Pb²⁺ Binds to Downstream Regulatory Element Antagonist Modulator (DREAM) and Modulates Its Interactions with Binding Partners: A Link between Neuronal Calcium Sensors and Pb²⁺ Neurotoxicity

Pb2+ exposure leads to diverse neurological disorders; however, the mechanism of Pb2+-induced neurotoxicity is not clearly understood. Here we demonstrate that Pb2+ binds to EF-hands in apo-DREAM (downstream regulatory element antagonist modulator) with a lower equilibrium dissociation constant (Kd = 20 ± 2 nM) than Ca2+ (Kd = 1 μM). Based on the Trp169 emission and CD spectra, we report that Pb2+ association triggers changes in the protein secondary and tertiary structures that are analogous to those previously observed for Ca2+-bound protein. The hydrophobic cavity in the C-terminal domain of DREAM is solvent exposed in the presence of Pb2+ as determined using a hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS). Pb2+ association with DREAM also modulates interactions between DREAM and its intracellular partners as evident from the fact that Pb2+-bound DREAM associates with peptide-based model systems, presenilin-1 helix-9 “PS1HL9” KV4.3­(70–90) “site-2” and KV4.3­(2–22) “site 1”. Namely, dissociation constants for Pb2+-bound DREAM interaction with PS1HL9 (Kd = 2.4 ± 0.1 μM), site-2 (Kd = 11.0 ± 0.5 μM) and site 1 (Kd = 5.0 ± 0.6 μM) are nearly identical to those observed for Ca2+ bound DREAM. Isothermal titration calorimetry data reveal that Pb2+ binds to two high-affinity sites in Ca2+ bound DREAM with the overall apparent constant of 4.81 ± 0.06 μM and its binding to Ca2+ bound DREAM is entropy-driven. Taking into account the structural and sequence similarity between DREAM and other neuronal calcium sensor (NCS) proteins, these results strongly indicate that DREAM and possibly other NCS proteins bind Pb2+ with a higher affinity than that for Ca2+ and Pb2+ interactions with NCS proteins can contribute to Pb2+-induced neurotoxicity

Amphiphilic Residues 29–44 of DREAM N‑Termini Mediate Calmodulin:DREAM Complex Formation

DREAM (downstream regulatory element antagonist modulator) is a neuronal calcium sensor that has been shown to modulate gene expression as well as to be involved in numerous neuronal processes. In this report, we show that association of calcium-bound calmodulin (CaM) with DREAM is mediated by a short amphipathic amino acid sequence located between residues 29 and 44 on DREAM. The association of CaM with a peptide analogous to DREAM(29–44) or to full-length DREAM protein is calcium-dependent with a dissociation constant of 136 nM or 3.4 μM, respectively. Thermodynamic and kinetic studies show that the observed decrease in affinity for the native protein is due to electrostatic interactions between the basic N-terminus and an electronegative surface on DREAM. These results are further supported by circular dichroism, binding studies, and molecular dynamics simulations. Additionally, fluorescence anisotropy decay measurements show a rotational correlation time of 10.8 ns for a complex of CaM with a DREAM(29–44) peptide, supporting a wraparound semispherical model with 1:1 stoichiometry. Furthermore, the interaction between an IEDANS-labeled CaM construct with DREAM is best modeled as a heterotetramer that adopts an elongated conformation with a correlation time of 45 ns in the presence of Ca²⁺. We also demonstrate that association of CaM with DREAM eliminates the nonspecific interaction of DREAM with the DRE double-stranded DNA sequence of the human <i>prodynorphin</i> gene. This work provides molecular insight into the CaM:DREAM complex and its potential role in modulation of gene expression

β‑Cyclodextrin Reverses Binding of Perfluorooctanoic Acid to Human Serum Albumin

Perfluorooctanoic acid (PFOA), a persistent organic pollutant known to cause adverse health effects, strongly binds to human serum albumin (HSA). β-Cyclodextrin (β-CD), a nontoxic cyclic sugar, strongly complexes PFOA in a host–guest complex and has been proposed for environmental remediation of PFOA. The interactions between HSA, PFOA, and β-CD were investigated in order to determine if β-CD can reverse the binding of PFOA to HSA, with potential therapeutic applications toward exposure to PFOA. ¹⁹F Nuclear magnetic resonance (NMR), circular dichroism, and fluorescence spectroscopies were used to study these interactions. Multiple PFOA binding sites to HSA, one with strong affinity and others with low affinity, are evident from changes in the fluorescence emission spectra of HSA and the fluorescence lifetimes of the single Trp residue in HSA with increasing PFOA concentration. Structural changes in the protein are also evident from changes in the circular dichroism spectra of HSA upon titration of PFOA. Addition of β-CD to PFOA and HSA reversed these changes, indicating that formation of the β-CD:PFOA host–guest complex is favored even in the presence of HSA. Equimolar β-CD to PFOA (1:1 β-CD:PFOA ratio) causes dissociation of the weakly bound PFOA from HSA, whereas excess β-CD relative to PFOA (5:1 β-CD:PFOA ratio) leads to the complete disassociation of the strongly bound PFOA molecule from HSA. The ¹⁹F NMR studies further suggest that the 2:1 β-CD:PFOA complex inhibits PFOA binding to HSA. These data demonstrate that β-CD has potential to be used in therapeutic applications for PFOA in human blood

