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Compounds that mimic the biological properties of glycosaminoglycans (GAGs) and can be more easily prepared than the native GAG oligosaccharides are highly demanded. Here, we present the synthesis of sulfated oligosaccharides displaying a perfluorinated aliphatic tag at the reducing end as GAG mimetics. The preparation of these molecules was greatly facilitated by the presence of the fluorinated tail since the reaction intermediates were isolated by simple fluorous solid-phase extraction. Fluorescence polarization competition assays indicated that the synthesized oligosaccharides interacted with two heparin-binding growth factors, midkine (MK) and FGF-2, showing higher binding affinities than the natural oligosaccharides, and can be therefore considered as useful GAG mimetics. Moreover, NMR experiments showed that the 3D structure of these compounds is similar to that of the native sequences, in terms of sugar ring and glycosidic linkage conformations. Finally, we also demonstrated that these derivatives are able to block the MK-stimulating effect on NIH3T3 cells growth.

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein-protein interactions with many cochaperones. One "hotspot" for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in class B J-domain proteins, such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70's EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70-CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70-DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein-protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded.

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein-protein interactions with many cochaperones. One "hotspot" for cochaperone binding is the EEVD motif, found at the extreme C terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat domain cochaperones, such as the E3 ubiquitin ligase CHIP. In addition, the EEVD motif also interacts with a structurally distinct domain that is present in class B J-domain proteins, such as DnaJB4. These observations suggest that CHIP and DnaJB4 might compete for binding to Hsp70's EEVD motif; however, the molecular determinants of such competition are not clear. Using a collection of EEVD-derived peptides, including mutations and truncations, we explored which residues are critical for binding to both CHIP and DnaJB4. These results revealed that some features, such as the C-terminal carboxylate, are important for both interactions. However, CHIP and DnaJB4 also had unique preferences, especially at the isoleucine position immediately adjacent to the EEVD. Finally, we show that competition between these cochaperones is important in vitro, as DnaJB4 limits the ubiquitination activity of the Hsp70-CHIP complex, whereas CHIP suppresses the client refolding activity of the Hsp70-DnaJB4 complex. Together, these data suggest that the EEVD motif has evolved to support diverse protein-protein interactions, such that competition between cochaperones may help guide whether Hsp70-bound proteins are folded or degraded.

Antimicrobial resistance threatens the eradication of infectious diseases and impairs the efficacy of available therapeutics. The bacterial SOS pathway is a conserved response triggered by genotoxic stresses and represents one of the principal mechanisms that lead to resistance. The RecA recombinase acts as a DNA-damage sensor inducing the autoproteolysis of the transcriptional repressor LexA, thereby derepressing SOS genes that mediate DNA repair, survival to chemotherapy, and hypermutation. The inhibition of such pathway represents a promising strategy for delaying the evolution of antimicrobial resistance. We report the identification, via llama immunization and phage display, of nanobodies that bind LexA with sub-micromolar affinity and block autoproteolysis, repressing SOS response in Escherichia coli. Biophysical characterization of nanobody-LexA complexes revealed that they act by trapping LexA in an inactive conformation and interfering with RecA engagement. Our studies pave the way to the development of new-generation antibiotic adjuvants for the treatment of bacterial infections., SCOPUS: ar.j, info:eu-repo/semantics/published

Targeting the protein-protein interaction (PPI) between the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and its repressor, Kelch-like ECH-associated protein 1 (Keap1), constitutes a promising strategy for treating diseases involving oxidative stress and inflammation. Here, a fragment-based drug discovery (FBDD) campaign resulted in novel, high-affinity (Ki = 280 nM), and cell-active noncovalent small-molecule Keap1-Nrf2 PPI inhibitors. We screened 2500 fragments using orthogonal assays-fluorescence polarization (FP), thermal shift assay (TSA), and surface plasmon resonance (SPR)-and validated the hits by saturation transfer difference (STD) NMR, leading to 28 high-priority hits. Thirteen co-structures showed fragments binding mainly in the P4 and P5 subpockets of Keap1's Kelch domain, and three fluorenone-based fragments featuring a novel binding mode were optimized by structure-based drug discovery. We thereby disclose several fragment hits, including their binding modes, and show how FBDD can be performed to find new small-molecule Keap1-Nrf2 PPI inhibitors.

14-3-3 proteins regulate many intracellular processes and their ability to bind in subtly different fashions to their numerous partner proteins provides attractive drug-targeting points for a range of diseases. Schnurri-3 is a suppressor of mouse bone formation and a candidate target for novel osteoporosis therapeutics, and thus it is of interest to determine whether it interacts with 14-3-3. In this work, potential 14-3-3 interaction sites on mammalian Schnurri-3 were identified by an in silico analysis of its protein sequence. Using fluorescence polarization, isothermal titration calorimetry and X-ray crystallography, it is shown that synthetic peptides containing either phosphorylated Thr869 or Ser542 can indeed interact with 14-3-3, with the latter capable of forming an interprotein disulfide bond with 14-3-3σ: a hitherto unreported phenomenon.

The deviant Ras activation machinery is found in approximately 30% of all human cancers. SOS1 is an important protagonist of this pathway that plays a key-role in aberrant cell proliferation and differentiation. Interaction of SOS1 with 14-3-3 proteins modulates SOS1 activity in Ras-MAPK signaling. In the present study, we analyze the 14-3-3/SOS1 protein-protein interaction (PPI) by different biochemical assays and report the high resolution crystal structure of a 13-mer motif of SOS1 bound to 14-3-3ζ. These structural and functional insights are important for the evaluation of this PPI interface for small-molecule stabilization as a new starting point for modulating the Ras-Raf-MAPK pathway.

