DMNP as an Effective Cage for Ln3+: A Spectroscopic and Thermodynamic Study.

Dimethoxynitrophenyl-EDTA (DMNP) is a popular calcium cage that is frequently used to investigate the role of Ca2+ in signaling processes in vivo. Lanthanides have been used in Ca2+ biomimetics due to similarities in coordination properties of Ln3+ and Ca2+ that may enable fluorescence and NMR studies of functional and structural properties of Ca2+ binding proteins. In this study, we show that Tb3+, Eu3+, and Nd3+ bind strongly to DMNP in a 1:1 ratio. Isothermal titration calorimetric measurements of Ca2+ displacement by Ln3+ in DMNP provide the equilibrium binding constants for Ln3+DMNP complexation with association constants, K11 = (1.2 ± 0.7) × 1012 M-1 for Eu3+, (2.5 ± 1.7) × 1012 M-1 for Nd3+, and (2.8 ± 0.8) × 1012 M-1 for Tb3+. The kinetics and thermodynamics of Ca2+, Mg2+, and Tb3+ release from DMNP were characterized using photothermal beam deflection (PBD). Ligand release from the DMNP cage was rapid and occurred within 10 µs upon cage photofragmentation and was associated with similar reaction volume and enthalpy changes that can be attributed to the photoreleased ion solvation. In the case of Ca2+DMNP photodissociation at subsaturating Ca2+ concentrations, we observed a slower phase with a lifetime of 300 µs that we attribute to Ca2+ rebinding to unphotolyzed DMNP. These results demonstrate that DMNP can serve as an effective photolabile cage for oxophilic Ln3+ that has similar coordination properties to Ca2+ and Mg2+.

Distinct mechanism of Tb3+ and Eu3+ binding to NCS1.

Lanthanides have been frequently used as biomimetic compounds for NMR and fluorescence studies of Ca2+ binding proteins due to having similar physical properties and coordination geometry to Ca2+ ions. Here we report that a member of the neuronal calcium sensor family, neuronal calcium sensor 1, complexes with two lanthanide ions Tb3+ and Eu3+. The affinity for Tb3+ is nearly 50 times higher than that for Ca2+ (Kd,Tb3+ = 0.002 ± 0.0001 µM and Kd, Ca2+ = 91 nM) whereas Eu3+ binding is notably weaker, Kd,Eu3+ = 26 ± 1 µM. Interestingly, despite having identical charge and similar ionic radii, Tb3+ and Eu3+ ions exhibit a distinct binding stoichiometry for NCS1 with one Eu3+ and two Tb3+ ions bound per NCS1 monomer, as demonstrated in fluorescence titration and mass spectrometry studies. These results suggest that the lanthanides' affinity for the individual EF hands is fine-tuned by a small variation in the ion charge density as well as EF hand binding loop amino acid sequence. As observed previously for other lanthanide:protein complexes, the emission intensity of Ln3+ is enhanced upon complexation with the protein, likely due to the displacement of water molecules by oxygen atoms from the coordinating amino acid residues. The overall shape of the Tb3+NCS1 and Eu3+NCS1 monomer shows high levels of similarity compared to the Ca2+ bound protein based on their collision cross section. However, the distinct occupation of EF hands impacts NCS1 oligomerization and affinity for the D2R peptide that mimics the NCS1 binding site on the D2R receptor. Specifically, the Tb3+NCS1 complex populates the dimer and has comparable affinity for the D2R peptide, whereas Eu3+ bound NCS1 remains in the monomeric form with a negligible affinity for the D2R peptide.

Fluorescent 5-Pyrimidine and 8-Purine Nucleosides Modified with an N-Unsubstituted 1,2,3-Triazol-4-yl Moiety.

The Cu(I)- or Ag(I)-catalyzed cycloaddition between 8-ethynyladenine or guanine nucleosides and TMSN3 gave 8-(1- H-1,2,3-triazol-4-yl) nucleosides in good yields. On the other hand, reactions of 5-ethynyluracil or cytosine nucleosides with TMSN3 led to the chemoselective formation of triazoles via Cu(I)-catalyzed cycloaddition or vinyl azides via Ag(I)-catalyzed hydroazidation. These nucleosides with a minimalistic triazolyl modification showed excellent fluorescent properties with 8-(1- H-1,2,3-triazol-4-yl)-2'-deoxyadenosine (8-TrzdA), exhibiting a quantum yield of 44%. The 8-TrzdA 5'-triphosphate was incorporated into duplex DNA containing a one-nucleotide gap by DNA polymerase ß.

The effects of solution additives and gas-phase modifiers on the molecular environment and conformational space of common heme proteins.

RATIONALE: The molecular environment is known to impact the secondary and tertiary structures of biomolecules both in solution and in the gas phase, shifting the equilibrium between different conformational and oligomerization states. However, there is a lack of studies monitoring the impacts of solution additives and gas-phase modifiers on biomolecules characterized using ion mobility techniques. METHODS: The effect of solution additives and gas-phase modifiers on the molecular environment of two common heme proteins, bovine cytochrome c and equine myoglobin, is investigated as a function of the time after desolvation (e.g., 100-500 ms) using nanoelectrospray ionization coupled to trapped ion mobility spectrometry with detection by time-of-flight mass spectrometry. Organic compounds used as additives/modifiers (methanol, acetonitrile, acetone) were either added to the aqueous protein solution before ionization or added to the ion mobility bath gas by nebulization. RESULTS: Changes in the mobility profiles are observed depending on the starting solution composition (i.e., in aqueous solution at neutral pH or in the presence of organic content: methanol, acetone, or acetonitrile) and the protein. In the presence of gas-phase modifiers (i.e., N2 doped with methanol, acetone, or acetonitrile), a shift in the mobility profiles driven by the gas-modifier mass and size and changes in the relative abundances and number of IMS bands are observed. CONCLUSIONS: We attribute the observed changes in the mobility profiles in the presence of gas-phase modifiers to a clustering/declustering mechanism by which organic molecules adsorb to the protein ion surface and lower energetic barriers for interconversion between conformational states, thus redefining the free energy landscape and equilibria between conformers. These structural biology experiments open new avenues for manipulation and interrogation of biomolecules in the gas phase with the potential to emulate a large suite of solution conditions, ultimately including conditions that more accurately reflect a variety of intracellular environments.

ß-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). ß-Cyclodextrin (ß-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 ß-CD were investigated in order to determine if ß-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 ß-CD to PFOA and HSA reversed these changes, indicating that formation of the ß-CD:PFOA host-guest complex is favored even in the presence of HSA. Equimolar ß-CD to PFOA (1:1 ß-CD:PFOA ratio) causes dissociation of the weakly bound PFOA from HSA, whereas excess ß-CD relative to PFOA (5:1 ß-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 ß-CD:PFOA complex inhibits PFOA binding to HSA. These data demonstrate that ß-CD has potential to be used in therapeutic applications for PFOA in human blood.

Insights from ion mobility-mass spectrometry, infrared spectroscopy, and molecular dynamics simulations on nicotinamide adenine dinucleotide structural dynamics: NAD+vs. NADH.

Nicotinamide adenine dinucleotide (NAD) is found in all living cells where the oxidized (NAD+) and reduced (NADH) forms play important roles in many enzymatic reactions. However, little is known about NAD+ and NADH conformational changes and kinetics as a function of the cell environment. In the present work, an analytical workflow is utilized to study NAD+ and NADH dynamics as a function of the organic content in solution using fluorescence lifetime spectroscopy and in the gas-phase using trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) and infrared multiple photon dissociation (IRMPD) spectroscopy. NAD solution time decay studies showed a two-component distribution, assigned to changes from a "close" to "open" conformation with the increase of the organic content. NAD gas-phase studies using nESI-TIMS-MS displayed two ion mobility bands for NAD+ protonated and sodiated species, while four and two ion mobility bands were observed for NADH protonated and sodiated species, respectively. Changes in the mobility profiles were observed for NADH as a function of the starting solution conditions and the time after desolvation, while NAD+ profiles showed no dependence. IRMPD spectroscopy of NAD+ and NADH protonated species in the 800-1800 and 3200-3700 cm-1 spectral regions showed common and signature bands between the NAD forms. Candidate structures were proposed for NAD+ and NADH kinetically trapped intermediates of the protonated and sodiated species, based on their collision cross sections and IR profiles. Results showed that NAD+ and NADH species exist in open, stack, and closed conformations and that the driving force for conformational dynamics is hydrogen bonding of the N-H-O and O-H-O forms with ribose rings.

Characterization of Intramolecular Interactions of Cytochrome c Using Hydrogen-Deuterium Exchange-Trapped Ion Mobility Spectrometry-Mass Spectrometry and Molecular Dynamics.

