Membrane surface charge modulates lipoprotein complex forming capability of peptides derived from the C-terminal domain of apolipoprotein E

Apolipoprotein E (apoE) plays a major role in the transport and metabolism of lipid by acting as a ligand for low density lipoprotein-receptors. The amphipathic helical regions of its C-terminal domain are necessary for the lipoprotein binding and assembly of nascent lipoprotein particles. Lipoproteins in the plasma are known to possess a net negative charge, determined by both its protein and lipid components, which regulates the metabolism of lipoproteins. The role of membrane surface charge on the interaction of apoE has not been studied previously. Also the importance of individual amphipathic helical regions of its C-terminal domain in binding to negatively charged lipid membrane is not addressed. In this study we have compared the interaction of four peptide segments of apoE C-terminal domain (apoE_(202-223), apoE_(223-244), apoE_(245-266), and apoE_(268-289)) with zwitterionic and negatively charged model membranes by employing UV-visible and fluorescence spectroscopy, circular dichroism, and native PAGE analysis. Our results show that the peptide sequence 202-223, 245-266 and 268-289 of apoE has higher affinity towards negatively charged lipid membrane and are independently capable of forming lipoprotein particles of 17 ± 2 nm Stokes diameter. The results suggest that surface charge of lipoprotein regulates its metabolism possibly by modulating the recruitment of apoE on its surface.     

Preferential binding of apolipoprotein E derived peptides with oxidized phospholipid

The physiological function of apolipoprotein E (apoE) includes transport and metabolism of lipids and its C-terminal domain harbors high affinity lipid-binding sites. Although the binding of apoE with non-oxidized phospholipid containing membranes has been characterized earlier, the interaction of apoE or its fragments with oxidized phospholipid containing membrane has never been studied. In this study we have compared the interaction of amphipathic helical peptide sequences derived from the C-terminal domain of apoE with membrane vesicles containing oxidized phospholipid, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), with membrane vesicles without PazePC. The interaction was studied by monitoring (a) fluorescence emission maxima of the peptides, (b) acrylamide quenching of the peptides tryptophan residues and (c) by measuring the equilibrium binding constants by resonance energy transfer (RET) analysis. Our result shows that peptide sequence 202-223, 245-266 and 268-289 of apoE has higher affinity towards membrane containing PazePC, compared to membrane without PazePC. Presence of 1mM divalent cation or 50 mM NaCl in the buffer decreased the binding of peptides to PazePC containing membrane vesicles suggesting possible involvement of the electrostatic interaction in the binding. These observations suggest that the preferential binding of apoE to oxidized phospholipid containing membrane may play a role in the anti-oxidative properties of apoE.     

In vitro and in vivo antimicrobial activity of a synthetic peptide derived from the C-terminal region of human chemokine CCL13 against Pseudomonas aeruginosa

Chemokines are important mediators of immunological responses during inflammation and under steady-state conditions. In addition to regulating cell migration, some chemotactic cytokines have direct effects on bacteria. Here, we characterized the antibacterial ability of the synthetic oligopeptide CCL13_57-75, which corresponds to the carboxyl-terminal region of the human chemokine CCL13. In vitro measurements indicated that CCL13_57-75 disrupts the cell membrane of Pseudomonas aeruginosa through a mechanism coupled to an unordered-helicoidal conformational transition. In a murine pneumonic model, CCL13_57-75 improved mouse survival and bacterial clearance and decreased neutrophil recruitment, proinflammatory cytokines and lung pathology compared with that observed in untreated infected animals. Overall, our study supports the ability of chemokines and/or chemokine-derived oligopeptides to act as direct defense agents against pathogenic bacteria and suggests their potential use as alternative antibiotics.     

Structure elucidation of platelet activating factor derived from human neutrophils

Platelet activating factor (PAF) synthesized by human neutrophils challenged by opsonized zymosan or calcium ionophore was isolated from cells and buffer using Bligh and Dyer extraction following the addition of tracer amounts of tritiated-PAF. The extract was subjected to TLC separation of phospholipid classes, followed by reverse phase HPLC for molecular species separation. All fractions were measured for radioactivity, biological activity and fast atom bombardment mass spectrometry. While the radioactive tracer PAF could be separated into three molecular species, PAF biological activity eluted as a single component which was characterized as 1-O-hexadecyl-2-acetyl-glycero-3-phosphocholine. The lack of molecular species heterogeneity of PAF produced in response to stimuli implies a higher degree of control of biosynthesis than previously suspected.     

