Showing papers in "Biochemistry in 2004"

LNA (Locked Nucleic Acid): High-Affinity Targeting of Complementary RNA and DNA†

Abstract: Locked nucleic acid (LNA) is a nucleic acid analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation. LNA oligonucleotides display unprecedented hybridization affinity toward complementary single-stranded RNA and complementary single- or double-stranded DNA. Structural studies have shown that LNA oligonucleotides induce A-type (RNA-like) duplex conformations. The wide applicability of LNA oligonucleotides for gene silencing and their use for research and diagnostic purposes are documented in a number of recent reports, some of which are described herein.

Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot.

Abstract: The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata B2, which has...

Anti-angiogenic, antioxidant, and anti-carcinogenic properties of a novel anthocyanin-rich berry extract formula

Abstract: Edible berry anthocyanins possess a broad spectrum of therapeutic and anti-carcinogenic properties. Berries are rich in anthocyanins, compounds that provide pigmentation to fruits and serve as natural antioxidants. Anthocyanins repair and protect genomic DNA integrity. Earlier studies have shown that berry anthocyanins are beneficial in reducing age-associated oxidative stress, as well as in improving neuronal and cognitive brain function. Six berry extracts (wild blueberry, bilberry, cranberry, elderberry, raspberry seeds, and strawberry) were studied for antioxidant efficacy, cytotoxic potential, cellular uptake, and anti-angiogenic (the ability to reduce unwanted growth of blood vessels, which can lead to varicose veins and tumor formation) properties. We evaluated various combinations of edible berry extracts and developed a synergistic formula, OptiBerry IH141, which exhibited high ORAC (Oxygen-Radical Absorbing Capacity) value, low cytotoxicity, and superior anti-angiogenic properties compared to the other combinations tested. Anti-angiogenic approaches to treat cancer represent a priority area in vascular tumor biology. OptiBerry significantly inhibited both H2O2- and TNF-alpha-induced VEGF (Vascular Endothelial Growth Factor) expression by human keratinocytes. VEGF is a key regulator of tumor angiogenesis. Matrigel assay using human microvascular endothelial cells showed that OptiBerry impaired angiogenesis. In an in vivo model of angiogenesis, OptiBerry significantly inhibited basal MCP-1 and inducible NF-kappaB transcriptions. Endothelioma cells pretreated with OptiBerry showed a diminished ability to form hemangioma and markedly decreased tumor growth by more than 50%. In essence, these studies highlight the novel anti-angiogenic, antioxidant, and anti-carcinogenic potential of a novel anthocyanin-rich berry extract formula, OptiBerry.

Heat-induced secondary structure and conformation change of bovine serum albumin investigated by Fourier transform infrared spectroscopy.

Abstract: Fourier transform infrared (FT-IR) spectra were measured for an aqueous solution (pD = 5.40) of defatted monomer bovine serum albumin (BSA) over a temperature range of 25-90 degrees C to investigate temperature-induced secondary structure and conformation changes. The curve fitting method combined with the Fourier self-deconvolution technique allowed us to explore details of the secondary structure and conformation changes in defatted BSA. Particularly striking in the FT-IR spectra was an observation of the formation of an irreversible intermolecular beta-sheet of BSA on heating above 70 degrees C. A band at 1630 cm(-1) in the spectra was assigned to short-segment chains connecting alpha-helical segments. The transition temperature for the short-segment chains connecting alpha-helical segments is lower by 17-18 degrees C, when compared to those of the alpha-helix, turn, and intermolecular beta-sheet structures of BSA, suggesting that the alpha-helix and turn structures of BSA are cooperatively denatured on heating. Moreover, the results give an important feature in heat-induced denaturation of BSA that the conformation changes occur twice around both 57 and 75 degrees C. The appearance of two peaks is interpreted by the collapse of the N-terminal BSA domain due to the crevice in the vicinity between domains I and II at low-temperature transition and by the change in cooperative unit composed of the other two BSA domains at high-temperature transition.

Pathway for polyarginine entry into mammalian cells.

Abstract: Cationic peptides known as protein transduction domains (PTDs) provide a means to deliver molecules into mammalian cells. Here, nonaarginine (R(9)), the most efficacious of known PTDs, is used to elucidate the pathway for PTD internalization. Although R(9) is found in the cytosol as well as the nucleolus when cells are fixed, this peptide is observed only in the endocytic vesicles of live cells. Colocalization studies with vesicular markers confirm that PTDs are internalized by endocytosis rather than by crossing the plasma membrane. The inability of R(9) to enter living cells deficient in heparan sulfate (HS) suggests that binding to HS is necessary for PTD internalization. This finding is consistent with the high affinity of R(9) for heparin (K(d) = 109 nM). Finally, R(9) is shown to promote the leakage of liposomes but only at high peptide:lipid ratios. These and other data indicate that the PTD-mediated delivery of molecules into live mammalian cells involves (1) binding to cell surface HS, (2) uptake by endocytosis, (3) release upon HS degradation, and (4) leakage from endocytic vesicles.

