Mapping Free Energy Pathways for ATP Hydrolysis in the E. coli ABC Transporter HlyB by the String Method

HlyB functions as an adenosine triphosphate (ATP)-binding cassette (ABC) transporter that enables bacteria to secrete toxins at the expense of ATP hydrolysis. Our previous work, based on potential energy profiles from combined quantum mechanical and molecular mechanical (QM/MM) calculations, has suggested that the highly conserved H-loop His residue H662 in the nucleotide binding domain (NBD) of E. coli HlyB may catalyze the hydrolysis of ATP through proton relay. To further test this hypothesis when entropic contributions are taken into account, we obtained QM/MM minimum free energy paths (MFEPs) for the HlyB reaction, making use of the string method in collective variables. The free energy profiles along the MFEPs confirm the direct participation of H662 in catalysis. The MFEP simulations of HlyB also reveal an intimate coupling between the chemical steps and a local protein conformational change involving the signature-loop residue S607, which may serve a catalytic role similar to an Arg-finger motif in many ATPases and GTPases in stabilizing the phosphoryl-transfer transition state

Dual Role of Electron-Accepting Metal-Carboxylate Ligands: Reversible Expansion of Exciton Delocalization and Passivation of Nonradiative Trap-States in Molecule-like CdSe Nanocrystals

This paper reports large bathochromic shifts of up to 260 meV in both the excitonic absorption and emission peaks of oleylamine (OLA)-passivated molecule-like (CdSe)34 nanocrystals caused by postsynthetic treatment with the electron accepting Cd(O2CPh)2 complex at room temperature. These shifts are found to be reversible upon removal of Cd(O2CPh)2 by N,N,N′,N′-tetramethylethylene-1,2-diamine. 1H NMR and FTIR characterizations of the nanocrystals demonstrate that the OLA remained attached to the surface of the nanocrystals during the reversible removal of Cd(O2CPh)2. On the basis of surface ligand characterization, X-ray powder diffraction measurements, and additional control experiments, we propose that these peak red shifts are a consequence of the delocalization of confined exciton wave functions into the interfacial electronic states that are formed from interaction of the LUMO of the nanocrystals and the LUMO of Cd(O2CPh)2, as opposed to originating from a change in size or reorganization of the inorganic core. Furthermore, attachment of Cd(O2CPh)2 to the OLA-passivated (CdSe)34 nanocrystal surface increases the photoluminescence quantum yield from 5% to an unprecedentedly high 70% and causes a 3-fold increase of the photoluminescence lifetime, which are attributed to a combination of passivation of nonradiative surface trap states and relaxation of exciton confinement. Taken together, our work demonstrates the unique aspects of surface ligand chemistry in controlling the excitonic absorption and emission properties of ultrasmall (CdSe)34 nanocrystals, which could expedite their potential applications in solid-state device fabrication

Elucidating the role of surface passivating ligand structural parameters in hole wave function delocalization in semiconductor cluster molecules

This article describes the mechanisms underlying electronic interactions between surface passivating ligands and (CdSe)34 semiconductor cluster molecules (SCMs) that facilitate band-gap engineering through the delocalization of hole wave functions without altering their inorganic core. We show here both experimentally and through density functional theory calculations that the expansion of the hole wave function beyond the SCM boundary into the ligand monolayer depends not only on the pre-binding energetic alignment of interfacial orbitals between the SCM and surface passivating ligands but is also strongly influenced by definable ligand structural parameters such as the extent of their π-conjugation [π-delocalization energy; pyrene (Py), anthracene (Anth), naphthalene (Naph), and phenyl (Ph)], binding mode [dithiocarbamate (DTC, –NH–CS2−), carboxylate (–COO−), and amine (–NH2)], and binding head group [–SH, –SeH, and –TeH]. We observe an unprecedentedly large ∼650 meV red-shift in the lowest energy optical absorption band of (CdSe)34 SCMs upon passivating their surface with Py-DTC ligands and the trend is found to be Ph- < Naph- < Anth- < Py-DTC. This shift is reversible upon removal of Py-DTC by triethylphosphine gold(I) chloride treatment at room temperature. Furthermore, we performed temperature-dependent (80–300 K) photoluminescence lifetime measurements, which show longer lifetime at lower temperature, suggesting a strong influence of hole wave function delocalization rather than carrier trapping and/or phonon-mediated relaxation. Taken together, knowledge of how ligands electronically interact with the SCM surface is crucial to semiconductor nanomaterial research in general because it allows the tuning of electronic properties of nanomaterials for better charge separation and enhanced charge transfer, which in turn will increase optoelectronic device and photocatalytic efficiencies

Identification of an L-Phenylalanine Binding Site Enhancing The Cooperative Responses of The Calcium Sensing Receptor to Calcium