Functional independence of a peptide with the sequence of human apolipoprotein A-I central region

Previous results [J. Biol. Chem. 276 (2001) 16978] indicated that an apolipoprotein A-I (apoAI) central region swings away from lipid contact in discoidal high density lipoproteins (HDL), but it is able to penetrate into the bilayer of lipid vesicles. In this work, we have studied the interaction with lipid membranes of a synthetic peptide with the sequence of apoAI region between residues 77 and 120 (AI 77-120). Like apoAI, AI 77-120 binds to phospholipid vesicles and shows selectivity for cholesterol-containing membranes. Moreover, AI 77-120 promotes cholesterol desorption from membranes in a similar fashion as apoAI and can stimulate cholesterol efflux from Chinese hamster ovary cells. AI 77-120 has a considerable alpha-helical content in water solution, and its secondary structure is not largely modified after binding to membranes. Both apoA-I and AI 77-120 are oligomeric in the lipid-bound state, suggesting that dimerization of the central domain could be required for the membrane binding activity of apoA-I in HDL.     

Biophysical studies of the interactions between 14-mer and 21-mer model amphipathic peptides and membranes: Insights on their modes of action

We have investigated the interactions between synthetic amphipathic peptides and zwitterionic model membranes. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers have been synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure as revealed by circular dichroism. To shed light on their mechanism of membrane interaction, different complementary biophysical techniques have been used such as circular dichroism, fluorescence, membrane conductivity measurement and NMR spectroscopy. Results obtained by these different techniques show that the 14-mer peptide is a membrane perturbator that facilitate the leakage of species such as calcein and Na ions, while the 21-mer peptide acts as an ion channel. ³¹P solid-state NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are greatly affected by the presence of the peptides. Similar results have also been obtained in mechanically oriented DLPC and DMPC bilayers where different acyl chain lengths seem to play a role in the interaction. On the other hand, ²H NMR experiments on multilamellar vesicles demonstrate that the acyl chain order is affected differently by the two peptides. Based on these studies, mechanisms of action are proposed for the 14-mer and 21-mer peptides with zwitterionic membranes.     

Combined multispectroscopic and molecular docking investigation on the interaction between strictosamide and human serum albumin

The interaction between strictosamide (STM) and human serum albumin (HSA) was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, three‐dimensional fluorescence spectroscopy, ultraviolet‐visible absorption spectroscopy, circular dichroism spectroscopy and molecular modeling under physiological pH 7.4. STM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding site number n and apparent binding constant K_a were determined at different temperatures by fluorescence quenching. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated as ?3.01 kJ/mol and 77.75 J/mol per K, respectively, which suggested that the hydrophobic force played major roles in stabilizing the HSA–STM complex. The distance r between donor and acceptor was obtained to be 4.10 nm according to Förster's theory. After the addition of STM, the synchronous fluorescence and three‐dimensional fluorescence spectral results showed that the hydrophobicity of amino acid residues increased and the circular dichroism spectral results showed that the α‐helix content of HSA decreased (from 61.48% to 57.73%). These revealed that the microenvironment and conformation of HSA were changed in the binding reaction. Furthermore, the study of molecular modeling indicated that STM could bind to site I of HSA and the hydrophobic interaction was the major acting force, which was in agreement with the binding mode study. Copyright © 2013 John Wiley & Sons, Ltd.     