Globular proteins, such as cytochrome c (cyt c), display an organized native conformation, maintained by a hydrogen bond interaction network. In the present work, the structural interrogation of kinetically trapped intermediates of cyt c was performed by correlating the ion-neutral collision cross section (CCS) and charge state with the starting solution conditions and time after desolvation using collision induced activation (CIA), time-resolved hydrogen/deuterium back exchange (HDX) and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). The high ion mobility resolving power of the TIMS analyzer allowed the identification of new ion mobility bands, yielding a total of 63 mobility bands over the +6 to +21 charge states and 20 mobility bands over the -5 to -10 charge states. Mobility selected HDX rates showed that for the same charge state, conformers with larger CCS present faster HDX rates in both positive and negative ion mode, suggesting that the charge sites and neighboring exchange sites on the accessible surface area define the exchange rate regardless of the charge state. Complementary molecular dynamic simulations permitted the generation of candidate structures and a mechanistic model of the folding transitions from native (N) to molten globule (MG) to kinetic intermediates (U) pathways. Our results suggest that cyt c major structural unfolding is associated with the distancing of the N- and C-terminal helices and subsequent solvent exposure of the hydrophobic, heme-containing cavity.

Characterization of the Photophysical, Thermodynamic, and Structural Properties of the Terbium(III)-DREAM Complex.

DREAM (also known as K(+) channel interacting protein 3 and calsenilin) is a calcium binding protein and an active modulator of KV4 channels in neuronal cells as well as a novel Ca(2+)-regulated transcriptional modulator. DREAM has also been associated with the regulation of Alzheimer's disease through the prevention of presenilin-2 fragmentation. Many interactions of DREAM with its binding partners (Kv4, calmodulin, DNA, and drugs) have been shown to be dependent on calcium. Therefore, understanding the structural changes induced by binding of metals to DREAM is essential for elucidating the mechanism of signal transduction and biological activity of this protein. Here, we show that the fluorescence emission and excitation spectra of the calcium luminescent analogue, Tb(3+), are enhanced upon binding to the EF-hands of DREAM due to a mechanism of energy transfer between Trp and Tb(3+). We also observe that unlike Tb(3+)-bound calmodulin, the luminescence lifetime of terbium bound to DREAM decays as a complex multiexponential (τaverage ∼ 1.8 ms) that is sensitive to perturbation of the protein structure and drug (NS5806) binding. Using isothermal calorimetry, we have determined that Tb(3+) binds to at least three sites with high affinity (Kd = 1.8 µM in the presence of Ca(2+)) and displaces bound Ca(2+) through an entropically driven mechanism (ΔH ∼ 12 kcal mol(-1), and TΔS ∼ 22 kcal mol(-1)). Furthermore, the hydrophobic probe 1,8-ANS shows that Tb(3+), like Ca(2+), triggers the exposure of a hydrophobic surface on DREAM, which modulates ligand binding. Analogous to Ca(2+) binding, Tb(3+) binding also induces the dimerization of DREAM. Secondary structural analyses using far-UV circular dichroism and trapped ion mobility spectrometry-mass spectrometry reveal that replacement of Ca(2+) with Tb(3+) preserves the folding state with minimal changes to the overall structure of DREAM. These findings pave the way for further investigation of the metal binding properties of DREAM using lanthanides as well as the study of DREAM-protein complexes by lanthanide resonance energy transfer or nuclear magnetic resonance.

Structures of the kinetically trapped i-motif DNA intermediates.