The ubiquitin-specific protease 8 (USP8)/14-3-3 protein-protein interaction has recently been shown to exert a significant role in the pathogenesis of Cushing's disease (CD). USP8 is a deubiquitinase that prevents epidermal growth factor receptor (EGFR) degradation. Impairment of 14-3-3 binding leads to a higher deubiquitination of EGFR and results in a higher EGFR signaling and an increased production of adrenocorticotropic hormone. Here we report the high-resolution crystal structure of the 14-3-3 binding motif of USP8 surrounding Ser718 in complex with 14-3-3ζ and characterize the interaction with fluorescence polarization and isothermal titration calorimetry. Furthermore, we analyze the effect of USP8 mutations identified in CD on binding to 14-3-3.

Heat shock factor 1 (HSF1) is a stress-responsive transcription factor that regulates expression of protein chaperones and cell survival factors. In cancer, HSF1 plays a unique role, hijacking the normal stress response to drive a cancer-specific transcriptional program. These observations suggest that HSF1 inhibitors could be promising therapeutics. However, HSF1 is activated through a complex mechanism, which involves release of a negative regulatory domain, leucine zipper 4 (LZ4), from a masked oligomerization domain (LZ1-3), and subsequent binding of the oligomer to heat shock elements (HSEs) in HSF1-responsive genes. Recent crystal structures have suggested that HSF1 oligomers are held together by extensive, buried contact surfaces, making it unclear whether there are any possible binding sites for inhibitors. Here, we have rationally designed a series of peptide-based molecules based on the LZ4 and LZ1-3 motifs. Using a plate-based, fluorescence polarization (FP) assay, we identified a minimal region of LZ4 that suppresses binding of HSF1 to the HSE. Using this information, we converted this peptide into a tracer and used it to understand how binding of LZ4 to LZ1-3 suppresses HSF1 activation. Together, these results suggest a previously unexplored avenue in the development of HSF1 inhibitors. Furthermore, the findings highlight how native interactions can inspire the design of inhibitors for even the most challenging protein-protein interactions (PPIs).

In the scanning model of translation initiation, the decoding site and latch of the 40S subunit must open to allow the recruitment and migration of messenger RNA (mRNA); however, the precise molecular details for how initiation factors regulate mRNA accommodation into the decoding site have not yet been elucidated. Eukaryotic initiation factor (eIF) 3j is a subunit of eIF3 that binds to the mRNA entry channel and A-site of the 40S subunit. Previous studies have shown that a reduced affinity of eIF3j for the 43S preinitiation complex (PIC) occurs on eIF4F-dependent mRNA recruitment. Because eIF3j and mRNA bind anticooperatively to the 43S PIC, reduced eIF3j affinity likely reflects a state of full accommodation of mRNA into the decoding site. Here, we have used a fluorescence-based anisotropy assay to quantitatively determine how initiation components coordinate their activities to reduce the affinity of eIF3j during the recruitment of mRNA to the 43S PIC. Unexpectedly, we show that a full reduction in eIF3j affinity for the 43S PIC requires an ATP-dependent, but unwinding-independent, activity of eIF4A. This result suggests that in addition to its helicase activity, eIF4A uses the free energy of ATP binding and hydrolysis as a regulatory switch to control the conformation of the 43S PIC during mRNA recruitment. Therefore, our results define eIF4A as a universal initiation factor in cap-dependent translation initiation that functions beyond its role in RNA unwinding.

Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

The retinoblastoma (Rb) tumor suppressor protein negatively regulates cell proliferation by binding and inhibiting E2F transcription factors. Rb inactivation occurs in cancer cells upon cyclin-dependent kinase (Cdk) phosphorylation, which induces E2F release and activation of cell cycle genes. We present a strategy for activating phosphorylated Rb with molecules that bind Rb directly and enhance affinity for E2F. We developed a fluorescence polarization assay that can detect the effect of exogenous compounds on modulating affinity of Rb for the E2F transactivation domain. We found that a peptide capable of disrupting the compact inactive Rb conformation increases affinity of the repressive Rb-E2F complex. Our results demonstrate the feasibility of discovering novel molecules that target the cell cycle and proliferation through directly targeting Rb rather than upstream kinase activity.

The retinoblastoma (Rb) tumor suppressor protein negatively regulates cell proliferation by binding and inhibiting E2F transcription factors. Rb inactivation occurs in cancer cells upon cyclin-dependent kinase (Cdk) phosphorylation, which induces E2F release and activation of cell cycle genes. We present a strategy for activating phosphorylated Rb with molecules that bind Rb directly and enhance affinity for E2F. We developed a fluorescence polarization assay that can detect the effect of exogenous compounds on modulating affinity of Rb for the E2F transactivation domain. We found that a peptide capable of disrupting the compact inactive Rb conformation increases affinity of the repressive Rb-E2F complex. Our results demonstrate the feasibility of discovering novel molecules that target the cell cycle and proliferation through directly targeting Rb rather than upstream kinase activity.

Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells and 72 h in NIH3T3 cells and only at 92 h in HEK293 cells. L6 showed a marked decrease in membrane fluidity at 72 h and starts to increase at 92 h. As expected, NIH3T3 cells have more rigid membrane at earlier time points. On the other hand, neurons tend to have the highest membrane fluidity at early time points emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein.

Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein.