Regulatory interaction of sodium channel IQ-motif with calmodulin C-terminal lobe

An increasing number of ion channels have been found to be regulated by the direct binding of calmodulin (CaM), but its structural features are mostly unknown. Previously, we identified the Ca(2+)-dependent and -independent interactions of CaM to the voltage-gated sodium channel via an IQ-motif sequence. In this study we used the trypsin-digested CaM fragments (TR(1)C and TR(2)C) to analyze the binding of Ca(2+)-CaM or Ca(2+)-free (apo) CaM with a sodium channel-derived IQ-motif peptide (NaIQ). Circular dichroic spectra showed that NaIQ peptide enhanced alpha-helicity of the CaM C-terminal lobe, but not that of the CaM N-terminal lobe in the absence of Ca(2+), whereas NaIQ enhanced the alpha-helicity of both the N- and C-terminal lobes in the presence of Ca(2+). Furthermore, the competitive binding experiment demonstrated that Ca(2+)-dependent CaM binding of target peptides (MLCKp or melittin) with CaM was markedly suppressed by NaIQ. The results suggest that IQ-motif sequences contribute to prevent target proteins from activation at low Ca(2+) concentrations and may explain a regulatory mechanism why highly Ca(2+)-sensitive target proteins are not activated in the cytoplasm.     

A combined spectroscopic and molecular docking approach to characterize binding interaction of megestrol acetate with bovine serum albumin

The binding interactions between megestrol acetate (MA) and bovine serum albumin (BSA) under simulated physiological conditions (pH 7.4) were investigated by fluorescence spectroscopy, circular dichroism and molecular modeling. The results revealed that the intrinsic fluorescence of BSA was quenched by MA due to formation of the MA–BSA complex, which was rationalized in terms of a static quenching procedure. The binding constant (K_b) and number of binding sites (n) for MA binding to BSA were 2.8 × 10⁵ L/mol at 310 K and about 1 respectively. However, the binding of MA with BSA was a spontaneous process due to the negative ∆G⁰ in the binding process. The enthalpy change (∆H⁰) and entropy change (∆S⁰) were – 124.0 kJ/mol and –295.6 J/mol per K, respectively, indicating that the major interaction forces in the binding process of MA with BSA were van der Waals forces and hydrogen bonding. Based on the results of spectroscopic and molecular docking experiments, it can be deduced that MA inserts into the hydrophobic pocket located in subdomain IIIA (site II) of BSA. The binding of MA to BSA leads to a slight change in conformation of BSA but the BSA retained its secondary structure, while conformation of the MA has significant change after forming MA–BSA complex, suggesting that flexibility of the MA molecule supports the binding interaction of BSA with MA. Copyright © 2014 John Wiley & Sons, Ltd.     

Formation of 5- and 6-Aminocytosine Nucleosides and Nucleotides from the Corresponding 5-Bromocytosine Derivatives: Synthesis and Reaction Mechanism

Abstract

An improved method for the synthesis of 5-aminocytidine (3a), 5-amino-2′-deoxycytidine (3b), and their 5′-monophosphates (3c,d) from the corresponding 5-bromo pyrimidines, using liquid ammonia, is described. The respective 6-aminocytosine derivatives (4a,b,c,d), minor products of the amination reaction, were isolated and characterized. A plausible mechanism is proposed to account for the formation of both 5-and 6-substituted products.     

Expression and characterization of the olfactomedin domain of human myocilin

The olfactomedin-domain has been first identified in olfactomedin, an extracellular matrix protein of the olfactory neuroepithelium. Members of this extracellular domain-family have since been shown to be present in several metazoan proteins, such as latrophilins, myocilins, and noelins, but their biological function is unknown. The olfactomedin-domain of myocilin is of considerable interest, since mutations affecting this domain are associated with primary open angle glaucoma. In order to define structural features of this domain-type we have expressed the olfactomedin-domain of human myocilin in Pichia pastoris. The olfactomedin-domain contains a single disulphide-bond connecting Cys-245 and Cys-433 residues; secondary structure predictions and circular dichroism studies indicate that it consists primarily of beta-strands. It is noteworthy that the majority of mutations associated with severe forms of glaucoma affect residues that reside in conserved secondary structural elements of the olfactomedin-domain or are otherwise critical for the integrity of this protein-fold.     

