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1.
J Chem Theory Comput ; 15(9): 5087-5102, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31402660

RESUMO

Circular dichroism (CD) spectroscopy is a highly sensitive but low-resolution technique to study the structure of proteins. Combined with molecular modeling or other complementary techniques, CD spectroscopy can provide essential information at higher resolution. To this end, we introduce a new computational method to calculate the electronic circular dichroism spectra of proteins from a structural model or ensemble using the average secondary structure composition and a precalculated set of basis spectra. The method is designed for model validation to estimate the error of a given protein structural model based on the measured CD spectrum. We compared the predictive power of our method to that of existing algorithms, namely, DichroCalc and PDB2CD, and found that it predicts CD spectra more accurately. Our results indicate that the derived basis sets are robust to both experimental errors in the reference spectra and the choice of the secondary structure classification algorithm. For over 80% of the globular reference proteins, our basis sets accurately predict the experimental spectrum solely from their secondary structure composition. For the remaining 20%, correcting for intensity normalization considerably improves the prediction power. Additionally, we show that the predictions for short peptides and an example complex of intrinsically disordered proteins strongly benefit from accounting for side-chain contributions and structural flexibility.


Assuntos
Dicroísmo Circular , Proteínas/química , Algoritmos , Modelos Moleculares , Conformação Proteica , Teoria Quântica
2.
PLoS One ; 14(6): e0216833, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31173589

RESUMO

Schwann cells myelinate selected axons in the peripheral nervous system (PNS) and contribute to fast saltatory conduction via the formation of compact myelin, in which water is excluded from between tightly adhered lipid bilayers. Peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS), are incurable demyelinating conditions that result in pain, decrease in muscle mass, and functional impairment. Many Schwann cell proteins, which are directly involved in the stability of compact myelin or its development, are subject to mutations linked to these neuropathies. The most abundant PNS myelin protein is protein zero (P0); point mutations in this transmembrane protein cause CMT subtype 1B and DSS. P0 tethers apposing lipid bilayers together through its extracellular immunoglobulin-like domain. Additionally, P0 contains a cytoplasmic tail (P0ct), which is membrane-associated and contributes to the physical properties of the lipid membrane. Six CMT- and DSS-associated missense mutations have been reported in P0ct. We generated recombinant disease mutant variants of P0ct and characterized them using biophysical methods. Compared to wild-type P0ct, some mutants have negligible differences in function and folding, while others highlight functionally important amino acids within P0ct. For example, the D224Y variant of P0ct induced tight membrane multilayer stacking. Our results show a putative molecular basis for the hypermyelinating phenotype observed in patients with this particular mutation and provide overall information on the effects of disease-linked mutations in a flexible, membrane-binding protein segment. Using neutron reflectometry, we additionally show that P0ct embeds deep into a lipid bilayer, explaining the observed effects of P0ct on the physical properties of the membrane.


Assuntos
Membrana Celular/metabolismo , Citoplasma/metabolismo , Mutação , Proteína P0 da Mielina/genética , Proteína P0 da Mielina/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Humanos , Bicamadas Lipídicas/metabolismo , Proteína P0 da Mielina/química , Fenótipo , Ligação Proteica , Dobramento de Proteína
3.
Life (Basel) ; 9(1)2019 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-30884807

RESUMO

The biomolecular homochirality in living organisms has been investigated for decades, but its origin remains poorly understood. It has been shown that circular polarized light (CPL) and other energy sources are capable of inducing small enantiomeric excesses (ees) in some primary biomolecules, such as amino acids or sugars. Since the first findings of amino acids in carbonaceous meteorites, a scenario in which essential chiral biomolecules originate in space and are delivered by celestial bodies has arisen. Numerous studies have thus focused on their detection, identification, and enantiomeric excess calculations in extraterrestrial matrices. In this review we summarize the discoveries in amino acids, sugars, and organophosphorus compounds in meteorites, comets, and laboratory-simulated interstellar ices. Based on available analytical data, we also discuss their interactions with CPL in the ultraviolet (UV) and vacuum ultraviolet (VUV) regions, their abiotic chiral or achiral synthesis, and their enantiomeric distribution. Without doubt, further laboratory investigations and upcoming space missions are required to shed more light on our potential extraterrestrial molecular origins.