The specialized light organ of the ponyfish supports the growth of the bioluminescent symbiont Photobacterium leiognathi. The bioluminescence of P. leiognathi is generated within a heteromeric protein complex composed of the bacterial luciferase and a 20-kDa lumazine binding protein (LUMP), which serves as a Förster resonance energy transfer (FRET) acceptor protein, emitting a cyan-colored fluorescence with an unusually long excited state lifetime of 13.6 ns. The long fluorescence lifetime and small mass of LUMP are exploited for the design of highly optimized encoded sensors for quantitative fluorescence anisotropy (FA) measurements of protein hydrodynamics. In particular, large differences in the FA values of the free and target-bound states of LUMP fusions appended with capture sequences of up to 20 kDa are used in quantitative FA imaging and analysis of target proteins. For example, a fusion protein composed of LUMP and a 5-kDa G protein binding domain is used as an FA sensor to quantify the binding of the GTP-bound cell division control protein 42 homolog (Cdc42) (21 kDa) in solution and within Escherichia coli. Additionally, the long fluorescence lifetime and the surface-bound fluorescent cofactor 6,7-dimethyl-8- (1'-dimethyl-ribityl) lumazine in LUMP are utilized in the design of highly optimized FRET probes that use Venus as an acceptor probe. The efficiency of FRET in a zero-length LUMP-Venus fusion is 62% compared to ∼ 31% in a related CFP-Venus fusion. The improved FRET efficiency obtained by using LUMP as a donor probe is used in the design of a FRET-optimized genetically encoded LUMP-Venus substrate for thrombin.

The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.

The archetypical fluorescent nucleoside analog, 2-aminopurine (2Ap), has been used in countless assays, though it suffers from very low quantum yield, especially when included in double strands, and from the fact that its residual emission frequently does not represent biologically relevant conformations. To conquer 2Ap's deficiencies, deoxythienoguanosine (d(th)G) was recently developed. Here, steady-state and time-resolved fluorescence spectroscopy was used to compare the ability of 2Ap and d(th)G, to substitute and provide relevant structural and dynamical information on a key G residue in the (-) DNA copy of the HIV-1 primer binding site, (-)PBS, both in its stem loop conformation and in the corresponding (-)/(+)PBS duplex. In contrast to 2Ap, this fluorescent nucleoside when included in (-)PBS or (-)/(+)PBS duplex fully preserves their stability and exhibits a respectable quantum yield and a simple fluorescence decay, with marginal amounts of dark species. In further contrast to 2Ap, the fluorescently detected d(th)G species reflect the predominantly populated G conformers, which allows exploring their relevant dynamics. Being able to perfectly substitute G residues, d(th)G will transform nucleic acid biophysics by allowing, for the first time, to selectively and faithfully monitor the conformations and dynamics of a given G residue in a DNA sequence.

The action of drugs an living systems is a membrane-based process. Their interaction at the membrane surface/interior and transport through it induces a change in the physical properties of the membrane, of which fluidity is among the most important ones. The lipophilic drugs can affect this fluidity directly through their incorporation into the lipid matrix, whereas the action of the hydrophilic drugs is confined mostly at the membrane-water interface. Using the liposomal membranes of dipalmitoyl phosphatidyl choline, a comparative study of the effect of a hydrophilic drug, chloroquine sulphate, and a lipophilic drug, hydrocortisone, on the membrane fluidity, measured in terms of anisotropy, is presented. From the membrane anisotropy-temperature curve, the associated enthalpy change has been calculated. Results show that the two drugs affect the membrane order in two different ways, indicating that there is a difference in the interaction of these two drugs with the liposomal membrane at the molecular level.

The action of drugs an living systems is a membrane-based process. Their interaction at the membrane surface/interior and transport through it induces a change in the physical properties of the membrane, of which fluidity is among the most important ones. The lipophilic drugs can affect this fluidity directly through their incorporation into the lipid matrix, whereas the action of the hydrophilic drugs is confined mostly at the membrane-water interface. Using the liposomal membranes of dipalmitoyl phosphatidyl choline, a comparative study of the effect of a hydrophilic drug, chloroquine sulphate, and a lipophilic drug, hydrocortisone, on the membrane fluidity, measured in terms of anisotropy, is presented. From the membrane anisotropy-temperature curve, the associated enthalpy change has been calculated. Results show that the two drugs affect the membrane order in two different ways, indicating that there is a difference in the interaction of these two drugs with the liposomal membrane at the molecular level.

O-GlcNAcylation is a reversible type of serine/threonine glycosylation on nucleocytoplasmic proteins in metazoa. Various genetic approaches in several animal models have revealed that O-GlcNAcylation is essential for embryogenesis. However, the dynamic changes in global O-GlcNAcylation and the underlying mechanistic biology linking them to embryonic development is not understood. One of the limiting factors towards characterizing changes in O-GlcNAcylation has been the limited specificity of currently available tools to detect this modification. In the present study, harnessing the unusual properties of an O-GlcNAcase (OGA) mutant that binds O-GlcNAc (O-N-acetylglucosamine) sites with nanomolar affinity, we uncover changes in protein O-GlcNAcylation as a function of Drosophila development.

目的建立一種利用雙堿基延伸法與熒光偏振技術(Fluorescence polarization,FP)快速檢測甲型H1N1流感病毒NA基因耐藥突變位點的新方法。方法從GenBank隨機下載30條甲型H1N1的NA(neuraminidase)基因序列,通過MEGA分析,利用Primer Premier軟件設計3條特異引物,其中一對引物通過RT-PCR用于擴增含有耐藥位點H275Y的NA基因,第3條引物是應用雙堿基延伸終止法與熒光偏振技術檢測NA基因對達菲類藥物耐藥位點H275Y的突變情況,再隨機選取50株甲型H1N1流感病毒進行檢測,與測序結果進行比對,驗證檢測結果的準確性。結果 50株甲型H1N1流感病毒的NA蛋白第275位氨基酸均為組氨酸,未發生H Y的替換,在靈敏度與特異性方面,與傳統的基因測序結果一致性均達100%。結論本方法操作簡便、敏感性及特異性高、判讀結果直觀明確、檢測費用低,能實現對耐藥位點突變的快速、準確檢測,可應用于與SNP相關疾病的快速分型和突變位點的實時監測和臨床檢測。A novel method based on two-base extension termination and fluorescence polarization was established for the detection of oseltamivir-resistant by a histidine to tyrosine mutation of residue 275 of the neuraminidase (NA) gene of influenza A/H1N1 2009. Firstly, 30 neuraminidase gene sequences coding for influenza A/H1N1 were randomly downloaded from GenBank and analyzed by software MEGA. Three specific primers for NA gene of influenza A/H1N1 were designed by Primer Premier--one pair of primer was used to amplify the NA gene containing the His275Tyr mutation region by RT-PCR, and the third primer was detected the H275Y mutation of oseltamivir-resistance by two-base extension termination and fluorescence polarization. Additionally, 50 sample of influenza A/H1N1 were tested using Sanger sequencing, which is the reference method for the novel one to verify the accuracy. Results indicated that the 275th amino acids of NA protein in 50 influenza A/H1N1 were histidine, while no replace found between H and Y. In comparison to Sanger sequencing, the sensitivity and specificity of the novel method were 100% (50/50) and 100% (50/50) respectively. The novel method is simpler, high in sensitivity and specificity, and low-cost. It can be used to achieve the rapid and accurate detection of drug-resistant mutation and applied into the other SNP related disease, which also need the fast classification, real-time monitoring and clinical testing of mutation.