Lipidic carriers of siRNA: differences in the formulation, cellular uptake, and delivery with plasmid DNA.

Abstract: RNA interference (RNAi) has become a popular tool for downregulating specific gene expression in many species, including mammalian cells [Novina, C. D., and Sharp, P. A. (2004) The RNAi revolution, Nature 430, 161-164]. Synthetic double-stranded RNA sequences (siRNA) of 21-23 nucleotides have been shown in particular to have the potential to silence specifically gene function in cultured mammalian cells. As a result, there has been a significant surge of interest in the application of siRNA in functional genomics programs as a means of deciphering specific gene function. However, for siRNA functional genomics studies to be valuable and effective, specific silencing of any given target gene is essential, devoid of nonspecific knockdown and toxic side effects. For this reason, we became interested in investigating cationic liposome/lipid-mediated siRNA delivery (siFection) as a meaningful and potentially potent way to facilitate effective functional genomics studies. Accordingly, a number of cationic liposome/lipid-based systems were selected, and their formulation with siRNA was studied, with particular emphasis on formulation parameters most beneficial for siRNA use in functional genomics studies. Cationic liposome/lipid-based systems were selected from a number of commercially available products, including lipofectAMINE2000 and a range of CDAN/DOPE systems formulated from different molar ratios of the cationic cholesterol-based polyamine lipid N(1)-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN) and the neutral helper lipid dioleoyl-L-alpha-phosphatidylethanolamine (DOPE). Parameters that were been investigated included the lipid:nucleic acid ratio of mixing, the extent of cationic liposome/lipid-nucleic acid complex (lipoplex) formation plus medium used, the lipoplex particle size, the mode of delivery, and dose-response effects. Results suggest that concentrations during siRNA lipoplex (LsiR) formation are crucial for maximum knockdown, but the efficacy of gene silencing is not influenced by the size of LsiR particles. Most significantly, results show that most commercially available cationic liposome/lipid-based systems investigated here mediate a significant nonspecific downregulation of the total cellular protein content at optimal doses for maximal specific gene silencing and knockdown. Furthermore, one pivotal aspect of using siRNA for functional genomics studies is the need for at least minimal cellular toxicity. Results demonstrate that CDAN and DOPE with and without siRNA confer low toxicity to mammalian cells, whereas lipofectAMINE2000 is clearly toxic both as a reagent and after formulation into LsiR particles. Interestingly, LsiR particles formulated from CDAN and DOPE (45:55, m/m; siFECTamine) seem to exhibit a slower cellular uptake than LsiR particles formulated from lipofectAMINE2000. Intracellularly, LsiR particles formulated from CDAN and DOPE systems also appear to behave differently, amassing in distinct but diffuse small nonlysosomal compartments for at least 5 h after siFection. By contrast, LsiR particles formulated from lipofectAMINE2000 accumulate in fewer larger intracellular vesicles.

Nickel superoxide dismutase structure and mechanism.

Abstract: The 1.30 A resolution crystal structure of nickel superoxide dismutase (NiSOD) identifies a novel SOD fold, assembly, and Ni active site. NiSOD is a hexameric assembly of right-handed 4-helix bundles of up-down-up-down topology with N-terminal hooks chelating the active site Ni ions. This newly identified nine-residue Ni-hook structural motif (His-Cys-X-X-Pro-Cys-Gly-X-Tyr) provides almost all interactions critical for metal binding and catalysis, and thus will likely be diagnostic of NiSODs. Conserved lysine residues are positioned for electrostatic guidance of the superoxide anion to the narrow active site channel. Apo structures show that the Ni-hook motif is unfolded prior to metal binding. The active site Ni geometry cycles from square planar Ni(II), with thiolate (Cys2 and Cys6) and backbone nitrogen (His1 and Cys2) ligands, to square pyramidal Ni(III) with an added axial His1 side chain ligand, consistent with electron paramagentic resonance spectroscopy. Analyses of the three NiSOD structures and comparisons to the Cu,Zn and Mn/Fe SODs support specific molecular mechanisms for NiSOD maturation and catalysis, and identify important structure-function relationships conserved among SODs.