Functional positive cooperative activation of the extracellular calcium ([Ca2+]o)-sensing receptor (CaSR), a member of the family C G protein-coupled receptors (GPCRs), by [Ca2+]o or amino acids elicits intracellular Ca2+ ([Ca2+]i) oscillations. Here, we report the central role of predicted Ca2+-binding Site 1 within the hinge region of the extracellular domain (ECD) of CaSR and its interaction with other Ca2+-binding sites within the ECD in tuning functional positive homotropic cooperativity caused by changes in [Ca2+]o. Next, we identify an adjacent L-Phe-binding pocket that is responsible for positive heterotropic cooperativity between [Ca2+]o and L- Phe in eliciting CaSR-mediated [Ca2+]i oscillations. The hetero-communication between Ca2+ and an amino acid globally enhances functional positive homotropic cooperative activation of CaSR in response to [Ca2+]o signaling by positively impacting multiple [Ca2+]o-binding sites within the ECD. Elucidation of the underlying mechanism provides important insights into the longstanding question of how the receptor transduces signals initiated by [Ca2+]o and amino acids into intracellular signaling events

Role of Ca2+ and L-Phe in Regulating Functional Cooperativity of Disease- Associated ‘‘Toggle’’ Calcium-Sensing Receptor Mutations

The Ca2+-sensing receptor (CaSR) regulates Ca2+ homeostasis in the body by monitoring extracellular levels of Ca2+ ([Ca2+]o) and amino acids. Mutations at the hinge region of the N-terminal Venus flytrap domain (VFTD) produce either receptor inactivation (L173P, P221Q) or activation (L173F, P221L) related to hypercalcemic or hypocalcemic disorders. In this paper, we report that both L173P and P221Q markedly impair the functional positive cooperativity of the CaSR as reflected by [Ca2+]o–induced [Ca2+]i oscillations, inositol-1-phosphate (IP1) accumulation and extracellular signal-regulated kinases (ERK1/2) activity. In contrast, L173F and P221L show enhanced responsiveness of these three functional readouts to [Ca2+]o. Further analysis of the dynamics of the VFTD mutants using computational simulation studies supports disruption in the correlated motions in the loss-offunction CaSR mutants, while these motions are enhanced in the gain-of-function mutants. Wild type (WT) CaSR was modulated by L-Phe in a heterotropic positive cooperative way, achieving an EC50 similar to those of the two activating mutations. The response of the inactivating P221Q mutant to [Ca2+]o was partially rescued by L-Phe, illustrating the capacity of the L-Phe binding site to enhance the positive homotropic cooperativity of CaSR. L-Phe had no effect on the other inactivating mutant. Moreover, our results carried out both in silico and in intact cells indicate that residue Leu173, which is close to residues that are part of the L-Phe-binding pocket, exhibited impaired heterotropic cooperativity in the presence of L-Phe. Thus, Pro221 and Leu173 are important for the positive homo- and heterotropic cooperative regulation elicited by agonist binding

Extracellular Calcium Modulates Actions of Orthosteric and Allosteric Ligands on Metabotropic Glutamate Receptor 1alpha

SUMMARY: Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors (GPCRs), is emerging as a potential drug target for various disorders including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca2+. However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca2+-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca2+-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we have found that extracellular Ca2+ enhances mGluR1α-mediated intracellular Ca2+ responses evoked by the orthosteric agonist, L-quisqualate. Conversely, extracellular Ca2+ diminishes the inhibitory effect of the mGluR1α orthosteric antagonist, (s)-MCPG. In addition, selective positive (Ro 67-4853) and negative (CPCCOEt) allosteric modulators of mGluR1α potentiate and inhibit responses to extracellular Ca2+, respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca2+-binding, including E325I, have significant effects on the modulation of responses to the orthosteric agonist, L-quisqualate, and the allosteric modulator Ro 67-4853 by extracellular Ca2+. These studies reveal that binding of extracellular Ca2+ to the predicted Ca2+-binding site in the ECD of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α

Cyclodextrin Complexes of Reduced Bromonoscapine in Guar Gum Microspheres Enhance Colonic Drug Delivery

Here, we report improved solubility and enhanced colonic delivery of reduced bromonoscapine (Red-Br-Nos), a cyclic ether brominated analogue of noscapine, upon encapsulation of its cyclodextrin (CD) complexes in bioresponsive guar gum microspheres (GGM). Phase−solubility analysis suggested that Red-Br-Nos complexed with β-CD and methyl-β-CD in a 1:1 stoichiometry, with a stability constant (Kc) of 2.29 × 103 M−1 and 4.27 × 103 M−1. Fourier transforms infrared spectroscopy indicated entrance of an O−CH2 or OCH3−C6H4−OCH3 moiety of Red-Br-Nos in the β-CD or methyl-β- CD cavity. Furthermore, the cage complex of Red-Br-Nos with β-CD and methyl-β-CD was validated by several spectral techniques. Rotating frame Overhauser enhancement spectroscopy revealed that the Ha proton of the OCH3−C6H4−OCH3 moiety was closer to the H5 proton of β-CD and the H3 proton of the methyl-β-CD cavity. The solubility of Red-Br-Nos in phosphate buffer saline (PBS, pH ∼ 7.4) was improved by ∼10.7-fold and ∼21.2-fold when mixed with β-CD and methyl-β-CD, respectively. This increase in solubility led to a favorable decline in the IC50 by ∼2-fold and ∼3-fold for Red-Br-Nos−β-CD-GGM and Red-Br-Nos−methyl-β-CD-GGM formulations respectively, compared to free Red-Br-Nos−β-CD and Red-Br-Nos−methyl-β-CD in human colon HT-29 cells. GGM-bearing drug complex formulations were found to be highly cytotoxic to the HT-29 cell line and further effective with simultaneous continuous release of Red-Br-Nos from microspheres. This is the first study to showing the preparation of drug-complex loaded GGMS for colon delivery of Red-Br-Nos that warrants preclinical assessment for the effective management of colon cancer