4.
J Phys Chem A ; 122(27): 5832-5847, 2018 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-29909620

RESUMO

The absorption spectrum of quinoline was measured in the gas phase between 3.5 and 10.7 eV using a synchrotron photon source. A large number of sharp and broad spectral features were observed, some of which have plasmon-type collective π-electron modes contributing to their intensities. Eight valence electronic transitions were assigned, considerably extending the number of π-π* transitions previously observed mainly in solution. The principal factor in solution red-shifts is found to be the Lorentz-Lorenz polarizability parameter. Rydberg bands, observed for the first time, are analyzed into eight different series, converging to the D0 ground and two excited electronic states, namely, D3 and D4, of the quinoline cation. The R1 series limit is 8.628 eV for the first ionization energy of quinoline, a value more precise than previously published. This value, combined with cation electronic transition data, provides precise energies, respectively, 10.623 and 11.355 eV, for the D3 and D4 states. The valence transition assignments are based on density functional theory (DFT) calculations as well as on earlier Pariser-Parr-Pople (P-P-P) self-consistent field linear combination of atomic orbitals molecular orbital results. The relative quality of the P-P-P and DFT data is discussed. Both are far from spectroscopic accuracy concerning electronic excited states but were nevertheless useful for our assignments. Our time-dependent DFT calculations of quinoline are excellent for its ground-state properties such as geometry, rotational constants, dipole moment, and vibrational frequencies, which agree well with experimental observations. Vibrational components of the valence and Rydberg transitions mainly involve C-H bend and C═C and C═N stretch modes. Astrophysical applications of the vacuum UV absorption of quinoline are briefly discussed.

5.
Phys Chem Chem Phys ; 20(22): 15273-15287, 2018 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-29790512

RESUMO

We present results of a combined vacuum ultraviolet (VUV) and infrared (IR) photoabsorption study of amorphous benzene : water mixtures and layers to investigate the benzene-water interaction in the solid phase. VUV spectra of 1 : 1, 1 : 10 and 1 : 100 benzene : water mixtures at 24 K reveal a concentration dependent shift in the energies of the 1B2u, 1B1u and 1E1u electronic states of benzene. All the electronic bands blueshift from pure amorphous benzene towards gas phase energies with increasing water concentration. IR results reveal a strong dOH-π benzene-water interaction via the dangling OH stretch of water with the delocalised π system of the benzene molecule. Although this interaction influences the electronic states of benzene with the benzene-water interaction causing a redshift in the electronic states from that of the free benzene molecule, the benzene-benzene interaction has a more significant effect on the electronic states of benzene. VUV spectra of benzene and water layers show evidence of non-wetting between benzene and water, characterised by Rayleigh scattering tails at wavelengths greater than 220 nm. Our results also show evidence of benzene-water interaction at the benzene-water interface affecting both the benzene and the water electronic states. Annealing the mixtures and layers of benzene and water show that benzene remains trapped in/under water ice until water desorption near 160 K. These first systematic studies of binary amorphous mixtures in the VUV, supported with complementary IR studies, provide a deeper insight into the influence of intermolecular interactions on intramolecular electronic states with significant implications for our understanding of photochemical processes in more realistic astrochemical environments.