Copper Mediates Dityrosine Cross-Linking of Alzheimer's Amyloid-β

Abstract: We have previously reported that amyloid Abeta, the major component of senile plaques in Alzheimer's disease (AD), binds Cu with high affinity via histidine and tyrosine residues [Atwood, C. S., et al. (1998) J. Biol. Chem. 273, 12817-12826; Atwood, C. S., et al. (2000) J. Neurochem. 75, 1219-1233] and produces H(2)O(2) by catalyzing the reduction of Cu(II) or Fe(III) [Huang, X., et al. (1999) Biochemistry 38, 7609-7616; Huang, X., et al. (1999) J. Biol. Chem. 274, 37111-37116]. Incubation with Cu induces the SDS-resistant oligomerization of Abeta [Atwood, C. S., et al. (2000) J. Neurochem. 75, 1219-1233], a feature characteristic of neurotoxic soluble Abeta extracted from the AD brain. Since residues coordinating Cu are most vulnerable to oxidation, we investigated whether modifications of these residues were responsible for Abeta cross-linking. SDS-resistant oligomerization of Abeta caused by incubation with Cu was found to induce a fluorescence signal characteristic of tyrosine cross-linking. Using ESI-MS and a dityrosine specific antibody, we confirmed that Cu(II) (at concentrations lower than that associated with amyloid plaques) induces the generation of dityrosine-cross-linked, SDS-resistant oligomers of human, but not rat, Abeta peptides. The addition of H2O2 strongly promoted Cu-induced dityrosine cross-linking of Abeta1-28, Abeta1-40, and Abeta1-42, suggesting that the oxidative coupling is initiated by interaction of H2O2 with a Cu(II) tyrosinate. The dityrosine modification is significant since it is highly resistant to proteolysis and is known to play a role in increasing structural strength. Given the elevated concentration of Cu in senile plaques, our results suggest that Cu interactions with Abeta could be responsible for causing the covalent cross-linking of Abeta in these structures.

Primary Structure Elements of Spider Dragline Silks and Their Contribution to Protein Solubility

Abstract: Spider silk proteins have mainly been investigated with regard to their contribution to mechanical properties of the silk thread. However, little is known about the molecular mechanisms of silk assembly. As a first step toward characterizing this process, we aimed to identify primary structure elements of the garden spider's (Araneus diadematus) major dragline silk proteins ADF-3 and ADF-4 that determine protein solubility. In addition, we investigated the influence of conditions involved in mediating natural thread assembly on protein aggregation. Genes encoding spider silk-like proteins were generated using a cloning strategy, which is based on a combination of synthetic DNA modules and PCR-amplified authentic gene sequences. Comparing secondary structure, solubility, and aggregation properties of the synthesized proteins revealed that single primary structure elements have diverse influences on protein characteristics. Repetitive regions representing the largest part of dragline silk proteins determined the solubility of the synthetic proteins, which differed greatly between constructs derived from ADF-3 and ADF-4. Factors, such as acidification and increases in phosphate concentration, which promote silk assembly in vivo generally decreased silk protein solubility in vitro. Strikingly, this effect was pronounced in engineered proteins comprising the carboxyl-terminal nonrepetitive regions of ADF-3 or ADF-4, indicating that these regions might play an important role in initiating assembly of spider silk proteins.

Impact of the Acidic C-Terminal Region Comprising Amino Acids 109−140 on α-Synuclein Aggregation in Vitro†

Abstract: The aggregation of alpha-synuclein, involved in the pathogenesis of several neurodegenerative disorders such as Parkinson's disease, is enhanced in vitro by biogenic polyamines binding to the highly charged C-terminal region aa109-140. In this study, we investigated the influence of this region on the aggregation kinetics, monitored by thioflavin T binding and static light scattering, and morphology, assessed by electron microscopy, fluorescence microscopy, and turbidity, by comparing the effect of various solution conditions on the wild-type protein, the disease related mutants A53T and A30P, and two truncated variants, syn(1-108) and syn(1-124), lacking the complete or the C-terminal half of the polyamine binding site. In the presence of the intact C-terminus, aggregation was strongly retarded in physiological buffer. This inhibition of aggregation was overridden by (i) addition of spermine or MgCl(2) or lowering of pH, leading to strong charge shielding in the C-terminus or (ii) by truncation of aa125-140 or aa109-140. Addition of MgCl(2) or spermine or acidification were not effective in promoting aggregation of syn(1-108). The impact of the disease-related mutations on the aggregation kinetics was dependent on the solution conditions, with the aggregation propensity order A53T approximately wt > A30P at low ionic strength, but A53T > wt approximately A30P at high ionic strength, with exceedingly potent promotion of aggregation by the A53T mutation in the presence of spermine. In contrast to full-length alpha-synuclein aggregates, those formed from syn(1-108) did not exhibit a pronounced polymorphism. The effects of the C-terminus on aggregation cannot be rationalized merely by a contribution to the protein net charge, but rather suggest a specific role of aa109-140 in the regulation of aggregation, presumably involving formation of intramolecular contacts.