Many classes of S-adenosylmethionine (SAM)-binding RNAs and proteins are of interest as potential drug targets in diverse therapeutic areas, from infectious diseases to cancer. In the former case, the SAM-I riboswitch is an attractive target because this structured RNA element is found only in bacterial mRNAs and regulates multiple genes in several human pathogens. Here, we describe the synthesis of stable and fluorescent analogs of SAM in which the fluorophore is introduced through a functionalizable linker to the ribose. A Cy5-labeled SAM analog was shown to bind several SAM-I riboswitches via in-line probing and fluorescence polarization assays, including one from Staphylococcus aureus that controls the expression of SAM synthetase in this organism. A fluorescent ligand displacement assay was developed and validated for high-throughput screening of compounds to target the SAM-I riboswitch class.

Many classes of S-adenosylmethionine (SAM)-binding RNAs and proteins are of interest as potential drug targets in diverse therapeutic areas, from infectious diseases to cancer. In the former case, the SAM-I riboswitch is an attractive target because this structured RNA element is found only in bacterial mRNAs and regulates multiple genes in several human pathogens. Here, we describe the synthesis of stable and fluorescent analogs of SAM in which the fluorophore is introduced through a functionalizable linker to the ribose. A Cy5-labeled SAM analog was shown to bind several SAM-I riboswitches via in-line probing and fluorescence polarization assays, including one from Staphylococcus aureus that controls the expression of SAM synthetase in this organism. A fluorescent ligand displacement assay was developed and validated for high-throughput screening of compounds to target the SAM-I riboswitch class.

Nuclear receptor coregulators are master regulators of transcription and selectively interact with the vitamin D receptor (VDR) to modulate cell differentiation, cell proliferation and calcium homeostasis. Herein, we report the syntheses and evaluation of highly potent and selective VDR-coactivator inhibitors based on a recently identified 3-indolylmethanamine scaffold. The most active compound, PS121912, selectively inhibited VDR-mediated transcription among eight other nuclear receptors tested. PS121912 is also selectively disrupting the binding between VDR and the third nuclear receptor interaction domain of the coactivator SRC2. Genetic studies revealed that PS121912 behaves like a VDR antagonist by repressing 1,25-(OH)2D3 activated gene transcription. In addition, PS121912 induced apoptosis in HL-60.

Anatoxin-a (ATX) is a potent neurotoxin produced by several species of Anabaena spp. Cyanobacteria blooms around the world have been increasing in recent years; therefore, it is urgent to develop sensitive techniques that unequivocally confirm the presence of these toxins in fresh water and cyanobacterial samples. In addition, the identification of different ATX analogues is essential to later determine its toxicity. In this paper we designed a fluorescent polarization (FP) method to detect ATXs in water samples. A nicotinic acetylcholine receptor (nAChR) labeled with a fluorescein derivative was used to develop this assay. Data showed a direct relationship between the amount of toxin in a sample and the changes in the polarization degree of the emitted light by the labeled nAChR, indicating an interaction between the two molecules. This method was used to measure the amount of ATX in three Anabaena spp. cultures. Results indicate that it is a good method to show ATXs presence in algal samples. In order to check the toxin profile of Anabaena cultures a LC-MS/MS method was also developed. Within this new method, ATX-a, retention time (RT) 5 min, and three other molecules with a mass m/z 180.1 eluting at 4.14 min, 5.90 min and 7.14 min with MS/MS spectra characteristic of ATX toxin group not previously identified were detected in the Anabaena spp. cultures. These ATX analogues may have an important role in the toxicity of the sample

Sister-chromatid cohesion is established during S phase when Eco1 acetylates cohesin. In budding yeast, Eco1 activity falls after S phase due to Cdk1-dependent phosphorylation, which triggers ubiquitination by SCF(Cdc4). We show here that Eco1 degradation requires the sequential actions of Cdk1 and two additional kinases, Cdc7-Dbf4 and the GSK-3 homolog Mck1. These kinases recognize motifs primed by previous phosphorylation, resulting in an ordered sequence of three phosphorylation events on Eco1. Only the latter two phosphorylation sites are spaced correctly to bind Cdc4, resulting in strict discrimination between phosphates added by Cdk1 and by Cdc7. Inhibition of Cdc7 by the DNA damage response prevents Eco1 destruction, allowing establishment of cohesion after S phase. This elaborate regulatory system, involving three independent kinases and stringent substrate selection by a ubiquitin ligase, enables robust control of cohesion establishment during normal growth and after stress.