6.
Biochim Biophys Acta Proteins Proteom ; 1866(7): 743-758, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29357311

RESUMO

Living organisms on the Earth almost exclusively use l-amino acids for the molecular architecture of proteins. The biological occurrence of d-amino acids is rare, although their functions in various organisms are being gradually understood. A possible explanation for the origin of biomolecular homochirality is the delivery of enantioenriched molecules via extraterrestrial bodies, such as asteroids and comets on early Earth. For the asymmetric formation of amino acids and their precursor molecules in interstellar environments, the interaction with circularly polarized photons is considered to have played a potential role in causing chiral asymmetry. In this review, we summarize recent progress in the investigation of chirality transfer from chiral photons to amino acids involving the two major processes of asymmetric photolysis and asymmetric synthesis. We will discuss analytical data on cometary and meteoritic amino acids and their potential impact delivery to the early Earth. The ongoing and future ambitious space missions, Hayabusa2, OSIRIS-REx, ExoMars 2020, and MMX, are scheduled to provide new insights into the chirality of extraterrestrial organic molecules and their potential relation to the terrestrial homochirality. This article is part of a Special Issue entitled: d-Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca.


Assuntos
Aminoácidos/química , Evolução Molecular , Meio Ambiente Extraterreno , Fotólise , Aminoácidos/síntese química , Luz , Estereoisomerismo
7.
J Phys Chem A ; 122(1): 184-191, 2018 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-29207234

RESUMO

p-Terphenyl is used as a component in a variety of optical devices. In this investigation, the electronic transitions of p-terphenyl are investigated by synchrotron radiation linear dichroism (SRLD) spectroscopy in the range 30000-58000 cm-1 (330-170 nm) on molecular samples aligned in stretched polyethylene, thereby extending the region investigated by polarization spectroscopy into the vacuum UV. The resulting partial absorbance curves reveal that the vacuum UV band system with a maximum at 55000 cm-1 (180 nm) is predominantly short axis-polarized. This result is of interest in the optical applications of p-terphenyl, for example as a wavelength shifter. The observed polarization spectra are compared with the results of quantum chemical model calculations. Convoluted versions of the transitions predicted with the semiempirical ZINDO method and with the long-range-corrected time-dependent density functional theory (TD-DFT) procedures TD-CAM-B3LYP, TD-LC-ωPBE, and TD-ωB97XD are in similar qualitative agreement with the observed partial absorbance curves throughout the investigated spectral regions, while TD-B3LYP fails to predict qualitatively the spectrum of p-terphenyl in the region above 40000 cm-1 (250 nm).

8.
Phys Chem Chem Phys ; 19(40): 27544-27555, 2017 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-28979950

RESUMO

We present the first high resolution vacuum ultraviolet photoabsorption study of amorphous benzene with comparisons to annealed crystalline benzene and the gas phase. Vapour deposited benzene layers were grown at 25 K and annealed to 90 K under conditions pertinent to interstellar icy dust grains and icy planetary bodies in our solar system. Three singlet-singlet electronic transitions in solid benzene correspond to the 1B2u, 1B1u and 1E1u states, redshifted by 0.05, 0.25 and 0.51 eV respectively with respect to the gas phase. The symmetry forbidden 1B2u ← 1A1g and 1B1u ← 1A1g transitions exhibit vibronic structure due to vibronic coupling and intensity borrowing from the allowed 1E1u ← 1A1g transition. Additionally the 1B2u ← 1A1g structure shows evidence of coupling between intramolecular vibrational and intermolecular lattice modes in crystalline benzene with Davydov crystal field splitting observed. The optically forbidden 0-0 electronic origin is clearly visible as a doublet at 4.69/4.70 eV in the crystalline solid and as a weak broadened feature at 4.67 eV in amorphous benzene. In the case of the 1B1u ← 1A1g transition the forbidden 0-0 electronic origin is only observed in crystalline benzene as an exciton peak at 5.77 eV. Thicker amorphous benzene samples show diffuse bands around 4.3, 5.0 and 5.4 eV that we tentatively assign to spin forbidden singlet-triplet 3B2u ← 1A1g, 3E1u ← 1A1g and 3B1u ← 1A1g transitions respectively, not previously reported in photoabsorption spectra of amorphous benzene. Furthermore, our results show clear evidence of non-wetting or 'islanding' of amorphous benzene, characterised by thickness-dependent Rayleigh scattering tails at wavelengths greater than 220 nm. These results have significant implications for our understanding of the physical and chemical properties and processes in astrochemical ices and highlight the importance of VUV spectroscopy.