DNA polymerase fidelity: kinetics, structure, and checkpoints.

Abstract: On careful examination of existing kinetic data for correct and incorrect dNTP incorporations by a variety of DNA polymerases, it is apparent that these enzymes resist a unified description. Instead, the picture that emerges is a rather complex one: for most polymerases, there is evidence for a noncovalent step preceding phosphoryl transfer, but there are less reliable data for determining whether the noncovalent step or phosphoryl transfer is rate-limiting during misincorporation. Although the structural conservation in the polymerase superfamily is probably reflected in a common set of intermediates along the reaction pathway, the energetics of these species vary even when closely related polymerases are compared. Consequently, some polymerases apparently show more discrimination between correctly paired and mispaired dNTPs in the binding step, and polymerases may differ in terms of which step of the reaction is rate-limiting in correct and incorrect insertion reactions. Because of the higher energy barrier in the misincorporation reaction, at least some of the intermediates both before and after the rate-limiting step in the misincorporation pathway will have higher energies than the corresponding intermediates in correct incorporation; consequently, these steps can serve as kinetic checkpoints.

Transthyretin aggregation under partially denaturing conditions is a downhill polymerization.

Abstract: The deposition of fibrils and amorphous aggregates of transthyretin (TTR) in patient tissues is a hallmark of TTR amyloid disease, but the molecular details of amyloidogenesis are poorly understood. Tetramer dissociation is typically rate-limiting for TTR amyloid fibril formation, so we have used a monomeric variant of TTR (M-TTR) to study the mechanism of aggregation. Amyloid formation is often considered to be a nucleation-dependent process, where fibril growth requires the formation of an oligomeric nucleus that is the highest energy species on the pathway. According to this model, the rate of fibril formation should be accelerated by the addition of preformed aggregates or "seeds", which effectively bypasses the nucleation step. Herein, we demonstrate that M-TTR amyloidogenesis at low pH is a complex, multistep reaction whose kinetic behavior is incompatible with the expectations for a nucleation-dependent polymerization. M-TTR aggregation is not accelerated by seeding, and the dependence of the reaction timecourse is first-order on the M-TTR concentration, consistent either with a dimeric nucleus or with a nonnucleated process where each step is bimolecular and essentially irreversible. These studies suggest that amyloid formation by M-TTR under partially denaturing conditions is a downhill polymerization, in which the highest energy species is the native monomer. Our results emphasize the importance of therapeutic strategies that stabilize the TTR tetramer and may help to explain why more than eighty TTR variants are disease-associated. The differences between amyloid formation by M-TTR and other amyloidogenic peptides (such as amyloid beta-peptide and islet amyloid polypeptide) demonstrate that these polypeptides do not share a common aggregation mechanism, at least under the conditions examined thus far.

Effects of sodium ions on DNA duplex oligomers: improved predictions of melting temperatures.

Abstract: Melting temperatures, Tm, were systematically studied for a set of 92 DNA duplex oligomers in a variety of sodium ion concentrations ranging from 69 mM to 1.02 M. The relationship between Tm and ln [Na+] was nonlinear over this range of sodium ion concentrations, and the observed melting temperatures were poorly predicted by existing algorithms. A new empirical relationship was derived from UV melting data that employs a quadratic function, which better models the melting temperatures of DNA duplex oligomers as sodium ion concentration is varied. Statistical analysis shows that this improved salt correction is significantly more accurate than previously suggested algorithms and predicts salt-corrected melting temperatures with an average error of only 1.6 °C when tested against an independent validation set of Tm measurements obtained from the literature. Differential scanning calorimetry studies demonstrate that this Tm salt correction is insensitive to DNA concentration. The Tm salt correction function ...