Sister-chromatid cohesion is established during S phase when Eco1 acetylates cohesin. In budding yeast, Eco1 activity falls after S phase due to Cdk1-dependent phosphorylation, which triggers ubiquitination by SCF(Cdc4). We show here that Eco1 degradation requires the sequential actions of Cdk1 and two additional kinases, Cdc7-Dbf4 and the GSK-3 homolog Mck1. These kinases recognize motifs primed by previous phosphorylation, resulting in an ordered sequence of three phosphorylation events on Eco1. Only the latter two phosphorylation sites are spaced correctly to bind Cdc4, resulting in strict discrimination between phosphates added by Cdk1 and by Cdc7. Inhibition of Cdc7 by the DNA damage response prevents Eco1 destruction, allowing establishment of cohesion after S phase. This elaborate regulatory system, involving three independent kinases and stringent substrate selection by a ubiquitin ligase, enables robust control of cohesion establishment during normal growth and after stress.

PSD-93 (chapsyn-110, DLG2) is a member of the family of membrane-associated guanylate kinase (MAGUK) proteins. The MAGUK proteins are involved in receptor localization and signalling pathways. The best characterized MAGUK protein, PSD-95, is known to be involved in NMDA receptor signalling via its PDZ domains. The PDZ domains of PSD-95 and PSD-93 are structurally very similar, but relatively little is known about the function of PSD-93. PSD-93 has been suggested to interact with GluD2 from the family of ionotropic glutamate receptors. Here, the interactions of four residues (GTSI) representing the extreme C-terminus of GluD2 with PSD-93 PDZ1 have been investigated in the crystalline phase. Two different binding modes of these residues were observed, suggesting that the peptide is not tightly bound to PSD-93 PDZ1. In accordance, the two N-terminal PSD-93 PDZ domains show no appreciable binding affinity for a GluD2-derived C-terminal octapeptide, whereas micromolar affinity was observed for a GluN2B-derived C-terminal octapeptide. This indicates that if present, the interactions between GluD2 and PSD-93 involve more than the extreme terminus of the receptor. In contrast, the tumour-suppressor protein SCRIB PDZ3 shows low micromolar affinity towards the GluD2-derived octapeptide, which is in agreement with previous findings using high-throughput assays.

Cílem práce bylo ověřit možnost využití fluorescenčních sond ke studiu vlivu orientace a uspořádání polymerních vodivých systémů PEDOT/PSS na elektrický odpor tenké vrstvy., Properties of thin layers of PEDOT/PSS on glass was studied using fluorescent probes polarization measurement. This permit assess of deposition methods and their impact on the supramolecular structure of polymer complex and also the electrical resistance of layers., Katedra polygrafie a fotofyziky, ing. T. Syrový,Ph.D. - Jaké parametry ovlivňují tloušku vrsty připravené technikou Spin Coating? Mohlo k vymizení fluorescence dojít v důsledku odpaření rozpouštědla? doc. Ing. P. Němec, Ph.D. - Jak jste vzorky skladoval? Jak víte, že po 4 dnech nevykazují fluorescenci? Jakým způsobem jste definoval anizotropii? Jakou experimentální technikou by se dalo pohlédnout na reálnou pozici zabudované sondy?

PSD-93 (chapsyn-110, DLG2) is a member of the family of membrane-associated guanylate kinase (MAGUK) proteins. The MAGUK proteins are involved in receptor localization and signalling pathways. The best characterized MAGUK protein, PSD-95, is known to be involved in NMDA receptor signalling via its PDZ domains. The PDZ domains of PSD-95 and PSD-93 are structurally very similar, but relatively little is known about the function of PSD-93. PSD-93 has been suggested to interact with GluD2 from the family of ionotropic glutamate receptors. Here, the interactions of four residues (GTSI) representing the extreme C-terminus of GluD2 with PSD-93 PDZ1 have been investigated in the crystalline phase. Two different binding modes of these residues were observed, suggesting that the peptide is not tightly bound to PSD-93 PDZ1. In accordance, the two N-terminal PSD-93 PDZ domains show no appreciable binding affinity for a GluD2-derived C-terminal octapeptide, whereas micromolar affinity was observed for a GluN2B-derived C-terminal octapeptide. This indicates that if present, the interactions between GluD2 and PSD-93 involve more than the extreme terminus of the receptor. In contrast, the tumour-suppressor protein SCRIB PDZ3 shows low micromolar affinity towards the GluD2-derived octapeptide, which is in agreement with previous findings using high-throughput assays.

We present a novel homogeneous (mix-incubate-read) droplet microfluidic assay for specific protein detection in picoliter volumes by fluorescence polarization (FP), for the first time demonstrating the use of FP in a droplet microfluidic assay. Using an FP-based assay we detect streptavidin concentrations as low as 500?nM and demonstrate that an FP assay allows us to distinguish droplets containing 5?mu M rabbit IgG from droplets without IgG with an accuracy of 95%, levels relevant for hybridoma screening. This adds to the repertoire of droplet assay techniques a direct protein detection method which can be performed entirely inside droplets without the need for labeling of the analyte molecules., QC 20110225. Updated from manuscript to article in journal.

Cellular membranes are heterogeneous in composition, and the prevailing theory holds that the structures responsible for this heterogeneity in vivo are small structures (10-200 nm), sterol- and sphingolipid-enriched, of different sizes, highly dynamic denominated rafts. Rafts are postulated to be platforms, which by sequestering different membrane components can compartmentalize cellular processes and regulate signaling pathways. Despite an enormous effort in this area, the existence of these domains is still under debate due to the characteristics of the structures itself: small in size and highly mobile, which from the technical point of view implies using techniques with high spatial and temporal resolution. In this report we measured rapid fluctuations of the normalized ratio of the emission intensity at two wavelengths of Laurdan, a membrane fluorescent dye sensitive to local membrane packing. We observed generalized polarization fluctuations in the plasma membrane of intact rabbit erythrocytes and Chinese hamster ovary cells that can be explained by the existence of tightly packed micro-domains moving in a more fluid background phase. These structures, which display different lipid packing, have different sizes; they are found in the same cell and in the entire cell population. The small size and characteristic high lipid packing indicate that these micro-domains have properties that have been proposed for lipid rafts.

Cellular membranes are heterogeneous in composition, and the prevailing theory holds that the structures responsible for this heterogeneity in vivo are small structures (10-200 nm), sterol- and sphingolipid-enriched, of different sizes, highly dynamic denominated rafts. Rafts are postulated to be platforms, which by sequestering different membrane components can compartmentalize cellular processes and regulate signaling pathways. Despite an enormous effort in this area, the existence of these domains is still under debate due to the characteristics of the structures itself: small in size and highly mobile, which from the technical point of view implies using techniques with high spatial and temporal resolution. In this report we measured rapid fluctuations of the normalized ratio of the emission intensity at two wavelengths of Laurdan, a membrane fluorescent dye sensitive to local membrane packing. We observed generalized polarization fluctuations in the plasma membrane of intact rabbit erythrocytes and Chinese hamster ovary cells that can be explained by the existence of tightly packed micro-domains moving in a more fluid background phase. These structures, which display different lipid packing, have different sizes; they are found in the same cell and in the entire cell population. The small size and characteristic high lipid packing indicate that these micro-domains have properties that have been proposed for lipid rafts.

Many areas of biological research increasingly perform large-scale analyses. In genomics the entire gene repertoire of an organism is analyzed. Proteomics attempts to understand the function and expression patterns of all proteins in a cell or organism. Cell biologists study large numbers of single cells to understand the heterogeneity of cell populations. In biotechnology and synthetic biology researchers search for new functional biomolecules in large libraries of biomolecular diversity e.g. for uses in medicine or bioprocessing. More and more all of these fields employ high throughput methods to achieve the scale of analysis necessary. Miniaturization and parallelization provide routes towards high throughput analysis, which have proven successful for microelectronics as well as for DNA sequencing. For the analysis of cells and biomolecules, native to an aqueous environment, miniaturization and parallelization hinges on the handling and parallel processing of very small amounts of water. Droplet microfluidics utilizes stable picoliter (water) droplets contained in inert fluorinated oils as compartments in which to isolate and analyze cells, molecules or reactions. These droplets can be manipulated, detected and analyzed at rates of thousands per second in microfluidic modules combining top-down microscale fabrication with the self-assembly of droplets of exact size. The studies constituting this thesis involve new droplet based biomolecular and single cell assays, manipulation techniques and device fabrication methods to extend the capabilities of droplet microfluidics for high throughput biological analysis. The first paper in the thesis describes a novel analysis method for studying the low abundant biomarkers present on the surface individual cells at resolutions not available by flow cytometry, the current gold standard of single cell analysis. The use of a fluorescent optical dye code enabled the analysis of several single cell samples concurrently, QC 20110225

The nuclear pore complex (NPC) perforates the nuclear envelope to facilitate selective transport between nucleus and cytoplasm. The NPC is composed of multiple copies of ∼30 different proteins, termed nucleoporins, whose arrangement within the NPC is an important unsolved puzzle in structural biology. Various alternative models for NPC architecture have been proposed but not tested experimentally in intact NPCs. We present a method using polarized fluorescence microscopy to investigate nucleoporin orientation in live yeast and mammalian cells. Our results support an arrangement of both yeast Nic96 and human Nup133-Nup107 in which their long axes are approximately parallel to the nuclear envelope plane. The method we developed can complement X-ray crystallography and electron microscopy to generate a high-resolution map of the entire NPC, and may be able to monitor nucleoporin rearrangements during nucleocytoplasmic transport and NPC assembly. This strategy can also be adapted for other macromolecular machines.

To comprehend the molecular processes that lead to the Fas death receptor clustering in lipid rafts, a 21-mer peptide corresponding to its single transmembrane domain (TMD) was reconstituted into mammalian raft model membranes composed of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol. The peptide membrane lateral organization and dynamics, and its influence on membrane properties, were studied by steady-state and time-resolved fluorescence techniques and by attenuated total reflection Fourier transformed infrared spectroscopy. Our results show that Fas TMD is preferentially localized in liquid-disordered membrane regions and undergoes a strong reorganization as the membrane composition is changed toward the liquid-ordered phase. This results from the strong hydrophobic mismatch between the length of the peptide hydrophobic stretch and the hydrophobic thickness of liquid-ordered membranes. The stability of nonclustered Fas TMD in liquid-disordered domains suggests that its sequence may have a protective function against nonligand-induced Fas clustering in lipid rafts. It has been reported that ceramide induces Fas oligomerization in lipid rafts. Here, it is shown that neither Fas TMD membrane organization nor its conformation is affected by ceramide. These results are discussed within the framework of Fas membrane signaling events., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

The nuclear pore complex (NPC) perforates the nuclear envelope to facilitate selective transport between nucleus and cytoplasm. The NPC is composed of multiple copies of ∼30 different proteins, termed nucleoporins, whose arrangement within the NPC is an important unsolved puzzle in structural biology. Various alternative models for NPC architecture have been proposed but not tested experimentally in intact NPCs. We present a method using polarized fluorescence microscopy to investigate nucleoporin orientation in live yeast and mammalian cells. Our results support an arrangement of both yeast Nic96 and human Nup133-Nup107 in which their long axes are approximately parallel to the nuclear envelope plane. The method we developed can complement X-ray crystallography and electron microscopy to generate a high-resolution map of the entire NPC, and may be able to monitor nucleoporin rearrangements during nucleocytoplasmic transport and NPC assembly. This strategy can also be adapted for other macromolecular machines.

