efficiency when compared with the closely related and more extensively studied rat 3α-HSD (AKR1C9). Specifically, AKR1C1-4 display 3α-, 17β- and 20α-HSD activities to varying degrees whereas AKR1C9 is positional- and stereo-specific for the 3α-HSD reaction. In addition, AKR1C1-4 isoforms have significantly lower catalytic efficiencies ( k cat/ K m) than AKR1C9 and this is largely due to a lower k cat. To understand these functional differences, human type 3 3α-HSD (AKR1C2) was studied as a representative human 3α-HSD. Comparison of the crystal structure of AKR1C2–NADP +–ursodeoxycholate ternary complex (3.0 Å) with that of the AKR1C9–NADP +–testosterone ternary complex (2.8 Å) demonstrates the expected conservancy in overall structure and active site topology. More interestingly, it reveals striking differences in the structure of the steroid binding pockets of the two enzymes and shows how ursodeoxycholate binds ‘backwards’ and ‘upside-down’ with respect to testosterone. This difference in steroid binding provides a structural basis for the broad positional specificity of AKR1C2 and the exquisite stereospecificity of AKR1C9. To determine why AKR1C2 has a much lower k cat than AKR1C9, the events associated with the binding of cofactor to both enzymes were studied by steady state fluorescence titration and stopped-flow experiments. Comparable K d values for E–NADP(H) and k obs values for the fluorescence transients were obtained for the two enzymes. These data are consistent with both enzymes binding NADP(H) in a conserved manner which is supported by the available crystal structures. The results suggest that cofactor binding or release for the human and rat 3α-HSDs are similar and do not account for the observed differences in k cat. (Supported by DK40715 and CA90744 to T.M.P.)

Evidence of heterogeneous 1-anilinonaphthalene-8-sulfonate binding to β-lactoglobulin from fluorescence spectroscopy

Steady-state and dynamic fluorescence titrations show that: (a) the complex between β-lactoglobulin (BLG) and 1-anilinonaphthalene-8-sulfonate (ANS) displays a heterogeneous equilibrium with large changes in the binding strength vs. pH and ion concentration; and (b) the fluorescence response of bound ANS reveals two separate lifetimes that suggest two different sites (or binding modes). While steady-state fluorescence titrations yield effective values of the binding constant and of the bound ANS quantum efficiency, it is shown that, by combining steady-state fluorescence and lifetime decay of ANS, it is possible to give quantitative estimates of the association constants for each site. When heading from the acid (pH∼2) to the native state (pH∼6) the main result is a very large reduction of the effective binding constant. This and the results of titrations vs. ionic strength suggest that electrostatic interactions are a major contribution to ANS binding to BLG.

Role of lysine 240 in the mechanism of yeast pyruvate kinase catalysis.

Site-directed mutagenesis was used to change Lys 240 of yeast pyruvate kinase (Lys 269 in muscle PK) to Met. K240M has an absolute requirement for FBP for catalysis. K240M is 100- and 1000-fold less active than wild-type YPK in the presence of Mn(2+) and Mg(2+), respectively. Steady-state fluorescence titration data suggest that the substrate PEP binds to K240M with the same affinity as it does to wild-type YPK. The rate of phosphoryl transfer in K240M has been decreased >1000-fold compared to wild-type YPK. The detritiation of 3-[(3)H]pyruvate catalyzed by YPK occurs at a rate significantly greater than the spontaneous rate. Detritiation of pyruvate by wild-type YPK occurs as a divalent metal- and FBP-dependent process requiring ATP. There is no detectable detritiation of pyruvate catalyzed by K240M. The solvent deuterium isotope effect on k(cat) is 2.7 +/- 0.2 and 1.6 +/- 0.1 for the wild type and for K240M YPK, respectively. This suggests that the isotope sensitive step in the PK reaction does not involve Lys 240 and that the enolpyruvate intermediate is still protonated by K240M. Isotope trapping was used to characterize enolpyruvate protonation by K240M. While there was enrichment of the methyl protons of pyruvate from labeled solvent formed by catalysis with muscle PK and wild-type YPK, only background levels of tritium were trapped with K240M. In K240M, the proton donor exchanges protons with the solvent at a higher rate relative to turnover than does the proton donor in wild-type YPK. The pH-rate profile of K240M exhibits the loss of a pK(a) value of 8. 8 observed with wild-type YPK. The above data and recent crystal structure data suggest that Lys 240 interacts with the phosphoryl group of phosphoenolpyruvate and helps to stabilize the pentavalent phosphate transition state during phosphoryl transfer. Phosphoryl transfer is highly coupled to proton transfer, or Lys 240 also affects enolate protonation.

Interactions of fluorescent cholinergic antagonists with the membrane-bound acetylcholine receptor

The synthesis of pyrenyl-choline derivatives is reported. The interactions of these compounds with the acetylcholine receptor has been studied in developing chick myotubes in culture and in the adult frog neuromuscular junction with electrophysiological techniques. In vivo, the compounds display the pharmacological characteristics of nicotinic antagonists. Similar conclusions can be derived from in vitro experiments using radiolabelled derivatives in equilibrium binding studies with the membrane-bound receptor from Torpedo, steady-state fluorescence titrations, and toxin kinetic inhibition studies. The rapid kinetics of receptor-probe interactions were studied with the temperature-jump method. The fluorescence emission of the probes, excited by energy transfer from the protein chromophores, enabled the observation of two relaxation processes. The concentration dependence of the relaxation times and associated amplitudes can most economically be accounted for in terms of a reaction mechanism consisting of two sequential ligand binding-isomerization steps. The two ligands find themselves in an environment of high electronic polarizability, having a dielectric constant of about 40, after rapidly binding to two sites which differ in their affinities roughly by a factor of 10 (Apparent Kd's 1.5 and 0.2 μM).