In the present work, the conformational dynamics and folding pathways of i-motif DNA were studied in solution and in the gas-phase as a function of the solution pH conditions using circular dichroism (CD), photoacoustic calorimetry analysis (PAC), trapped ion mobility spectrometry-mass spectrometry (TIMS-MS), and molecular dynamics (MD). Solution studies showed at thermodynamic equilibrium the existence of a two-state folding mechanism, whereas during the pH = 7.0 → 4.5 transition a fast and slow phase (ΔHfast + ΔHslow = 43 ± 7 kcal mol-1) with a volume change associated with the formation of hemiprotonated cytosine base pairs and concomitant collapse of the i-motif oligonucleotide into a compact conformation were observed. TIMS-MS experiments showed that gas-phase, kinetically trapped i-motif DNA intermediates produced by nanoESI are preserved, with relative abundances depending on the solution pH conditions. In particular, a folded i-motif DNA structure was observed in nanoESI-TIMS-MS for low charge states in both positive and negative ion mode (e.g., z = ±3 to ±5) at low pH conditions. As solution pH increases, the cytosine neutralization leads to the loss of cytosine-cytosine+ (C·CH+) base pairing in the CCC strands and in those conditions we observe partially unfolded i-motif DNA conformations in nanoESI-TIMS-MS for higher charge states (e.g., z = -6 to -9). Collisional induced activation prior to TIMS-MS showed the existence of multiple local free energy minima, associated with the i-motif DNA unfolding at z = -6 charge state. For the first time, candidate gas-phase structures are proposed based on mobility measurements of the i-motif DNA unfolding pathway. Moreover, the inspection of partially unfolded i-motif DNA structures (z = -7 and z = -8 charge states) showed that the presence of inner cations may or may not induce conformational changes in the gas-phase. For example, incorporation of ammonium adducts does not lead to major conformational changes while sodium adducts may lead to the formation of sodium mediated bonds between two negatively charged sides inducing the stabilization towards more compact structures in new local, free energy minima in the gas-phase.

Synthesis of 8-(1,2,3-triazol-1-yl)-7-deazapurine nucleosides by azide-alkyne click reactions and direct C-H bond functionalization.

Treatment of toyocamycin or sangivamycin with 1,3-dibromo-5,5-dimethylhydantoin in MeOH (r.t./30 min) gave 8-bromotoyocamycin and 8-bromosangivamycin in good yields. Nucleophilic aromatic substitution of 8-bromotoyocamycin with sodium azide provided novel 8-azidotoyocamycin. Strain promoted click reactions of the latter with cyclooctynes resulted in the formation of the 1,2,3-triazole products. Iodine-mediated direct C8-H bond functionalization of tubercidin with benzotriazoles in the presence of tert-butyl hydroperoxide gave the corresponding 8-benzotriazolyltubercidin derivatives. The 8-(1,2,3-triazol-1-yl)-7-deazapurine derivatives showed moderate quantum yields and a large Stokes shifts of ~ 100 nm.

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 Ca(2+). 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 prodynorphin gene. This work provides molecular insight into the CaM:DREAM complex and its potential role in modulation of gene expression.

Modulation of the voltage-gated potassium channel (Kv4.3) and the auxiliary protein (KChIP3) interactions by the current activator NS5806.

KChIP3 (potassium channel interacting protein 3) is a calcium-binding protein that binds at the N terminus of the Kv4 voltage-gated potassium channel through interactions at two contact sites and has been shown to regulate potassium current gating kinetics as well as channel trafficking in cardiac and neuronal cells. Using fluorescence spectroscopy, isothermal calorimetry, and docking simulations we show that the novel potassium current activator, NS5806, binds at a hydrophobic site on the C terminus of KChIP3 in a calcium-dependent manner, with an equilibrium dissociation constant of 2-5 µM in the calcium-bound form. We further determined that the association between KChIP3 and the hydrophobic N terminus of Kv4.3 is calcium-dependent, with an equilibrium dissociation constant in the apo-state of 70 ± 3 µM and 2.7 ± 0.1 µM in the calcium-bound form. NS5806 increases the affinity between KChIP3 and the N terminus of Kv4.3 (Kd = 1.9 ± 0.1 µM) in the presence and absence of calcium. Mutation of Tyr-174 or Phe-218 on KChIP3 abolished the enhancement of Kv4.3 site 1 binding in the apo-state, highlighting the role of these residues in drug and K4.3 binding. Kinetic studies show that NS5806 decreases the rate of dissociation between KChIP3 and the N terminus of KV4.3. Overall, these studies support the idea that NS5806 directly interacts with KChIP3 and modulates the interactions between this calcium-binding protein and the T1 domain of the Kv4.3 channels through reorientation of helix 10 on KChIP3.

Application of ANS fluorescent probes to identify hydrophobic sites on the surface of DREAM.

