Binding Events of (S)-N-(3-Oxo-octanoyl)-homoserine Lactone with Agrobacterium tumefaciens Mutant Cells Studied by Saturation Transfer Difference NMR

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Quorum-sensing is a widely studied communication phenomenon in bacteria, which involves the production and detection of signaling substances in relation with cell density and colony behavior. Herein, the membrane binding interactions of the signal (S)-N-(3-oxo-octanoyl)-HSL with A. tumefaciens NTL4(pZLR4) cells were studied using saturation transfer difference NMR spectroscopy (STD-NMR). The substance epitope map was obtained showing that the hydrophobic acyl chain is the most important interacting site for the signal and the cell membrane. Results were interpreted upon comparisons with a simpler system, using liposomes as membrane models. Some insights on the use of beta-cyclodextrin as acyl-HSL carrier were also provided.224702708Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)FAPESP [05/02934-4, 07/52389-8]CAPES [141841/2006-0

Topology of a ternary complex (proparacaine-beta-cyclodextrin-liposome) by STD NMR

The topologies of proparacaine (PPC) in fl-cycloclextrin (fl-CID), PPC in egg phosphaticly1choline (EPQ liposomes and PPC in fl-CD in EPC were investigated using NMR experiments (I D ROESY and saturation transfer difference (STD)). This is the first description of the STID technique applied to PPC - EPC -P-CD system, revealing that not only PPC was imbedded in EPC bilayer, but fl-CD was also interacting with liposome vesicles. These results are novel and were rationalized as the spontaneous formation of a ternary complex with some fl-CD molecules bound to external liposome vesicles surfaces. Copyright (c) 2008 John Wiley & Sons, Ltd.46983283

Liposome-Prilocaine Interaction Mapping Evaluated through STD NMR and Molecular Dynamics Simulations

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)We have examined the interaction of the neutral and protonated species of the local anesthetic prilocaine (PLC) with phosphatidylcholine (PC) bilayers combining experimental ((1)H NMR) and theoretical (molecular dynamics simulations) approaches. DOSY experiments allowed the determination of the association constants of protonated (K(a) = 9 L/mol) and neutral (K(a) = 21 L/mol) PLC to egg PC liposomes. Saturation transfer difference (STD) experiments showed a different trend depending on pH: At high pH the PLC hydrogen saturation was essentially uniform and at pH 5.5 the experiments show an enhancement of the aromatic moiety hydrogen saturation, with respect to the tail. Molecular dynamics simulations, performed with PLC molecules on planar bilayers of palmitoyloleyl-PC, revealed a preferential orientation for the protonated PLC species at the polar interface of the bilayer, and a nonoriented and deeper insertion for neutral PLC. Such preferential location of protonated and neutral PLC inside the bilayer can be described as different transient sites which could modulate the access of these molecules to their binding site(s) in the voltage-gated sodium channel, justifying differences in the anesthetic's potency upon ionization.113823652370Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [06/02523-7, 06/00121-9]CNPq [141841/2006-0

Prilocaine-cyclodextrin-liposome: effect of pH variations on the encapsulation and topology of a ternary complex using H-1 NMR

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)A better comprehension of the prilocaine (PLC)-beta-cyclodextrin (beta-CD) complex liberation to membranes was provided by studying the architectural supramolecular arrangements of PLC, beta-CD and egg phosphatidylcholine (EPC) liposomes, a membrane model. The topologies and possible interactions of mixtures of PLC, beta-CD and EPC liposomes were investigated by nuclear magnetic resonances combining experimental H-1-NMR (1D ROESY, STD and DOSY) at different pHs. The results indicate that in the mixture PLC/beta-CD/EPC at pH 10 the PLC molecules are almost totally embedded into the liposomes and little interaction was observed between PLC and beta-CD. However, at pH 5.5 not only was PLC imbedded in the EPC bilayer, but PLC was also interacting with beta-CD. These results were rationalized as a spontaneous PLC release from beta-CD to liposomes vesicles, whereas the PLC/EPC complex formation was higher at pH 10 than pH 5.5. Copyright (C) 2011 John Wiley & Sons, Ltd.496295300Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)ANPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

beta-Lactam Antibiotics Epitope Mapping with STD NMR Spectroscopy: a Study of Drug-Human Serum Albumin Interaction

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Molecular recognition events are key issues in many biological processes. STD NMR (saturation transfer difference nuclear magnetic resonance spectroscopy) is one of the techniques used to understand such biological interactions. Herein, we have investigated the interactions of four beta-lactam antibiotics belonging to two classes (cephalosporins and penicillins) with human serum albumin (HSA) by H-1 STD NMR revealing that the interaction between the aromatic moiety and HSA is responsible for the binding efficiency. Thus, the structural differences from the five to six-membered thio ring in penicillins and cephalosporins do not seem to influence antibiotic-albumin interactions.233403+Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq