Roles of the Conserved Amino Acid Residues in Reduced Human Defensin 5: Cysteine and Arginine Are Indispensable for Its Antibacterial Action and LPS Neutralization

Antimicrobial peptides (AMPs) that are able to neutralize toxins are promising antibiotics. In this study we investigated the role of structurally conserved amino acids in reduced human defensin 5 (HD5_RED), which is an endogenous peptide with antibacterial action and the ability to neutralize lipopolysaccharide (LPS). Cys residues and high Arg content, rather than Gly¹⁸ and Arg⁶–Glu¹⁴, were found to be indispensable for HD5_RED binding to lipid A, for penetrating the bacterial outer and inner membranes, and for eliminating bacteria. Otherwise, all the conserved sites were requisite for HD5_RED to block the interaction between LPS and LPS-binding protein and to suppress the TLR4–NF-κB signaling pathway initiated by LPS. Accordingly, we designed the acetamidomethylated ^AcmCys-E21R-HD5_RED, which was much more potent than HD5_RED at eliminating bacteria and which can neutralize LPS. ^AcmCys-E21R-HD5_RED was also found to exhibit a synergistic effect with ciprofloxacin in killing multidrug-resistant Acinetobacter baumannii. The results of this study, in which multifunctional AMPs were designed based on structure–activity research, may help in the development of more peptide antibiotics.     

Designing a metal-binding site in the scaffold of Escherichia coli KDO8PS

KDO8PS (3-deoxy-d-manno-octulosonate-8-phosphate synthase) and DAH7PS (3-deoxy-d-arabino-heptulosonic acid-7-phosphate synthase) enzymes catalyse analogous condensation reactions between phosphoenolpyruvate and arabinose 5-phosphate or erythrose 4-phosphate, respectively. All known DAH7PS and some of KDO8PS enzymes (Aquifex aeolicus KDO8PS) require a metal ion for activity whereas another class of KDO8PS (including Escherichia coli KDO8PS) does not. Based on sequence alignment of all known KDO8PS and DAH7PS enzymes, we identified a single amino acid residue that might define the metal dependence of KDO8PS activity. One of the four metal-binding residues, a cysteine, is conserved only among metal-binding KDO8PS and DAH7PS enzymes and is replaced by an asparagine residue in other KDO8PS enzymes. We introduced a metal binding site into E.coli KDO8PS by a single N26C and a double M25P N26C mutation, which led to an increased k(cat) of the enzymes in the presence of activating Mn(2+) ions. The M25P N26C mutant of E.coli KDO8PS had a value of k(cat)/K(M) in the presence of Mn(2+) ions four times higher than A.aeolicus KDO8PS. KDO8PS and DAH7PS may have evolved from a common ancestor protein that required a divalent metal ion for activity. A non-metal-binding KDO8PSs may have evolved from an ancestor protein that was able to bind Mn(2+) but no longer required Mn(2+) to function and eventually lost one of metal-binding residues.     

Effect of Amino Acid Substitutions on 70S Ribosomal Binding,Cellular Uptake,and Antimicrobial Activity of Oncocin Onc112

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates for the treatment of infections caused by high-priority human pathogens. Their mode of action consists of (I) passive diffusion across the outer membrane, (II) active transport through the inner membrane, and (III) inhibition of protein biosynthesis by blocking the exit tunnel of the 70S ribosome. We tested whether in vitro data on ribosomal binding and bacterial uptake could predict the antibacterial activity of PrAMPs against Gram-negative and Gram-positive bacteria. Ribosomal binding and bacterial uptake rates were measured for 47 derivatives of PrAMP Onc112 and compared to the minimal inhibitory concentrations (MIC) of each peptide. Ribosomal binding was evaluated for ribosome extracts from four Gram-negative bacteria. Bacterial uptake was assessed by quantifying each peptide in the supernatants of bacterial cultures. Oncocin analogues with a higher net positive charge appeared to be more active, although their ribosome binding and uptake rates were not necessarily better than for Onc112. The data suggest a complex mode of action influenced by further factors improving or reducing the antibacterial activity, including diffusion through membranes, transport mechanism, secondary targets, off-target binding, intracellular distribution, and membrane effects. Relying only on in vitro binding and uptake data may not be sufficient for the rational development of more active analogues.     

检测脑膜炎球菌菌苗中内毒素方法的研究

作者应用聚丙烯酰胺凝胶电泳结合银染(SDS—PAGE银染)和2—酮基—3—脱氧辛糖(KDO)方法来测定脑膜炎球菌外膜蛋白和多糖菌苗中的脂多糖(LPS)。这二种方法能比较真实地测定出存在于蛋白或多糖制品中的LPS实际含量。其结果和家兔热原质试验一致。KDO法并能定量测定蛋白样品中的LPS含量。     

Membrane Binding of Parkinson's Protein α-Synuclein: Effect of Phosphorylation at Positions 87 and 129 by the S to D Mutation Approach

Human α-synuclein, a protein relevant in the brain with so-far unknown function, plays an important role in Parkinson's disease. The phosphorylation state of αS was related to the disease, prompting interest in this process. The presumed physiological function and the disease action of αS involves membrane interaction. Here, we study the effect of phosphorylation at positions 87 and 129, mimicked by the mutations S87A, S129A (nonphosphorylated) and S87D, S129D (phosphorylated) on membrane binding. Local binding is detected by spin-label continuous-wave electron paramagnetic resonance. For S87A/D, six positions (27, 56, 63, 69, 76, and 90) are probed; and for S129A/D, three (27, 56, and 69). Binding to large unilamellar vesicles of 100 nm diameter of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in a 1 : 1 composition is not affected by the phosphorylation state of S129. For phosphorylation at S87, local unbinding of αS from the membrane is observed. We speculate that modulating the local membrane affinity by phosphorylation could tune the way αS interacts with different membranes; for example, tuning its membrane fusion activity.     

Molecularly imprinted polymers by phase inversion technique for the selective recognition of saccharides of biomedical interest in aqueous solutions

The purpose of this work was the preparation and characterization of polymeric membranes for the selective recognition of saccharides using molecular imprinting technology associated with phase inversion. A system able to bind saccharides with high selectivity is particularly important in the pharmaceutical sector, since some of these compounds are constituents of molecules which can exert serious toxic effects even at very low concentrations. Two polymeric matrices were prepared using poly(ethylene‐co‐vinyl alcohol) copolymers, with an ethylene molar content of 32% and 44%, and were imprinted with two different saccharide molecules: maltose and 2‐keto‐3‐deoxy‐d ‐manno‐octulosonate (KDO). Matrices imprinted against maltose and KDO showed an easy template extraction, high binding capability and satisfactory selectivity, particularly for the matrix with an ethylene molar content of 44%. © 2017 Society of Chemical Industry     

Synthesis and Anti-Cancer Activity of New Pyrazolinyl-Indole Derivatives: Pharmacophoric Interactions and Docking Studies for Identifying New EGFR Inhibitors

Newly designed series of indole-containing pyrazole analogs, pyrazolinylindoles, were synthesized, and their structures were confirmed based on the spectral data of the ¹H NMR, ¹³C NMR, and HR-MS analyses. Preliminary anti-cancer activity testings were carried out by the National Cancer Institute, United States of America (NCI, USA). Compounds HD02, HD05, and HD12 demonstrated remarkable cytotoxic activities against nine categories of cancer types based cell line panels which included leukemia, colon, breast, melanoma, lungs, renal, prostate, CNS, and ovarian cancer cell lines. The highest cytotoxic effects were exhibited by the compounds HD02 [1-(5-(1-H-indol-3-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-phenylethanone], HD05 [1-(3-(4-chlorophenyl)-5-(1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)-2-phenoxyethanone], and HD12 [(3-(4-chlorophenyl)-5-(1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone] against some of the 56 types of NCI-based cell lines in different panels. Compound HD05 showed the maximum range of cancer cell growth inhibitions against all categories of the cell lines in all nine panels. On average, in comparison to the referral standard, imatinib, at a dose level of 10 µM, the HD05 showed significant activity against leukemia in the range of 78.76%, as compared to the imatinib at 9% of cancer cells’ growth inhibitions. Molecular docking simulation studies were performed in silico on the epidermal growth factor receptor (EGFR) tyrosine kinase, in order to validate the activity.     

Identification of Bacterial Membrane Selectivity of Romo1-Derived Antimicrobial Peptide AMPR-22 via Molecular Dynamics

The abuse or misuse of antibiotics has caused the emergence of extensively drug-resistant (XDR) bacteria, rendering most antibiotics ineffective and increasing the mortality rate of patients with bacteremia or sepsis. Antimicrobial peptides (AMPs) are proposed to overcome this problem; however, many AMPs have attenuated antimicrobial activities with hemolytic toxicity in blood. Recently, AMPR-11 and its optimized derivative, AMPR-22, were reported to be potential candidates for the treatment of sepsis with a broad spectrum of antimicrobial activity and low hemolytic toxicity. Here, we performed molecular dynamics (MD) simulations to clarify the mechanism of lower hemolytic toxicity and higher efficacy of AMPR-22 at an atomic level. We found four polar residues in AMPR-11 bound to a model mimicking the bacterial inner/outer membranes preferentially over eukaryotic plasma membrane. AMPR-22 whose polar residues were replaced by lysine showed a 2-fold enhanced binding affinity to the bacterial membrane by interacting with bacterial specific lipids (lipid A or cardiolipin) via hydrogen bonds. The MD simulations were confirmed experimentally in models that partially mimic bacteremia conditions in vitro and ex vivo. The present study demonstrates why AMPR-22 showed low hemolytic toxicity and this approach using an MD simulation would be helpful in the development of AMPs.     

An Organic Liquid Membrane System for the Separation and Production of Histamine

Abstract

The recovery of histamine (Hm) produced by histidine decarboxylase (HD) from histidine (His) by using an organic liquid membrane system was investigated. The system was composed of two aqueous phases (phase I of pH 4.5 and phase II of pH 7.2) separated by a third organic chloroform phase containing di-(2-ethylhexyl) phosphoric acid (EHP) as a carrier. His could not be moved from phase I of pH 4.5, the optimum pH of HD into the organic layer. On the other hand, Hm was moved from phase I into the organic layer, and Hm was released from the organic layer into phase II. The removal of Hm from phase I into the organic layer increased at dose dependency with EHP concentration up to 50 mM and decreased at concentrations above 50 mM. According to these results, the recovery of Hm from His by HD was investigated. In this experiment phase I was pH 4.5 containing 1 mM His, 40 μg/mL pyridoxal-5-phosphate, and a suitable concentration of HD; and phase II was set at pH 7.2. 170 μM Hm (3 h later) and 760 μM (8 h later) were transported into phase II by using 120 μg/mL HD and 50 mM EHP, as confirmed by HPLC.     

Hydrothermal degradation of adsorbed sulfur mustard on activated carbon

Thermal and hydrothermal degradations of adsorbed sulfur mustard (HD) on activated carbon particles from a chemical protective over-garment were studied. Carbon loaded with 5 wt.% HD was heated in a closed reactor at temperatures up to 160 °C for 0.5–6 h and analyzed by solid-state ¹³C MAS NMR. On dry carbon at room temperature, HD was stable for months. On a thoroughly pre-wetted carbon, adsorbed HD partially degraded to thiodiglycol (TDG) and TDG-sulfoxide (TDG-SO) within 2–3 months. Heating dry HD-loaded carbon to 160 °C caused partial degradation within 4 h to 1,4-thioxane, along with 1,4-dithiane and vinyl sulfides. Complete degradation within 2.5 h to the same products occurred upon hydrothermal treatment of the HD-loaded carbon, using a water/carbon ratio of 0.3:1. With higher water/carbon ratios of 0.6:1–5:1 at temperatures of 120 °C and above, adsorbed HD hydrolyzed rapidly within 0.5 h. The latter reaction led to the formation of TDG concurrent with either thioxane (at 160 °C) or TDG-SO and TDG-dimer (at 120 °C). The mechanisms of the observed degradation processes are discussed.     

Spin-label EPR on alpha-synuclein reveals differences in the membrane binding affinity of the two antiparallel helices

The putative function of the Parkinson's disease-related protein alpha-Synuclein (alphaS) is thought to involve membrane binding. Therefore, the interaction of alphaS with membranes composed of zwitterionic (POPC) and anionic (POPG) lipids was investigated through the mobility of spin labels attached to the protein. Differently labelled variants of alphaS were produced, containing a spin label at positions 9, 18 (both helix 1), 69, 90 (both helix 2), and 140 (C terminus). Protein binding to POPC/POPG vesicles for all but alphaS140 resulted in two mobility components with correlation times of 0.5 and 3 ns, for POPG mole fractions >0.4. Monitoring these components as a function of the POPG mole fraction revealed that at low negative-charge densities helix 1 is more tightly bound than helix 2; this indicates a partially bound form of alphaS. Thus, the interaction of alphaS with membranes of low charge densities might be initiated at helix 1. The local binding information thus obtained gives a more differentiated picture of the affinity of alphaS to membranes. These findings contribute to our understanding of the details and structural consequences of alphaS-membrane interactions.     

伤寒沙门氏菌外膜蛋白的分离和鉴定

用 Triton X-100处理伤寒沙门氏菌外膜,经3次超离后从上清液中获得外膜蛋白(OMPs)。SDS-PAGE 显示,OMPs 蛋白带的分子量在17～70KD 间,其中分子量为36～41KD的主要蛋白占总 OMPs 的50～60%。经测定2-酮-3-脱氧辛酸(KDO)的含量间接测得,OMPs 中脂多糖(LPS)的污染量约为4%。用 OMPs 免疫家兔,抗体效价琼脂双扩散法为1:64,ELISA 法为1:12 500。免疫印迹法显示,兔抗-OMP 血清主要与 OMPs 中的主要蛋白发生反应。     

Differential Cellular Balance of Olfactory and Vomeronasal Epithelia in a Transgenic BACHD Rat Model of Huntington’s Disease

Background. For neurodegenerative diseases such as Huntington’s disease (HD), early diagnosis is essential to treat patients and delay symptoms. Impaired olfaction, as observed as an early symptom in Parkinson´s disease, may also constitute a key symptom in HD. However, there are few reports on olfactory deficits in HD. Therefore, we aimed to investigate, in a transgenic rat model of HD: (1) whether general olfactory impairment exists and (2) whether there are disease-specific dynamics of olfactory dysfunction when the vomeronasal (VNE) and main olfactory epithelium (MOE) are compared. Methods. We used male rats of transgenic line 22 (TG22) of the bacterial artificial chromosome Huntington disease model (BACHD), aged 3 days or 6 months. Cell proliferation, apoptosis and macrophage activity were examined with immunohistochemistry in the VNE and MOE. Results. No differences were observed in cellular parameters in the VNE between the groups. However, the MOE of the 6-month-old HD animals showed a significantly increased number of mature olfactory receptor neurons. Other cellular parameters were not affected. Conclusions. The results obtained in the TG22 line suggest a relative stability in the VNE, whereas the MOE seems at least temporarily affected.     

Aroyl-pyrrolyl hydroxyamides: influence of pyrrole C4-phenylacetyl substitution on histone deacetylase inhibition

The novel aroyl-pyrrolyl hydroxyamides 4 a-a' are analogues of the lead compound 3-(1-methyl-4-phenylacetyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide (2) and are active as HDAC inhibitors. The benzene ring of 2 was substituted with a wide range of electron-donating and electron-withdrawing groups, and the effect was evaluated on three HDACs from maize, namely HD2, HD1-B (a class I HDAC), and HD1-A (a class II HDAC). Inhibition studies show that the benzene 3' and, to a lesser extent, 4' positions of 2 were the most suitable for the introduction of substituents, with the 3'-chloro (in 4 b) and the 3'-methyl (in 4 k) derivatives being the most potent compounds, reaching the same activity as SAHA. Inhibition data for 4 b,k against mouse HDAC1 were consistent with those observed in the maize enzyme. The substituent insertion on the benzene ring of 2 (compounds 4 a-a') abated the slight (3-fold) selectivity for class II HDACs displayed by 2. Compound 4 b showed interesting, dose-dependent antiproliferative and cytodifferentiation properties against human acute promyelocytic leukemia HL-60 cells.     

Influence of Substitutions in the Binding Motif of Proline-Rich Antimicrobial Peptide ARV-1502 on 70S Ribosome Binding and Antimicrobial Activity

Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs, we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding motif D³KPRPYLPRP¹² with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluorescein-labeled (Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant (K_d) of 201 ± 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome. Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of wild-type and ΔsbmA and ΔmdtM knockout mutants indicated that substitutions in the ribosomal binding motif altered also the bacterial uptake, which was generally improved by incorporation of hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502 analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding, inhibition of translation, and bacterial uptake.     

Synergistic Action of Antimicrobial Lung Proteins against Klebsiella pneumoniae

As key components of innate immunity, lung antimicrobial proteins play a critical role in warding off invading respiratory pathogens. Lung surfactant protein A (SP-A) exerts synergistic antimicrobial activity with the N-terminal segment of the SP-B proprotein (SP-B^N) against Klebsiella pneumoniae K2 in vivo. However, the factors that govern SP-A/SP-B^N antimicrobial activity are still unclear. The aim of this study was to identify the mechanisms by which SP-A and SP-B^N act synergistically against K. pneumoniae, which is resistant to either protein alone. The effect of these proteins on K. pneumoniae was studied by membrane permeabilization and depolarization assays and transmission electron microscopy. Their effects on model membranes of the outer and inner bacterial membranes were analyzed by differential scanning calorimetry and membrane leakage assays. Our results indicate that the SP-A/SP-B^N complex alters the ultrastructure of K. pneumoniae by binding to lipopolysaccharide molecules present in the outer membrane, forming packing defects in the membrane that may favor the translocation of both proteins to the periplasmic space. The SP-A/SP-B^N complex depolarized and permeabilized the inner membrane, perhaps through the induction of toroidal pores. We conclude that the synergistic antimicrobial activity of SP-A/SP-B^N is based on the capability of this complex, but not either protein alone, to alter the integrity of bacterial membranes.     

Nucleobase amino acids incorporated into the HIV-1 nucleocapsid protein increased the binding affinity and specificity for a hairpin RNA

L-alpha-amino acids with a nucleobase in the side chain (nucleobase amino acids; NBAs) were used to enhance the function of RNA-binding proteins that recognize structured RNA. These NBAs were utilized in the three-dimensional structure of the protein to enhance RNA binding affinity and specificity as a result of selective recognition of NBAs by RNA bases. NBA units were incorporated at various positions into the HIV-1 nucleocapsid protein NCp7 (residues 1-55), which contains two CCHC-type (Cys-X(2)-Cys-X(4)-His-X(4)-Cys-type; X=an amino acid residue) zinc knuckle domains. The binding ability was evaluated by using the stem-loop (SL)3 region of HIV-1 Psi-RNA. Visible light absorption measurements revealed that two zinc ions bound strongly and quantitatively to the NBA-NCp7 molecule and to the wild-type NCp7 protein. This result indicates that the incorporation of NBA units composed of L-alpha-amino acids did not influence the formation of the specific structure of NCp7. Binding analysis with fluorescein-labeled SL3 RNA revealed that incorporation of NBA units into the NCp7 protein at appropriate positions increased its RNA binding affinity and specificity. An NBA-NCp7 protein that possessed cytosine and guanine NBA units at positions 13 and 46, respectively, showed a binding affinity for SL3 RNA ninefold higher than that of wild-type NCp7 as a result of the specific and cooperative interaction of the NBA units with RNA bases. These results clearly demonstrate that inclusion of NBA units in the three-dimensional structure of an RNA-binding protein is a useful strategy for enhancing the function of the protein.     