To comprehend the molecular processes that lead to the Fas death receptor clustering in lipid rafts, a 21-mer peptide corresponding to its single transmembrane domain (TMD) was reconstituted into mammalian raft model membranes composed of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol. The peptide membrane lateral organization and dynamics, and its influence on membrane properties, were studied by steady-state and time-resolved fluorescence techniques and by attenuated total reflection Fourier transformed infrared spectroscopy. Our results show that Fas TMD is preferentially localized in liquid-disordered membrane regions and undergoes a strong reorganization as the membrane composition is changed toward the liquid-ordered phase. This results from the strong hydrophobic mismatch between the length of the peptide hydrophobic stretch and the hydrophobic thickness of liquid-ordered membranes. The stability of nonclustered Fas TMD in liquid-disordered domains suggests that its sequence may have a protective function against nonligand-induced Fas clustering in lipid rafts. It has been reported that ceramide induces Fas oligomerization in lipid rafts. Here, it is shown that neither Fas TMD membrane organization nor its conformation is affected by ceramide. These results are discussed within the framework of Fas membrane signaling events., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

Ribonucleotide reductase (RNR) is a viable target for new drugs against the causative agent of tuberculosis, Mycobacterium tuberculosis. Previous work has shown that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. Here the synthesis and binding affinity, evaluated by competitive fluorescence polarization, of several truncated and N-protected peptides are described. The protected single-amino acid Fmoc-Trp shows binding affinity comparable to the N-acetylated heptapeptide, making it an attractive candidate for further development of non-peptidic RNR inhibitors.

We present a novel homogeneous assay for detecting biomolecule interactions in microdroplets by fluorescence polarization (FP) for the first time. The FP assay allows the detection of target biomolecules directly after incubation without removing the detection reagent by separation or washing, making the assay amenable to automation. Using this assay we evaluate protein-protein and drug-DNA interactions. We detect these interactions at concentrations as low as 100nM and 69 pM respectively. This is a proof-of-concept homogeneous labeling assay in droplets for detecting bio-macromolecules., QC 20140815

Oritavancin, a lipoglycopeptide with marked bactericidal activity against vancomycin-resistant Staphylococcus aureus and enterococci, induces calcein release from CL:POPE and POPG:POPE liposomes, an effect enhanced by an increase in POPG:POPE ratio and decreased when replacing POPG by DPPG (Domenech et al., Biochim Biophys Acta. 2009; 1788:1832-40). Using vesicles prepared from lipids extracted from Staphylococcus aureus, we showed that oritavancin induces holes, erosion of the edges, and decrease of the thickness of the supported lipid bilayers (Atomic Force Microscopy). Oritavancin also induced an increase of membrane permeability (calcein release) on a time- and dose-dependent manner. These effects were probably related to the ability of the drug to bind to lipid bilayers as showed by 8-anilino-1- naphthalene sulfonic acid (ANS) assay. Interaction of oritavancin with phospholipids at the level of their glycerol backbone and hydrophobic domain was studied by monitoring changes of Laurdan excitation generalized polarization (GP(ex)) and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy upon temperature increase. Oritavancin increased GP(ex) values and the transition temperature, indicating a more ordered structure at the level of the glycerol backbone. Oritavancin slightly decreased DPH fluorescence depolarization intensities, suggesting an increase in fluidity at the level of acyl chains. Together, our data confirm the interaction of oritavancin with lipids and the potential role of a rigidifying effect at the level of glycerol backbone for membrane permeabilization. This work shows how AFM and biophysical methods may help in characterizing drug-membrane interactions, and sheds further light on the mode of action of oritavancin.

Current methods for locating and recovering submerged oil spills are inadequate. Detection methods are often improvised on-scene, and recovery techniques are labor intensive and not always successful. The U.S. Coast Guard Research and Development Center has embarked on a multi-year project to develop a complete approach for dealing with spills of submerged oils. This report describes the assessment of detection techniques using sonar, laser fluorometry, real-time mass spectrometry, and in-situ fluorometry to locate oil sitting on the sea floor. Evaluation of four proof-of-concept devices was conducted at the Oil and Hazardous Material Simulated Environmental Test Tank (OHMSETT), now called The National Oil Spill Response Test Facility, in Leonardo, NJ, between November 2007 and February 2008. Further testing of two of these prototype devices, plus three additional detection systems, was conducted at OHMSETT in January 2009. This report contains the results of these tests and recommendations for Federal On-scene Coordinators when responding to spills of heavy oil (contained in Appendix E)., Prepared in collaboration with Potomac Management Group, Inc., McLean, VA. The original document contains color images.

This thesis describes structural and binding studies in enzymes from two different organisms: ribonucleotide reductase from Mycobacterium tuberculosis (RNR) and lipase A from Candida antarctica (CalA). RNR is viable as a target for new drugs against the causative agent of tuberculosis. The biologically active form of RNR is a heterotetramer with an α2β2 substructure. Here we show that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. An N-terminal truncation, an alanine scan and a novel statistical molecular design approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied. A full-length acetylated heptapeptide was necessary for inhibition, and Trp5 and Phe7 were also essential. Exchanging the acetyl for the N-terminal Fmoc protective-group increased the binding potency ten-fold. Based on this, several truncated and N-protected peptides were evaluated in a competitive fluorescence polarization assay. The single-amino acid Fmoc-Trp inhibits the RNR holoenzyme formation with a dissociation constant of 12µM, making it an attractive candidate for further development of non-peptidic inhibitors Lipases are enzymes with major biotechnological applications. We report the x-ray structure of CalA, the first member of a novel family of lipases. The fold includes a well-defined lid as well as a classical α/β hydrolase domain. The structure is that of the closed/inactive state of the enzyme, but loop movements near Phe431 will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during i