Ground- and Excited-State Properties of Zn(II) Tetrakis(4-tetramethylpyridyl) Pophyrin Specifically Encapsulated within a Zn(II) HKUST Metal–Organic Framework

We have examined the photophysical properties of Zn(II) tetramethylpyridyl porphyrin (ZnT4MPyP) specifically encapsulated within the cubioctahedral cavities of a ZnHKUST metal– organic framework. The encapsulated ZnT4MPyP exhibits a Soret maxima at ∼458 nm that is bathochromically shifted relative to ZnT4PyP in ethanol solution (Soret maxima centered at 440 nm). The corresponding emission spectra of the encapsulated porphyrin exhibit resolvable bands centered at 636 and 677 nm relative to a single broad emission band of the ZnT4MPyP in ethanol solution centered at 636 nm with a shoulder situated near ∼660 nm. The fluorescence lifetime of the encapsulated porphyrin is also perturbed relative to that of the free porphyrin in solution (1.88 ns for the encapsulated porphyrin relative to 1.2 ns in solution). These results are consistent with the ZnT4MPyP being in a more constrained environment in which the peripheral pyridyl groups have restricted rotational motion. The ZnT4MPyP triplet lifetime is also affected by encapsulation, giving rise to a longer lifetime (τ ≈ 3.3 ms) relative to that for the free porphyrin in solution (τ ≈ 1 ms). The triplet-state results indicate that nonplanar vibrational modes of the porphyrin leading to intersystem crossing are retained by encapsulation of the porphyrin but that either the density of vibrational states or the specific nonplanar modes coupling the singlet and triplet states may be perturbed, resulting in the longer observed lifetime

Ground- and Excited-State Properties of Zn(II) Tetrakis(4-tetramethylpyridyl) Pophyrin Specifically Encapsulated within a Zn(II) HKUST Metal–Organic Framework

We have examined the photophysical properties of Zn(II) tetramethylpyridyl porphyrin (ZnT4MPyP) specifically encapsulated within the cubioctahedral cavities of a ZnHKUST metal– organic framework. The encapsulated ZnT4MPyP exhibits a Soret maxima at ∼458 nm that is bathochromically shifted relative to ZnT4PyP in ethanol solution (Soret maxima centered at 440 nm). The corresponding emission spectra of the encapsulated porphyrin exhibit resolvable bands centered at 636 and 677 nm relative to a single broad emission band of the ZnT4MPyP in ethanol solution centered at 636 nm with a shoulder situated near ∼660 nm. The fluorescence lifetime of the encapsulated porphyrin is also perturbed relative to that of the free porphyrin in solution (1.88 ns for the encapsulated porphyrin relative to 1.2 ns in solution). These results are consistent with the ZnT4MPyP being in a more constrained environment in which the peripheral pyridyl groups have restricted rotational motion. The ZnT4MPyP triplet lifetime is also affected by encapsulation, giving rise to a longer lifetime (τ ≈ 3.3 ms) relative to that for the free porphyrin in solution (τ ≈ 1 ms). The triplet-state results indicate that nonplanar vibrational modes of the porphyrin leading to intersystem crossing are retained by encapsulation of the porphyrin but that either the density of vibrational states or the specific nonplanar modes coupling the singlet and triplet states may be perturbed, resulting in the longer observed lifetime

Role of Ionic Strength and pH in Modulating Thermodynamic Profiles Associated with CO Escape from Rice Nonsymbiotic Hemoglobin 1

Type 1 nonsymbiotic hemoglobins are found in a wide variety of land plants and exhibit very high affinities for exogenous gaseous ligands. These proteins are presumed to have a role in protecting plant cells from oxidative stress under etiolated/hypoxic conditions through NO dioxygenase activity. In this study we have employed photoacoustic calorimetry, time-resolved absorption spectroscopy, and classical molecular dynamics simulations in order to elucidate thermodynamics, kinetics, and ligand migration pathways upon CO photodissociation from WT and a H73L mutant of type 1 nonsymbiotic hemoglobin from <i>Oryza sativa</i> (rice). We observe a temperature dependence of the resolved thermodynamic parameters for CO photodissociation from CO-rHb1 which we attribute to temperature dependent formation of a network of electrostatic interactions in the vicinity of the heme propionate groups. We also observe slower ligand escape from the protein matrix under mildly acidic conditions in both the WT and H73L mutant (τ = 134 ± 19 and 90 ± 15 ns). Visualization of transient hydrophobic channels within our classical molecular dynamics trajectories allows us to attribute this phenomenon to a change in the ligand migration pathway which occurs upon protonation of the distal His73, His117, and His152. Protonation of these residues may be relevant to the functioning of the protein in vivo given that etiolation/hypoxia can cause a decrease in intracellular pH in plant cells