The Effects of Chimeric Antigen Receptor (CAR) Hinge Domain Post-Translational Modifications on CAR-T Cell Activity

To improve the efficacy and safety of chimeric antigen receptor (CAR)-expressing T cell therapeutics through enhanced CAR design, we analysed CAR structural factors that affect CAR-T cell function. We studied the effects of disulphide bonding at cysteine residues and glycosylation in the HD on CAR-T function. We used first-generation CAR[V/28/28/3z] and CAR[V/8a/8a/3z], consisting of a mouse vascular endothelial growth factor receptor 2 (VEGFR2)-specific single-chain variable fragment tandemly linked to CD28- or CD8α-derived HD, transmembrane domain (TMD) and a CD3ζ-derived signal transduction domain (STD). We constructed structural variants by substituting cysteine with alanine and asparagine (putative N-linked glycosylation sites) with aspartate. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] formed homodimers, the former through a single HD cysteine residue and the latter through the more TMD-proximal of the two cysteine residues. The absence of disulphide bonds did not affect membrane CAR expression but reduced antigen-specific cytokine production and cytotoxic activity. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] harboured one N-linked glycosylation site, and CAR[V/8a/8a/3z] underwent considerable O-linked glycosylation at an unknown site. Thus, N-linked glycosylation of CAR[V/28/28/3z] promotes stable membrane CAR expression, while having no effect on the expression or CAR-T cell activity of CAR[V/8a/8a/3z]. Our findings demonstrate that post-translational modifications of the CAR HD influence CAR-T cell activity, establishing a basis for future CAR design.     

EcDBS1R4, an Antimicrobial Peptide Effective against Escherichia coli with In Vitro Fusogenic Ability

Discovering antibiotic molecules able to hold the growing spread of antimicrobial resistance is one of the most urgent endeavors that public health must tackle. The case of Gram-negative bacterial pathogens is of special concern, as they are intrinsically resistant to many antibiotics, due to an outer membrane that constitutes an effective permeability barrier. Antimicrobial peptides (AMPs) have been pointed out as potential alternatives to conventional antibiotics, as their main mechanism of action is membrane disruption, arguably less prone to elicit resistance in pathogens. Here, we investigate the in vitro activity and selectivity of EcDBS1R4, a bioinspired AMP. To this purpose, we have used bacterial cells and model membrane systems mimicking both the inner and the outer membranes of Escherichia coli, and a variety of optical spectroscopic methodologies. EcDBS1R4 is effective against the Gram-negative E. coli, ineffective against the Gram-positive Staphylococcus aureus and noncytotoxic for human cells. EcDBS1R4 does not form stable pores in E. coli, as the peptide does not dissipate its membrane potential, suggesting an unusual mechanism of action. Interestingly, EcDBS1R4 promotes a hemi-fusion of vesicles mimicking the inner membrane of E. coli. This fusogenic ability of EcDBS1R4 requires the presence of phospholipids with a negative curvature and a negative charge. This finding suggests that EcDBS1R4 promotes a large lipid spatial reorganization able to reshape membrane curvature, with interesting biological implications herein discussed.     

Unusually strong positive cooperativity in binding of peptides to latent membrane protein-1 DNA fragments of the Epstein-Barr viral gene

The DNA-binding preferences of two oligopeptide amides, (His-Pro-Arg-Lys)(3)NH(2) (HR-12) and (Ser-Pro-Arg-Lys)(3)NH(2) (SP-12), have been examined by quantitative DNase I footprinting studies. Two different DNA fragments were investigated: a pair of 5'-(32)P-labeled duplexes from pBR322 with one or other of the complementary strands labeled and a corresponding pair of 5'-(32)P-labeled duplexes representing fragments of the latent membrane protein (LMP-1) gene from a pathogenic Epstein-Barr virus variant derived from nasopharyngeal carcinoma. The major objective was to examine molecular recognition and cooperative features associated with sequence-selective binding of synthetic peptides to the LMP-1 fragments. At various binding sites on the pBR322 fragments, Hill coefficients (n(H)) ranging from 1.9-2.2 were observed; these results indicate modest positive cooperativity between binding sites for both peptides. By contrast, unusually high values of n(H), ranging from 4.0-9.3, were observed at various binding sites on the LMP-1 fragments. Allosteric models can be constructed to interpret the observed cooperative interactions between different DNA recognition sites in the LMP-1 gene upon binding of the peptide ligands. It is noteworthy that these models feature a novel network of cooperativity interconnecting multiple DNA allosteric sites. The evidence of sequence selectivity and strong cooperativity discovered in this work may prove to be a general feature of peptide interactions with some nucleic acids.