9.
Science ; 355(6321): 141, 2017 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-28082555

RESUMO

We detected ribose and related sugars in the organic residues of simulated interstellar ices using multidimensional gas chromatography. Kawai questions the formation of sugar compounds in the ices and suggests that they arise from a classical formose reaction during sample workup for analysis. We disagree with this hypothesis and present additional data to argue that Kawai's criticism does not apply.


Assuntos
Gelo/análise , Ribose , Meio Ambiente Extraterreno , Compostos Orgânicos , Raios Ultravioleta
10.
Langmuir ; 32(27): 6882-94, 2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27315326

RESUMO

Nanostructured bicontinuous lipidic cubic phases are used for the encapsulation of proteins in a range of applications such as in meso crystallization of transmembrane proteins and as drug delivery vehicles. The retention of the nanoscale order of the cubic phases subsequent to protein incorporation, as well as retention of the protein structure and function, is essential for all of these applications. Herein synthetic peptides (WALP21, WALPS53, and WALPS73) with a common α-helical hydrophobic domain, but varying hydrophilic loop size, were designed to systematically examine the effect of peptide structure and charge on bicontinuous cubic phases. The effect of the cubic phases on the secondary structure of the peptides was also investigated. The incorporation of the WALP peptides in cubic phases formed by a range of lipids showed that hydrophobic mismatch of the peptides with the lipid bilayers, the hydrophilic domain size, and peptide charge were all significant factors determining the response of the lipid nanomaterial to protein insertion. As charge repulsion had the most significant effect on the phase transitions observed, we suggest that buffer pH and salt concentration must be carefully considered to ensure cubic mesophase retention. Importantly, the WALP peptides were found to have a different conformation depending on the local lipid environment. Such structural changes could potentially affect membrane protein function, which is crucial for both current and prospective applications.


Assuntos
Bicamadas Lipídicas/química , Modelos Moleculares , Nanoestruturas/química , Peptídeos/química , Estrutura Secundária de Proteína
11.
Science ; 352(6282): 208-12, 2016 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-27124456

RESUMO

Ribose is the central molecular subunit in RNA, but the prebiotic origin of ribose remains unknown. We observed the formation of substantial quantities of ribose and a diversity of structurally related sugar molecules such as arabinose, xylose, and lyxose in the room-temperature organic residues of photo-processed interstellar ice analogs initially composed of H2O, CH3OH, and NH3 Our results suggest that the generation of numerous sugar molecules, including the aldopentose ribose, may be possible from photochemical and thermal treatment of cosmic ices in the late stages of the solar nebula. Our detection of ribose provides plausible insights into the chemical processes that could lead to formation of biologically relevant molecules in suitable planetary environments.


Assuntos
Radiação Cósmica , Gelo , Origem da Vida , RNA/química , Ribose/síntese química , Ácido Acético/química , Amônia/química , Arabinose/síntese química , Meio Ambiente Extraterreno , Meteoroides , Pentoses/síntese química , Processos Fotoquímicos , Raios Ultravioleta , Xilose/síntese química
12.
BMC Struct Biol ; 15: 4, 2015 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-25886849

RESUMO

BACKGROUND: Type IV pili are widely expressed among Gram-negative bacteria, where they are involved in biofilm formation, serve in the transfer of DNA, motility and in the bacterial attachment to various surfaces. Type IV pili in Shewanella oneidensis are also supposed to play an important role in extracellular electron transfer by the attachment to sediments containing electron acceptors and potentially forming conductive nanowires. RESULTS: The potential nanowire type IV pilin PilBac1 from S. oneidensis was characterized by a combination of complementary structural methods and the atomic structure was determined at a resolution of 1.67 Å by X-ray crystallography. PilBac1 consists of one long N-terminal α-helix packed against four antiparallel ß-strands, thus revealing the core fold of type IV pilins. In the crystal, PilBac1 forms a parallel dimer with a sodium ion bound to one of the monomers. Interestingly, our PilBac1 crystal structure reveals two unusual features compared to other type IVa pilins: an unusual position of the disulfide bridge and a straight α-helical section, which usually exhibits a pronounced kink. This straight helix leads to a distinct packing in a filament model of PilBac1 based on an EM model of a Neisseria pilus. CONCLUSIONS: In this study we have described the first structure of a pilin from Shewanella oneidensis. The structure possesses features of the common type IV pilin core, but also exhibits significant variations in the α-helical part and the D-region.