Photophysical Characterization of a Benzo-Fused Analogue of Brooker’s Merocyanine: Solvent Polarity and pH Effects

The photophysical properties of 4-[2-(6-hydroxy-2-naphthalenyl)-ethenyl]-1-methyl-pyridinium (<b>HNEP</b>^<b>+</b>) and its deprotonated form (<b>NEP</b>), a benzofused derivative of Brooker’s merocyanine (<b>BM</b>), were investigated through a combined spectroscopic and computational approach. Despite their structural similarities and similar p<i>K</i>_a values, <b>HNEP</b>^<b>+</b>/<b>NEP</b> and <b>BMH</b>^<b>+</b>/<b>BM</b> differ in the extent of charge delocalization in the ground and excited states. <b>NEP</b> exhibits the spectral characteristics of a charge transfer species in solvents in which <b>BM</b> exists in a charge-delocalized quinoid; however, quantum chemical calculations show that the CT absorption of <b>NEP</b> is not necessarily a consequence of the zwitterionic character. <b>HNEP</b>^<b>+</b> displays larger Stokes shifts than <b>BMH</b>^<b>+</b>, and <b>NEP</b> demonstrates enhanced solvatochromism relative to <b>BM</b> as a consequence of benzofusion

Emission Switching of 4,6-Diphenylpyrimidones: Solvent and Solid State Effects

The photophysics of 1-ethyl-4,6-bis­(4-methoxyphenyl)-2­(1H)-pyrimidone (1) and 1-ethyl-4,6-bis­(4-(dimethylamino)­phenyl)-2­(1H)-pyrimidone (2) were investigated to determine the mechanisms of emission switching in response to protonation. UV–vis and steady state emission spectroscopy of the protonated and unprotonated forms across a range of solvents reveal the polarity dependence of the vertical excitation energies. Emission lifetimes and quantum yields show the solvent dependency of the excited states. Emission enhancements were observed in polyethylene glycol solutions and in the solid state (both thin film and single crystal), demonstrating the role of intramolecular rotation in thermal relaxation of the excited states. TD-DFT calculations provide insights into the excited state geometries and the role of intramolecular charge transfer. The collected data show that emission of diphenylpyrimidones can be modulated by four factors, including the identity of the electron-donating auxochrome, protonation state, solvent polarity, and viscosity

Effects of Turn Stability on the Kinetics of Refolding of a Hairpin in a β-sheet

As part of our continuing study of the effects of the turn sequence on the conformational stability as well as the mechanism of folding of a β-sheet structure, we have undertaken a parallel investigation of the solution structure, conformational stability, and kinetics of refolding of the β-sheet VFIVDGOTYTEVDPGOKILQ. The latter peptide is an analogue of the original Gellman β-sheet VFITSDPGKTYTEVDPGOKILQ, wherein the TSDPGK turn sequence in the first hairpin has been replaced by VDGO. Thermodynamics studies revealed comparable conformational stability of the two peptides. However, unlike the Gellman peptide, which showed extremely rapid refolding of the first hairpin, early kinetic events associated with the refolding of the corresponding hairpin in the VDGO mutant were found to be significantly slower. A detailed study of the conformation of the modified peptide suggested that hydrophobic interactions might be contributing to its stability. Accordingly, we surmise that the early kinetic events are sensitive to whether the formation of the hairpin is nucleated at the turn or by sequestering of the hydrophobic residues across the strand, before structural rearrangements to produce the nativelike topology. Nucleation of the hairpin at the turn is expected to be intrinsically rapid for a strong turn. However, if the process must involve collapse of hydrophobic side chains, the nucleation should be slower as solvent molecules must be displaced to sequester the hydrophobic residues. These findings reflect the contribution of different forces toward nucleation of hairpins in the mechanism of folding of β-sheets

Emission Tuning of Fluorescent Kinase Inhibitors: Conjugation Length and Substituent Effects

Fluorescent <i>N</i>-phenyl-4-aminoquinazoline probes targeting the ATP-binding pocket of the ERBB family of receptor tyrosine kinases are reported. Extension of the aromatic quinazoline core with fluorophore “arms” through substitution at the 6- position of the quinazoline core with phenyl, styryl, and phenylbutadienyl moieties was predicted by means of TD-DFT calculations to produce probes with tunable photoexcitation energies and excited states possessing charge-transfer character. Optical spectroscopy identified several synthesized probes that are nonemissive in aqueous solutions and exhibit emission enhancements in solvents of low polarity, suggesting good performance as turn-on fluorophores. Ligand-induced ERBB2 phosphorylation assays demonstrate that despite chemical modification to the quinazoline core these probes still function as ERBB2 inhibitors in MCF7 cells. Two probes were found to exhibit ERBB2-induced fluorescence, demonstrating the utility of these probes as turn-on, fluoroescent kinase inhibitors