Differential scanning calorimetric and spectroscopic studies on the unfolding of Momordica charantia lectin. Similar modes of thermal and chemical denaturation

Thermal stability of Momordica charantia seed lectin (MCL) was investigated as a function of protein concentration, pH, scan rate, and at different ligand concentrations by using high-sensitivity differential scanning calorimetry (DSC). The DSC endotherm obtained at pH 7.4 consists of two entities with transition temperatures at ca. 333.7 K, and 338 K. The unfolding process is irreversible and could be described by a three-state model. For MCL tetramer ΔH_c/ΔH_v ratio is close to 4 for the first transition and ∼2 for the second transition, suggesting that four and two cooperative units are involved in the first and second transitions, respectively. In the presence of lactose both transitions shifted to higher temperatures, suggesting that ligand binds preferentially to the native conformation of MCL. Endotherms recorded as a function of pH indicate that MCL is more stable at lower pH. Chemical unfolding of MCL, induced by Gdn.HCl, was investigated by monitoring the intrinsic fluorescence properties of the protein. The results obtained indicate that chemical denaturation of MCL can also be described by a three-state process, involving an intermediate populated at ∼3–4 M Gdn.HCl. These observations suggest that the chemical and thermal unfolding processes are similar in that both of them proceed via an intermediate. The far UV and near UV CD spectra of MCL were nearly identical at different pH values and indicate that its secondary and tertiary structure do not change significantly with pH, suggesting that the structure of the protein is stable over a wide pH range.     

Calorimetric and spectroscopic examination of the solution phase structures of prekallikrein binding domain peptides of high molecular weight kininogen

Unique sequence-binding sites are exposed on the surface of high molecular weight kininogen which complex prekallikrein or factor XI with high affinity and specificity. A sequence comprising 31 residues of the mature kininogen molecule (Asp565-Lys595) retains full binding activity for prekallikrein (K _D =20 nM) and assumes a complex folded structure in solution which is stabilized by long-range interactions between N- and C-terminal residues. The sequence Trp569-Lys595 (27 residues) shows only 28% of this binding affinity and lacks the key structural features required for protein recognition (Scarsale, J. N., and Harris, R. B.,J. Prot. Chem. 9, 647–659, 1990). We were thus able to predict that N- or C-terminal truncations of the binding-site sequence would disrupt the conformational integrity required for binding. Two new peptides of 20- and 22- residues have now been synthesized and their solution phase structures examined. These peptides are N- and C-terminal truncations, respectively, of the 27-residue sequence and correspond to the sequences Asp576-Lys595 and Trp569-Asp590 of high molecular weight kininogen. The results of fluorescence emission and circular dichroism (CD) spectroscopies in the range 25–90°C and from differential scanning calorimetry (DSC) all substantiate the idea that the C-terminal truncation peptide binds prekallikrein 35-fold poorer than the 31-residue peptide because it is relatively unoredered and possesses a less stable structure. Surprisingly, the N-terminal truncation peptide (20-mer) shows structural stability even at elevated temperatures and, like the 31-residue peptide, undergoes cold-induced denaturation observable in the DSC. 2D-NMR analysis of the 20-residue peptide revealed two distinct structures; one conformer possesses a more compact, folded structure than the other. However, the predicted structures assumed by either conformer are very different from those of either the 31- or 27-residue peptides. Hence, the binding affinity of the 20-residue peptide is 60-fold poorer than that for the 31-residue peptide because it assumes a nonproductive binding conformation(s).     

Insights into the oligomerization process of the C-terminal domain of human plasma membrane Ca-ATPase

Plasma membrane calcium pumps (PMCAs) sustain a primary transport system for the specific removal of cytosolic calcium ions from eukaryotic cells. PMCAs are characterized by the presence of a C-terminal domain referred to as a regulatory domain. This domain is target of several regulatory mechanisms: activation by Ca²⁺-calmodulin complex and acidic phospholipids, phosphorylation by kinase A and C, proteolysis by calpain and oligomerization. As far as oligomerization is concerned, the C-terminal domain seems to be crucial for this process. We have cloned the C-terminal domain of the human PMCA isoform 1b, and characterized its properties in solution. The expressed protein maintains its tendency to oligomerize in aqueous solutions, but it is dissociated by amphipathic molecules such as diacylglycerol and sodium dodecyl sulphate. The presence of sodium dodecyl sulphate stabilizes the domain as a compact structure in monomeric form retaining the secondary structure elements, as shown by small angle neutron scattering and circular dichroism measurements. The importance of oligomerization for the regulation of PMCA activity and intracellular calcium concentration is discussed.     