Toward an Understanding of the Mechanism of Nonphotochemical Quenching in Green Plants

Abstract: Oxygenic photosynthesis in plants involves highly reactive intermediates and byproducts that can damage the photosynthetic apparatus and other chloroplast constituents. The potential for damage is exacerbated when the amount of absorbed light exceeds the capacity for light energy utilization in photosynthesis, a condition that can lead to decreases in photosynthetic efficiency. A feedback de-excitation mechanism (qE), measured as a component of nonphotochemical quenching of chlorophyll fluorescence, regulates photosynthetic light harvesting in excess light in response to a change in thylakoid lumen pH. qE involves de-excitation of the singlet excited state of chlorophyll in the light-harvesting antenna of photosystem II, thereby minimizing the deleterious effects of high light via thermal dissipation of excess excitation energy. While the physiological importance of qE has been recognized for many years, a description of its physical mechanism remains elusive. We summarize recent biochemical and spectroscopic results that have brought us closer to the goal of a mechanistic understanding of this fundamental photosynthetic regulatory process.

Assignment of endogenous substrates to enzymes by global metabolite profiling.

Abstract: Enzymes regulate biological processes through the conversion of specific substrates to products. Therefore, of fundamental interest for every enzyme is the elucidation of its natural substrates. Here, we describe a general strategy for identifying endogenous substrates of enzymes by untargeted liquid chromatography−mass spectrometry (LC−MS) analysis of tissue metabolomes from wild-type and enzyme-inactivated organisms. We use this method to discover several brain lipids regulated by the mammalian enzyme fatty acid amide hydrolase (FAAH) in vivo, including known signaling molecules (e.g., the endogenous cannabinoid anandamide) and a novel family of nervous system-enriched natural products, the taurine-conjugated fatty acids. Remarkably, the relative hydrolytic activity that FAAH exhibited for lipid metabolites in vitro was not predictive of the identity of specific FAAH substrates in vivo. Thus, global metabolite profiling establishes unanticipated connections between the proteome and metabolome that enabl...

Lithium, a Common Drug for Bipolar Disorder Treatment, Regulates Amyloid-β Precursor Protein Processing

Abstract: Lithium is one of the most widely used mood-stabilizing agents for the treatment of bipolar disorder Although the underlying mechanism(s) of this mood stabilizer remains controversial, recent evidence linking lithium to neurotrophic/neuroprotective effects (Choi and Sung (2000) 1475, 225−230; Davies et al (2000) 351, 95−105) suggests novel benefits of this drug in addition to mood stabilization Here, we report that both lithium as well as valproic acid (VPA) inhibit β-amyloid peptide (Aβ) production in HEK293 cells stably transfected with Swedish amyloid precursor protein (APP)751 and in the brains of the PDAPP (APPV717F) Alzheimer's disease transgenic mouse model at clinically relevant plasma concentrations Both lithium and VPA are known to be glycogen synthase kinase-3 (GSK3) inhibitors Our studies reveal that GSK3β is a potential downstream kinase, which modulates APP processing because inhibition of GSK3 activity by either a dominant negative GSK3β kinase-deficient construct or GSK3β antisense ol

Singlet oxygen inhibits the repair of photosystem II by suppressing the translation elongation of the D1 protein in Synechocystis sp. PCC 6803.

Abstract: Singlet oxygen, generated during photosynthesis, is a strong oxidant that can, potentially, damage various molecules of biological importance. We investigated the effects in vivo of singlet oxygen on the photodamage to photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. Increases in intracellular concentrations of singlet oxygen, caused by the presence of photosensitizers, such as rose bengal and ethyl eosin, stimulated the apparent photodamage to PSII. However, actual photodamage to PSII, as assessed in the presence of chloramphenicol, was unaffected by the production of singlet oxygen. These observations suggest that singlet oxygen produced by added photosensitizers acts by inhibiting the repair of photodamaged PSII. Labeling of proteins in vivo revealed that singlet oxygen inhibited the synthesis of proteins de novo and, in particular, the synthesis of the D1 protein. Northern blotting analysis indicated that the accumulation of psbA mRNAs, which encode the D1 protein, was unaffected by the production of singlet oxygen. Subcellular localization of polysomes with bound psbA mRNAs suggested that the primary target of singlet oxygen might be the elongation step of translation.

Disruption of the LOV-Jα helix interaction activates phototropin kinase activity

Abstract: Light plays a crucial role in activating phototropins, a class of plant photoreceptors that are sensitive to blue and UV-A wavelengths. Previous studies indicated that phototropin uses a bound flavin mononucleotide (FMN) within its light-oxygen-voltage (LOV) domain to generate a protein-flavin covalent bond under illumination. In the C-terminal LOV2 domain of Avena sativa phototropin 1, formation of this bond triggers a conformational change that results in unfolding of a helix external to this domain called Jalpha [Harper, S. M., et al. (2003) Science 301, 1541-1545]. Though the structural effects of illumination were characterized, it was unknown how these changes are coupled to kinase activation. To examine this, we made a series of point mutations along the Jalpha helix to disrupt its interaction with the LOV domain in a manner analogous to light activation. Using NMR spectroscopy and limited proteolysis, we demonstrate that several of these mutations displace the Jalpha helix from the LOV domain independently of illumination. When placed into the full-length phototropin protein, these point mutations display constitutive kinase activation, without illumination of the sample. These results indicate that unfolding of the Jalpha helix is the critical event in regulation of kinase signaling for the phototropin proteins.