This thesis describes structural and binding studies in enzymes from two different organisms: ribonucleotide reductase from Mycobacterium tuberculosis (RNR) and lipase A from Candida antarctica (CalA). RNR is viable as a target for new drugs against the causative agent of tuberculosis. The biologically active form of RNR is a heterotetramer with an α2β2 substructure. Here we show that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. An N-terminal truncation, an alanine scan and a novel statistical molecular design approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied. A full-length acetylated heptapeptide was necessary for inhibition, and Trp5 and Phe7 were also essential. Exchanging the acetyl for the N-terminal Fmoc protective-group increased the binding potency ten-fold. Based on this, several truncated and N-protected peptides were evaluated in a competitive fluorescence polarization assay. The single-amino acid Fmoc-Trp inhibits the RNR holoenzyme formation with a dissociation constant of 12µM, making it an attractive candidate for further development of non-peptidic inhibitors Lipases are enzymes with major biotechnological applications. We report the x-ray structure of CalA, the first member of a novel family of lipases. The fold includes a well-defined lid as well as a classical α/β hydrolase domain. The structure is that of the closed/inactive state of the enzyme, but loop movements near Phe431 will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during i

We demonstrate the use of statistical molecular design (SMD) in the selection of peptide libraries aimed to systematically investigate antigen-antibody binding spaces. Earlier, we derived two novel antibodies by mutating the complementarity-determining region of the anti-p24 (HIV-1) single chain Fv antibody, CB4-1 that had lost their affinity for a p24 epitope-homologous peptide by 8- and 60-fold. The present study was devoted to explore how peptide libraries can be designed under experimental design criteria for effective screening of peptide antigens. Several small peptide-antigen libraries were selected using SMD principles and their activities were evaluated by their binding to SPOT-synthesized peptide membranes and by fluorescence polarization (FP). The approach was able to reveal the most critical residues required for antigen binding, and finally to increase the binding activity by proper modifications of amino acids in the peptide antigen. A model of the active peptide binding pocket formed by the mutated scFv and the antigen was compatible with the information gained from the experimental data. Our results suggest that SMD approaches can be used to explore peptide antigen features essential for their interactions with antibodies.

The interactions between a drug and lipids may be critical for the pharmacological activity. We previously showed that the ability of a fluoroquinolone antibiotic, the ciprofloxacin, to induce disorder and modify the orientation of the acyl chains is related to its propensity to be expelled from a monolayer upon compression [1]. Here, we compared the binding of ciprofloxacin on DPPC and DPPG liposomes (or mixtures of phospholipids [DOPC:DPPC], and [DOPC:DPPG]) using quasi-elastic light scattering and steady-state fluorescence anisotropy. We also investigated ciprofloxacin effects on the transition temperature (T(m)) of lipids and on the mobility of phosphate head groups using Attenuated Total Reflection Fourier Transform Infrared-Red Spectroscopy (ATR-FTIR) and (31)P Nuclear Magnetic Resonance (NMR) respectively. In the presence of ciprofloxacin we observed a dose-dependent increase of the size of the DPPG liposomes whereas no effect was evidenced for DPPC liposomes. The binding constants K(app) were in the order of 10(5) M(-1) and the affinity appeared dependent on the negative charge of liposomes: DPPG>DOPC:DPPG (1:1; M:M)>DPPC>DOPC:DPPC (1:1; M:M). As compared to the control samples, the chemical shift anisotropy (Deltasigma) values determined by (31)P NMR showed an increase of 5 and 9 ppm for DPPC:CIP (1:1; M:M) and DPPG:CIP (1:1; M:M) respectively. ATR-FTIR experiments showed that ciprofloxacin had no effect on the T(m) of DPPC but increased the order of the acyl chains both below and above this temperature. In contrast, with DPPG, ciprofloxacin induced a marked broadening effect on the transition with a decrease of the acyl chain order below its T(m) and an increase above this temperature. Altogether with the results from the conformational analysis, these data demonstrated that the interactions of ciprofloxacin with lipids depend markedly on the nature of their phosphate head groups and that the major effects of ciprofloxacin on bacterial membranes should be

The interactions between a drug and lipids may be critical for the pharmacological activity. We previously showed that the ability of a fluoroquinolone antibiotic, ciprofloxacin, to induce disorder and modify the orientation of the acyl chains is related to its propensity to be expelled from a monolayer upon compression [1]. Here, we compared the binding of ciprofloxacin on DPPC and DPPG liposomes (or mixtures of phospholipids [DOPC:DPPC], and [DOPC:DPPG]) using quasi-elastic light scattering and steady-state fluorescence anisotropy. We also investigated ciprofloxacin effects on the transition temperature (T(m)) of lipids and on the mobility of phosphate head groups using Attenuated Total Reflection Fourier Transform Infrared-Red Spectroscopy (ATR-FTIR) and (31)P Nuclear Magnetic Resonance (NMR) respectively. In the presence of ciprofloxacin we observed a dose-dependent increase of the size of the DPPG liposomes whereas no effect was evidenced for DPPC liposomes. The binding constants K(app) were in the order of 10(5) M(-1) and the affinity appeared dependent on the negative charge of liposomes: DPPG>DOPC:DPPG (1:1; M:M)>DPPC>DOPC:DPPC (1:1; M:M). As compared to the control samples, the chemical shift anisotropy (Deltasigma) values determined by (31)P NMR showed an increase of 5 and 9 ppm for DPPC:CIP (1:1; M:M) and DPPG:CIP (1:1; M:M) respectively. ATR-FTIR experiments showed that ciprofloxacin had no effect on the T(m) of DPPC but increased the order of the acyl chains both below and above this temperature. In contrast, with DPPG, ciprofloxacin induced a marked broadening effect on the transition with a decrease of the acyl chain order below its T(m) and an increase above this temperature. Altogether with the results from the conformational analysis, these data demonstrated that the interactions of ciprofloxacin with lipids depend markedly on the nature of their phosphate head groups and that ciprofloxacin interacts preferentially with anionic lipid compounds, lik, Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published