Fluorescence-coupled CD conformational monitoring of filament formation of tau microtubule-binding repeat domain

To clarify the contribution of the three- or four-repeated peptide moiety in tau microtubule-binding domain (MBD) to paired helical filament (PHF) formation, conformational transition accompanied by heparin-induced filament formation was investigated stepwise for four repeat peptides (R1-R4), one three-repeated R1-R3-R4 peptide (3RMBD), and one four-repeated R1-R2-R3-R4 peptide (4RMBD) using a combination of thioflavin S fluorescence and circular dichroism (CD) measurements in a neutral buffer (pH 7.6). The comparison of the fluorescence profile of each repeat peptide with those of 3RMBD and 4RMBD showed the synergistic contribution of R1-R4 to PHF formation of MBD. The CD spectrum measured as a function of filament formation time indicates that: (i) two conformational transitions occur for the filament formations of R3 (from the random structure to the beta-sheet structure) and 3RMBD (from the random structure to the alpha-helix structure), (ii) the filament formations of R2 and 4RMBD proceed via the synchronized conformational transitions of the alpha-helix and random structures, and (iii) the filament formation of 4RMBD is dependent on the aggregation behavior of R2. These data are useful for elucidating the MBD conformational transition in tau PHF formation.     

NMR studies of the low-density lipoprotein receptor-binding peptide of apolipoprotein E bound to dodecylphosphocholine micelles.

Circular dichroism and NMR spectroscopy have been used to determine the structure of the low-density lipoprotein (LDL) receptor-binding peptide, comprising residues 130-152, of the human apolipoprotein E. This peptide has little persistent three-dimensional structure in solution, but when bound to micelles of dodecylphosphocholine (DPC) it adopts a predominantly alpha-helical structure. The three-dimensional structure of the DPC-bound peptide has been determined by using 1H-NMR spectroscopy: the structure derived from NOE-based distance constraints and restrained molecular dynamics is largely helical. The derived phi and psi angle order parameters show that the helical structure is well defined but with some flexibility that causes the structures not to be superimposable over the full peptide length. Deuterium exchange experiments suggest that many peptide amide groups are readily accessible to the solvent, but those associated with hydrophobic residues exchange more slowly, and this helix is thus likely to be positioned on the surface of the DPC micelles. In this conformation the peptide has one hydrophobic face and two that are rich in basic amino acid side chains. The solvent-exposed face of the peptide contains residues previously shown to be involved in binding to the LDL receptor.     

肺炎链球菌血红素结合脂蛋白PiuA的表达、纯化和表征

【背景】肺炎链球菌是社区获得性肺炎最常见的致病菌之一,它也会引起脑膜炎、鼻窦炎、中耳炎、菌血症等一系列疾病,对人类(特别是儿童、老人、免疫缺陷患者)健康造成重大威胁。铁是肺炎链球菌生存和感染所必需的元素之一,其中血红素转运系统PiuABCD是肺炎链球菌最重要的铁转运系统。【目的】克隆、表达和纯化肺炎链球菌血红素转运系统脂蛋白PiuA,并在体外表征PiuA蛋白的血红素结合特性。【方法】将肺炎链球菌D39菌株中的piuA(spd_1652)基因连接到载体pBAD-HisA上,在大肠杆菌Top10菌株中进行异源表达,然后运用Ni-NTA亲和层析纯化PiuA-His蛋白,并用肠激酶切掉His标签获得不含标签的PiuA蛋白,最后运用圆二色谱、紫外光谱和荧光光谱表征PiuA蛋白的血红素结合特性。【结果】构建了pBAD/HisA-PiuA重组表达载体,获得了纯度大于95%的PiuA蛋白,圆二色谱显示PiuA蛋白与Hemin结合后,其二级结构不发生改变;紫外光谱结果显示PiuA蛋白具有血红素结合能力;荧光光谱结果显示apo-PiuA蛋白与Hemin结合常数K=3.4×10~5 L/mol。【结论】肺炎链球菌血红素转运系统脂蛋白PiuA能够特异地结合血红素,为肺炎链球菌的生存和感染提供必需的铁源,PiuA蛋白的体外表征结果为针对PiuABCD血红素转运系统设计抗菌药物奠定了基础。     