Cyclodextrin Complexes of Reduced Bromonoscapine in Guar Gum Microspheres Enhance Colonic Drug Delivery

Here, we report improved solubility and enhanced colonic delivery of reduced bromonoscapine (Red-Br-Nos), a cyclic ether brominated analogue of noscapine, upon encapsulation of its cyclodextrin (CD) complexes in bioresponsive guar gum microspheres (GGM). Phase–solubility analysis suggested that Red-Br-Nos complexed with β-CD and methyl-β-CD in a 1:1 stoichiometry, with a stability constant (Kc) of 2.29 × 103 M–1 and 4.27 × 103 M–1. Fourier transforms infrared spectroscopy indicated entrance of an O–CH2 or OCH3–C6H4–OCH3 moiety of Red-Br-Nos in the β-CD or methyl-β-CD cavity. Furthermore, the cage complex of Red-Br-Nos with β-CD and methyl-β-CD was validated by several spectral techniques. Rotating frame Overhauser enhancement spectroscopy revealed that the Ha proton of the OCH3–C6H4–OCH3moiety was closer to the H5 proton of β-CD and the H3 proton of the methyl-β-CD cavity. The solubility of Red-Br-Nos in phosphate buffer saline (PBS, pH ∼ 7.4) was improved by ∼10.7-fold and ∼21.2-fold when mixed with β-CD and methyl-β-CD, respectively. This increase in solubility led to a favorable decline in the IC50 by ∼2-fold and ∼3-fold for Red-Br-Nos−β-CD-GGM and Red-Br-Nos–methyl-β-CD-GGM formulations respectively, compared to free Red-Br-Nos−β-CD and Red-Br-Nos–methyl-β-CD in human colon HT-29 cells. GGM-bearing drug complex formulations were found to be highly cytotoxic to the HT-29 cell line and further effective with simultaneous continuous release of Red-Br-Nos from microspheres. This is the first study to showing the preparation of drug-complex loaded GGMS for colon delivery of Red-Br-Nos that warrants preclinical assessment for the effective management of colon cancer

Molecular Cycloencapsulation Augments Solubility and Improves Therapeutic Index of Brominated Noscapine in Prostate Cancer Cells

We have previously shown that a novel microtubule-modulating noscapinoid, EM011 (9-Br-Nos), displays potent anticancer activity by inhibition of cellular proliferation and induction of apoptosis in prostate cancer cells and preclinical mice models. However, physicochemical and cellular barriers encumber the development of viable formulations for future clinical translation. To circumvent these limitations, we have synthesized EM011-cyclodextrin inclusion complexes to improve solubility and enhance therapeutic index of EM011. Phase solubility analysis indicated that EM011 formed a 1:1 stoichiometric complex with β-CD and methyl-β-CD, with a stability constant (Kc) of 2.42 × 10–3 M and 4.85 × 10–3 M, respectively. Fourier transform infrared spectroscopy suggested the penetrance of either a O–CH2 or OCH3–C6H4–OCH3 moiety of EM011 in the β-CD or methyl-β-CD cavity. In addition, multifarious techniques, namely, differential scanning calorimetry, powder X-ray diffraction, scanning electron microscopy, NMR spectroscopy, and computational studies validated the cage complex of EM011 with β-CD and methyl-β-CD. Moreover, rotating frame overhauser enhancement spectroscopy showed that the Ha proton of the OCH3–C6H4–OCH3 moiety was in close proximity with H3 proton of the β-CD or methyl-β-CD cavity. Furthermore, we found that the solubility of EM011 in phosphate buffer saline (pH 7.4) was enhanced by ∼11 fold and ∼21 fold upon complexation with β-CD and methyl-β-CD, respectively. The enhanced dissolution of the drug CD-complexes in aqueous phase remarkably decreased their IC50 to 28.5 μM (9-Br-Nos-β-CD) and 12.5 μM (9-Br-Nos-methyl-β-CD) in PC-3 cells compared to free EM011 (∼200 μM). This is the first report to demonstrate the novel construction of cylcodextrin-based nanosupramolecular vehicles for enhanced delivery of EM011 that warrants in vivo evaluation for the superior management of prostate cancer