DREAM (calsenilin or KChIP-3) is a calcium sensor involved in regulation of diverse physiological processes by interactions with multiple intracellular partners including DNA, Kv4 channels, and presenilin, however the detailed mechanism of the recognition of the intracellular partners remains unclear. To identify the surface hydrophobic surfaces on apo and Ca(2+)DREAM as a possible interaction sites for target proteins and/or specific regulators of DREAM function the binding interactions of 1,8-ANS and 2,6-ANS with DREAM were characterized by fluorescence and docking studies. Emission intensity of ANS-DREAM complexes increases upon Ca(2+) association which is consistent with an overall decrease in surface polarity. The dissociation constants for ANS binding to apoDREAM and Ca(2+)DREAM were determined to be 195±20µM and 62±4µM, respectively. Fluorescence lifetime measurements indicate that two ANS molecules bind in two independent binding sites on DREAM monomer. One site is near the exiting helix of EF-4 and the second site is located in the hydrophobic crevice between EF-3 and EF-4. 1,8-ANS displacement studies using arachidonic acid demonstrate that the hydrophobic crevice between EF-3 and EF-4 serves as a binding site for fatty acids that modulate functional properties of Kv4 channel:KChIP complexes. Thus, the C-terminal hydrophobic crevice may be involved in DREAM interactions with small hydrophobic ligands as well as other intracellular proteins.

Strain Promoted Click Chemistry of 2- or 8-Azidopurine and 5-Azidopyrimidine Nucleosides and 8-Azidoadenosine Triphosphate with Cyclooctynes. Application to Living Cell Fluorescent Imaging.

Strain-promoted click chemistry of nucleosides and nucleotides with an azido group directly attached to the purine and pyrimidine rings with various cyclooctynes in aqueous solution at ambient temperature resulted in efficient formation (3 min to 3 h) of fluorescent, light-up, triazole products. The 2- and 8-azidoadenine nucleosides reacted with fused cyclopropyl cyclooctyne, dibenzylcyclooctyne, or monofluorocyclooctyne to produce click products functionalized with hydroxyl, amino, N-hydroxysuccinimide, or biotin moieties. The 5-azidouridine and 5-azido-2'-deoxyuridine were similarly converted to the analogous triazole products in quantitative yields in less than 5 min. The 8-azido-ATP quantitatively afforded the triazole product with fused cyclopropyl cyclooctyne in aqueous acetonitrile (3 h). The novel triazole adducts at the 2- or 8-position of adenine or 5-position of uracil rings induce fluorescence properties which were used for direct imaging in MCF-7 cancer cells without the need for traditional fluorogenic reporters. FLIM of the triazole click adducts demonstrated their potential utility for dynamic measuring and tracking of signaling events inside single living cancer cells.

Flavin adenine dinucleotide structural motifs: from solution to gas phase.

Flavin adenine dinucleotide (FAD) is involved in important metabolic reactions where the biological function is intrinsically related to changes in conformation. In the present work, FAD conformational changes were studied in solution and in gas phase by measuring the fluorescence decay time and ion-neutral collision cross sections (CCS, in a trapped ion mobility spectrometer, TIMS) as a function of the solvent conditions (i.e., organic content) and gas-phase collisional partner (i.e., N2 doped with organic molecules). Changes in the fluorescence decay suggest that FAD can exist in four conformations in solution, where the abundance of the extended conformations increases with the organic content. TIMS-MS experiments showed that FAD can exist in the gas phase as deprotonated (M = C27H31N9O15P2) and protonated forms (M = C27H33N9O15P2) and that multiple conformations (up to 12) can be observed as a function of the starting solution for the [M + H](+) and [M + Na](+)molecular ions. In addition, changes in the relative abundances of the gas-phase structures were observed from a "stack" to a "close" conformation when organic molecules were introduced in the TIMS cell as collision partners. Candidate structures optimized at the DFT/B3LYP/6-31G(d,p) were proposed for each IMS band, and results showed that the most abundant IMS band corresponds to the most stable candidate structure. Solution and gas-phase experiments suggest that the driving force that stabilizes the different conformations is based on the interaction of the adenine and isoalloxazine rings that can be tailored by the "solvation" effect created with the organic molecules.

Emission tuning of fluorescent kinase inhibitors: conjugation length and substituent effects.

Fluorescent N-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.

Detail study on the interaction between perfluorooctanoic acid (PFOA) with human hemoglobin (Hb).