Transferrin-modified liposomes equipped with a pH-sensitive fusogenic peptide: an artificial viral-like delivery system.

Abstract: Liposomes are one of the most promising systems for selective cellular targeting via introduction of specific ligands for cell-surface receptors. After being taken up by the cells, these liposomes usually follow intracellular pathways of receptor-mediated endocytosis. Control of intracelluar trafficking is required for optimized drug delivery. In this study, we elucidated the intracellular fate of transferrin-modified liposomes and succeeded in altering it by introducing the pH-sensitive fusogenic peptide, GALA (WEAALAEALAEALAEHLAEALAEALEALAA). Transferrins that are chemically attached to a liposomal surface (Tf-L) were internalized via receptor-mediated endocytosis more slowly than unmodified transferrins. In contrast to the recyclable nature of transferrin, liposome-attached transferrins together with encapsulated rhodamines were retained in vesicular compartments. When GALA was introduced into liposomal membranes using a cholesteryl moiety for anchoring (Chol-GALA), rhodamines were efficiently released...

Real-Time Probing of Membrane Transport in Living Microbial Cells Using Single Nanoparticle Optics and Living Cell Imaging†

Abstract: Membrane transport plays a leading role in a wide spectrum of cellular and subcellular pathways, including multidrug resistance (MDR), cellular signaling, and cell-cell communication. Pseudomonas aeruginosa is renowned for its intriguing membrane transport mechanisms, such as the interplay of membrane permeability and extrusion machinery, leading to selective accumulation of specific intracellular substances and MDR. Despite extensive studies, the mechanisms of membrane transport in living microbial cells remain incompletely understood. In this study, we directly measure real-time change of membrane permeability and pore sizes of P. aeruginosa at the nanometer scale using the intrinsic color index (surface plasmon resonance spectra) of silver (Ag) nanoparticles as the nanometer size index probes. The results show that Ag nanoparticles with sizes ranging up to 80 nm are accumulated in living microbial cells, demonstrating that these Ag nanoparticles transport through the inner and outer membrane of the cells. In addition, a greater number of larger intracellular Ag nanoparticles are observed in the cells as chloramphenicol concentration increases, suggesting that chloramphenicol increases membrane permeability and porosity. Furthermore, studies of mutants (nalB-1 and DeltaABM) show that the accumulation rate of intracellular Ag nanoparticles depends on the expression level of the extrusion pump (MexAB-OprM), suggesting that the extrusion pump plays an important role in controlling the accumulation of Ag nanoparticles in living cells. Moreover, the accumulation kinetics measured by Ag nanoparticles are similar to those measured using a small fluorescent molecule (EtBr), eliminating the possibility of steric and size effects of Ag nanoparticle probes. Susceptibility measurements also suggest that a low concentration of Ag nanoparticles (1.3 pM) does not create significant toxicity for the cells, further validating that single Ag nanoparticles (1.3 pM) can be used as biocompatible nanoprobes for the study of membrane transport kinetics in living microbial cells.

Energetics of pore formation induced by membrane active peptides

Abstract: Antimicrobial peptides are known to form pores in cell membranes. We study this process in model bilayers of various lipid compositions. We use two of the best-studied peptides, alamethicin and melittin, to represent peptides making two types of pores, that is, barrel-stave pores and toroidal pores. In both cases, the key control variable is the concentration of the bound peptides in the lipid bilayers (expressed in the peptide-lipid molar ratio, P/L). The method of oriented circular dichroism (OCD) was used to monitor the peptide orientation in bilayers as a function of P/L. The same samples were scanned by X-ray diffraction to measure the bilayer thickness. In all cases, the bilayer thickness decreases linearly with P/L and then levels off after P/L exceeds a lipid-dependent critical value, (P/L)*. OCD spectra showed that the helical peptides are oriented parallel to the bilayers as long as P/L < (P/L)*, but as P/L increases over (P/L)*, an increasing fraction of peptides changed orientation to become perpendicular to the bilayer. We analyzed the data by assuming an internal membrane tension associated with the membrane thinning. The free energy containing this tension term leads to a relation explaining the P/L-dependence observed in the OCD and X-ray diffraction measurements. We extracted the experimental parameters from this thermodynamic relation. We believe that they are the quantities that characterize the peptide-lipid interactions related to the mechanism of pore formation. We discuss the meaning of these parameters and compare their values for different lipids and for the two different types of pores. These experimental parameters are useful for further molecular analysis and are excellent targets for molecular dynamic simulation studies.