Biophysical characterization of the interaction of p21 with calmodulin: a mechanistic study

p21 is a protein with important roles in cell proliferation, cell cycle regulation and apoptosis. Several studies have demonstrated that its intracellular localization plays an important role in the functional regulation and binding of calmodulin favors its nuclear translocation. However, the detail mechanism of the interaction with p21 and calmodulin is not well understood. In this report, peptides derived from the C-terminal of p21 that cover the binding domain of calmodulin were used to investigate the association of p21 with calmodulin. We found p21(141-164) interaction with Ca(2+)-saturated dansyl-labelled calmodulin caused a significant increase in dansyl fluorescence intensity and a blue shift of the maximum emission from 510 to 475 nm. The Trp fluorescence intensities of mutated p21(141-164) peptides (F150W, Y151W and F159W) increased upon binding to Ca(2+)-saturated calmodulin and fluorescence maxima were blue shifted from 350 nm to 330 nm. The results suggested p21(141-164) is most likely buried in the hydrophobic binding tunnel of calmodulin. Both dansyl and Trp fluorescence titrations generated dissociation constants around 0.1 muM and a stoichiometry of 1:1, which was further confirmed by nondenaturing gel band shift electrophoresis. Fluorescence titrations and Trp fluorescence quenching results indicated electrostatic interaction is involved in this association. Upon binding to calmodulin, p21(141-164) remained largely unstructured and showed only about 15% alpha-helix. In contrast to other calmodulin binding peptide, the dominant force in the association of p21(141-164) with calmodulin may be electrostatic interaction. Our results would be helpful for understanding the molecular details of p21 and calmodulin interaction.     

Characterization of a putative fusogenic sequence in the E2 hepatitis G virus protein

With the aim of better understanding the fusion process mediated by the envelope proteins of the hepatitis G virus (HGV/GBV-C), we have investigated the interaction with model membranes of two overlapping peptides [(267-284) and (279-298)] belonging to the E2 structural protein. The peptides were compared for their ability to perturb lipid bilayers by means of different techniques such as differential scanning calorimetry and fluorescence spectroscopy. Furthermore, the conformational behaviour of the peptides in different membrane environments was studied by Fourier-transform infrared spectroscopy and circular dichroism. The results showed that only the E2(279-298) peptide sequence was able to bind with high affinity to negatively charged membranes, to permeabilize efficiently negative lipid bilayers, to induce haemolysis, and to promote inter-vesicle fusion. This fusogenic activity could be related to the induced peptide conformation upon interaction with the target membrane.     

Binding properties of human telomeric quadruplex multimers: a new route for drug design

Human telomeric G-quadruplex structures are known to be promising targets for an anticancer therapy. In the past decade, several research groups have been focused on the design of new ligands trying to optimize the interactions between these small molecules and the G-quadruplex motif. In most of these studies, the target structures were the single quadruplex units formed by short human DNA telomeric sequences (typically 21-26 nt). However, the 3′-terminal single-stranded human telomeric DNA is actually 100-200 bases long and can form higher-order structures by clustering several consecutive quadruplex units (multimers). Despite the increasing number of structural information on longer DNA telomeric sequences, very few data are available on the binding properties of these sequences compared with the shorter DNA telomeric sequences.In this paper we use a combination of spectroscopic (CD, UV and fluorescence) and calorimetric techniques (ITC) to compare the binding properties of the (TTAGGG)₈TT structure formed by two adjacent quadruplex units with the binding properties of the (AG₃TT)₄ single quadruplex structure. The three side-chained triazatruxene derivative azatrux and TMPyP4 cationic porphyrin were used as quadruplex ligands. We found that, depending on the drug, the number of binding sites per quadruplex unit available in the multimer structure was smaller or greater than the one expected on the basis of the results obtained from individual quadruplex binding studies. This work suggests that the quadruplex units along a multimer structure do not behave as completely independent. The presence of adjacent quadruplexes results in a diverse binding ability not predictable from single quadruplex binding studies. The existence of quadruplex-quadruplex interfaces in the full length telomeric overhang may provide an advantageous factor in drug design to enhance both affinity and selectivity for DNA telomeric quadruplexes.