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are often referred to as legacy perfluoroalkyl substances (PFAS). Human exposure to PFAS leads to severe negative health impacts including cancers, infertility, and dysfunction in the kidneys. Steady-state absorbance, fluorescence, and circular dichroism (CD) methods were used to study the interactions between PFOA and Hb. The results demonstrate the presence of multiple PFOA binding sites on the Hb protein. The detailed analysis of the ferric hemoglobin protein (met Hb) absorbance data as a function of PFOA concentration indicates the presence of at least two binding sites with equilibrium dissociation constants of 0.8 ± (0.2) × 10-6 M and 63 ± (15) × 10-5 M. A competitive binding study with 1,8-ANS showed PFOA can bind to the same binding site as 1,8-ANS on the Hb protein. The titration curve for PFOA binding to Hb in its CO bound form (CO-Hb) yields a single equilibrium dissociation constant of 139 ± (20) × 10-6 M. PFOA binding at low concentrations occurs at the high-affinity sites leading to the destabilization of the protein structure as reflected by changes in the CD spectrum. PFOA interactions with Hb also interfere with the kinetics of CO association to this protein. The rate for CO association to Hb increases at low PFOA concentrations, whereas at elevated PFOA concentrations, the ligand association is biphasic as a new kinetic process with a different rate constant was observed. Overall, this study provides a detailed explanation of PFOA-induced structural and conformational changes to the Hb protein based on the spectroscopy data.

Conformational dynamics in human neuroglobin: effect of His64, Val68, and Cys120 on ligand migration.

Neuroglobin belongs to the family of hexacoordinate hemoglobins and has been implicated in the protection of neuronal tissue under hypoxic and ischemic conditions. Here we present transient absorption and photoacoustic calorimetry studies of CO photodissociation and bimolecular rebinding to neuroglobin focusing on the ligand migration process and the role of distal pocket residues (His64 and Val68) and two Cys residues (Cys55 and Cys120). Our results indicate that His64 has a minor impact on the migration of CO between the distal heme pocket and protein exterior, whereas the Val68 side chain regulates the transition of the photodissociated ligand between the distal pocket and internal hydrophobic cavities, which is evident from the increased geminate quantum yield in this mutated protein (Φ(gem) = 0.32 for WT and His64Gln, and Φ(gem) = 0.85 for Val68Phe). The interface between helix G and the A-B loop provides an escape pathway for the photodissociated ligand, which is evident from a decrease in the reaction enthalpy for the transition between the CO-bound hNgb and five-coordinate hNgb in the Cys120Ser mutant (ΔH = -3 ± 4 kcal mol(-1)) compared to that of the WT protein (ΔH = 20 ± 4 kcal mol(-1)). The extensive electrostatic/hydrogen binding network that includes heme propionate groups, Lys67, His64, and Tyr44 not only restricts the heme binding but also modulates the energetics of binding of CO to the five-coordinate hNgb as substitution of His64 with Gln leads to an endothermic association of CO with the five-coordinate hNgb (ΔH = 6 ± 3 kcal mol(-1)).

Time resolved thermodynamics associated with ligand photorelease in heme peroxidases and globins: Open access channels versus gated ligand release.

Heme proteins represent a diverse class of biomolecules responsible for an extremely diverse array of physiological functions including electron transport, monooxygenation, ligand transport and storage, cellular signaling, respiration, etc. An intriguing aspect of these proteins is that such functional diversity is accomplished using a single type of heme macrocycle based upon iron protoporphyrin IX. The functional diversity originates from a delicate balance of inter-molecular interactions within the protein matrix together with well choreographed dynamics that modulate the heme electronic structure as well as ligand entry/exit pathways from the bulk solvent to the active site. Of particular interest are the dynamics and energetics associated with the entry/exit of ligands as this process plays a significant role in regulating the rates of heme protein activity. Time-resolved photoacoustic calorimetry (PAC) has emerged as a powerful tool through which to probe the underlying energetics associated with small molecule dissociation and release to the bulk solvent in heme proteins on time scales from tens of nanoseconds to several microseconds. In this review, the results of PAC studies on various classes of heme proteins are summarized highlighting how different protein structures affect the thermodynamics of ligand migration. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

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 (HNEP(+)) and its deprotonated form (NEP), a benzofused derivative of Brooker's merocyanine (BM), were investigated through a combined spectroscopic and computational approach. Despite their structural similarities and similar pK(a) values, HNEP(+)/NEP and BMH(+)/BM differ in the extent of charge delocalization in the ground and excited states. NEP exhibits the spectral characteristics of a charge transfer species in solvents in which BM exists in a charge-delocalized quinoid; however, quantum chemical calculations show that the CT absorption of NEP is not necessarily a consequence of the zwitterionic character. HNEP(+) displays larger Stokes shifts than BMH(+), and NEP demonstrates enhanced solvatochromism relative to BM as a consequence of benzofusion.