Mechanism for the hydrolysis of organophosphates by the bacterial phosphotriesterase.

Abstract: Phosphotriesterase (PTE) from Pseudomonas diminuta is a zinc metalloenzyme that hydrolyzes a variety of organophosphorus compounds. The kinetic parameters of Zn/Zn PTE, Cd/Cd PTE, and a mixed-metal Zn/Cd hybrid PTE were obtained with a variety of substrates to determine the role of each metal ion in binding and catalysis. pH-rate profiles for the hydrolysis of diethyl p-nitrophenyl phosphate (I) and diethyl p-chlorophenyl phosphate (II) demonstrated that the ionization of a single group in the pH range of 5−10 was critical for substrate turnover. The pKa values determined from the kinetic assays were dependent on the identity of the metal ion that occupied the α site within the binuclear metal center. These results suggest that the hydrolytic nucleophile is activated as a hydroxide via the ionization of a water molecule attached to the α-metal ion. The kinetic constants for the hydrolysis of II and diethyl p-chlorophenyl thiophosphate (IV) were determined for the metal substituted forms of PTE. The kineti...

Formation of amyloid fibers triggered by phosphatidylserine-containing membranes

Abstract: Protein misfolding has been shown to be the direct cause of a number of highly devastating diseases such as Alzheimer's disease, Parkinson's disease, and Creutzfeldt-Jacob syndrome, affecting the aging population globally. The deposition in tissues of amyloid fibrils is a characteristic of all these diseases, and the mechanisms by which these protein aggregates form continue to be intensively investigated. In only a fraction of cases is an underlying mutation responsible, and accordingly, what initiates amyloid formation in vivo is the major question that is addressed. In this study, we show that membranes containing phosphatidylserine (PS), a negatively charged phospholipid, induce a rapid formation of fibers by a variety of proteins, viz., lysozyme, insulin, glyceraldehyde-3-phosphate dehydrogenase, myoglobin, transthyretin, cytochrome c, histone H1, and α-lactalbumin. Congo red staining of these fibers yields the characteristic light green birefringence of amyloid, and fluorescent lipid tracers further...

Photobiological principles of therapeutic applications of laser radiation.

Abstract: Laser therapy based on the stimulating and healing action of light of low-intensity lasers (LIL), along with laser surgery and photodynamic therapy, has been lately widely applied in the irradiation of human tissues in the absence of exogenous photosensitizers. Besides LIL, light-emitting diodes are used in phototherapy (photobiostimulation) whose action, like that of LIL, depends on the radiation wavelength, dose, and distribution of light intensity in time but, according to all available data, does not depend on the coherence of radiation.

Perturbation of the hydrophobic core of lipid bilayers by the human antimicrobial peptide LL-37.

Abstract: LL-37 is a cationic, amphipathic alpha-helical antimicrobial peptide found in humans that kills cells by disrupting the cell membrane. To disrupt membranes, antimicrobial peptides such as LL-37 must alter the hydrophobic core of the bilayer. Differential scanning calorimetry and deuterium ((2)H) NMR experiments on acyl chain perdeuterated lipids demonstrate that LL-37 inserts into the hydrophobic region of the bilayer and alters the chain packing and cooperativity. The results show that hydrophobic interactions between LL-37 and the hydrophobic acyl chains are as important for the ability of this peptide to disrupt lipid bilayers as its electrostatic interactions with the polar headgroups. The (2)H NMR data are consistent with the previously determined surface orientation of LL-37 (Henzler Wildman, K. A., et al. (2003) Biochemistry 42, 6545) with an estimated 5-6 A depth of penetration of the hydrophobic face of the amphipathic helix into the hydrophobic interior of the bilayer. LL-37 also alters the material properties of lipid bilayers, including the area per lipid, hydrophobic thickness, and coefficient of thermal expansion in a manner that varies with lipid type and temperature. Comparison of the effect of LL-37 on 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC-d(31)) and 1,2-dimyristoyl-phosphatidylcholine (DMPC-d(54)) at different temperatures demonstrates the importance of bilayer order in determining the type and extent of disordering and disruption of the hydrophobic core by LL-37. One possible explanation, which accounts for both the (2)H NMR data presented here and the known surface orientation of LL-37 under identical conditions, is that bilayer order influences the depth of insertion of LL-37 into the hydrophobic/hydrophilic interface of the bilayer, altering the balance of electrostatic and hydrophobic interactions between the peptide and the lipids.

Assembly and stability of nisin-lipid II pores.

Abstract: The peptide antibiotic nisin was the first reported example of an antibiotic that kills bacteria via targeted pore formation. The specific target of nisin is Lipid II, an essential intermediate in the bacterial cell-wall synthesis. High-affinity binding of the antibiotic to Lipid II is followed by rapid permeabilization of the membrane. Here, we investigated the assembly and stability of nisin−Lipid II pore complexes by means of pyrene fluorescence and circular dichroism. We demonstrated that nisin uses all available Lipid II molecules in the membrane to form pore complexes. The pore complexes have a uniform structure and consist of 8 nisin and 4 Lipid II molecules. Moreover, the pores displayed a remarkable stability, because they were able to resist the solubilization of the membrane environment by mild detergents. Similar experiments with [N20P/M21P]nisin showed that the hinge region is essential for the assembly into stable pore complexes. The new insights were used to propose a refined model for nisi...

A distinct utility of the amide III infrared band for secondary structure estimation of aqueous protein solutions using partial least squares methods.

Abstract: Fourier transform infrared spectroscopy is becoming an increasingly important method to study protein secondary structure. The amide I region of the protein infrared spectrum is the widely used region, whereas the amide III region has been comparatively neglected due to its low signal. Since there is no water interference in the amide III region and, more importantly, the different secondary structures of proteins have more resolved differences in their amide III spectra, it is quite promising to use the amide III region to determine protein secondary structure. In our current study, a partial least squares (PLS) method was used to predict protein secondary structures from the protein IR spectra. The IR spectra of aqueous solutions of 16 different proteins of known crystal structure have been recorded, and the amide I, the amide III, and the amide I combined with the amide III region of these proteins were used to set up the calibration set for the PLS algorithm. Our results correlate quite well with the data from X-ray studies, and the prediction from the amide III region is better than that from amide I or combined amide I and amide III regions.

A general framework for development and data analysis of competitive high-throughput screens for small-molecule inhibitors of protein-protein interactions by fluorescence polarization.

Abstract: Equilibrium binding experiments are widely used for the accurate characterization of binding and competitive binding behavior in biological systems. Modern high-throughput discovery efforts in chemical biology rely heavily upon this principle. Here, we derive exact analytical expressions for general competitive binding models which can also explain a commonly encountered phenomenon in these types of experiments, anticooperative incomplete displacement. We explore the effects of nonspecific binding behavior and parameter misestimation. All expressions are derived in terms of total concentrations determined a priori. We discuss a general framework for high-throughput screening assays based on fluorescence polarization and strategies for assay development, sensitivity regimes, data quality control, analysis, and ranking. Theoretical findings are visualized by simulations using realistic parameter sets. Our results are the basis for the discovery of small-molecule inhibitors of the protein−protein interaction...

Structural Details on the Binding of Antihypertensive Drugs Captopril and Enalaprilat to Human Testicular Angiotensin I-Converting Enzyme

Abstract: Angiotensin converting enzyme (ACE) plays a critical role in the circulating or endocrine renin−angiotensin system (RAS) as well as the local regulation that exists in tissues such as the myocardium and skeletal muscle. Here we report the high-resolution crystal structures of testis ACE (tACE) in complex with the first successfully designed ACE inhibitor captopril and enalaprilat, the Phe-Ala-Pro analogue. We have compared these structures with the recently reported structure of a tACE−lisinopril complex [Natesh et al. (2003) Nature 421, 551−554]. The analyses reveal that all three inhibitors make direct interactions with the catalytic Zn2+ ion at the active site of the enzyme: the thiol group of captopril and the carboxylate group of enalaprilat and lisinopril. Subtle differences are also observed at other regions of the binding pocket. These are compared with N-domain models and discussed with reference to published biochemical data. The chloride coordination geometries of the three structures are disc...

Oligomerization of G protein-coupled receptors: past, present, and future.

Abstract: G protein-coupled receptor (GPCR)-mediated signal transduction has been studied for more than a century. Despite the intense focus on this class of proteins, a molecular understanding of what constitutes the functional form of the receptor is still uncertain. GPCRs have traditionally been conceptualized as monomeric proteins, and this view has changed little over the years until relatively recently. Recent biochemical and biophysical studies have challenged this traditional concept, and point instead to a mechanistic view of signal transduction wherein the receptor functions as an oligomer. Cooperative interactions within such an oligomeric array may be critical for the propagation of an external signal across the cell membrane and to the G protein, and may therefore underlie the mechanistic basis of signaling.