Assuntos
Proteínas de Fímbrias/química , Fímbrias Bacterianas/química , Shewanella/genética , Cristalografia por Raios X , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Modelos Moleculares , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Shewanella/química , Shewanella/metabolismo
13.
Biochim Biophys Acta ; 1854(8): 930-8, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25839998

RESUMO

Integrin α(X)ß(2) (also known as complement receptor 4, p150,95, or CD11c/CD18) is expressed in the cell membrane of myeloid leukocytes. α(X)ß(2) has been reported to bind a large number of structurally unrelated ligands, often with a shared molecular character in the presence of polyanionic stretches in poorly folded proteins or glucosaminoglycans. Nevertheless, it is unclear what chemical sources of polyanionicity enable the binding by α(X)ß(2). Osteopontin (OPN) is an intrinsically disordered protein, which facilitates phagocytosis via the integrin α(X)ß(2). Unlike for other integrins, neither the RGD nor the SVVYGLR motifs account for this binding, and the molecular basis of OPN binding by α(X)ß(2) remains uncharacterized. Here, we show that the monovalent interactions between the ligand-binding domain of α(X)ß(2) and OPN, its fragments, or caseins are weak, with dissociation constants higher than 10(-5)M but with high apparent stoichiometries. From comparison with cell adhesion studies, the discrimination between α(X)ß(2) ligands and non-ligands appears to rely on these apparent stoichiometries in a way, which involves glutamate rather than aspartate side chains. Surprisingly, the extensive, negatively charged phosphorylation of OPN is not contributing to α(X)ß(2) binding. Furthermore, synchrotron radiation circular spectroscopy excludes that the phosphorylation affects the general folding of OPN. Taken together, our quantitative analyses reveal a mode of ligand recognition by integrin α(X)ß(2), which seem to differ in principles considerably from other OPN receptors.


Assuntos
Integrina alfaXbeta2/química , Osteopontina/química , Dobramento de Proteína , Motivos de Aminoácidos , Adesão Celular , Humanos , Integrina alfaXbeta2/metabolismo , Leucócitos/química , Leucócitos/metabolismo , Osteopontina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína
14.
Orig Life Evol Biosph ; 45(1-2): 149-61, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25773582

RESUMO

All life on Earth is characterized by its asymmetry - both the genetic material and proteins are composed of homochiral monomers. Understanding how this molecular asymmetry initially arose is a key question related to the origins of life. Cometary ice simulations, L-enantiomeric enriched amino acids in meteorites and the detection of circularly polarized electromagnetic radiation in star-forming regions point to a possible interstellar/protostellar generation of stereochemical asymmetry. Based upon our recently recorded anisotropy spectra g(λ) of amino acids in the vacuum-UV range, we subjected amorphous films of racemic (13)C-alanine to far-UV circularly polarized synchrotron radiation to probe the asymmetric photon-molecule interaction under interstellar conditions. Optical purities of up to 4% were reached, which correlate with our theoretical predictions. Importantly, we show that chiral symmetry breaking using circularly polarized light is dependent on both the helicity and the wavelength of incident light. In order to predict such stereocontrol, time-dependent density functional theory was used to calculate anisotropy spectra. The calculated anisotropy spectra show good agreement with the experimental ones. The European Space Agency's Rosetta mission, which successfully landed Philae on comet 67P/Churyumov-Gerasimenko on 12 November 2014, will investigate the configuration of chiral compounds and thereby obtain data that are to be interpreted in the context of the results presented here.


Assuntos
Alanina/química , Evolução Química , Raios Ultravioleta , Anisotropia , Meio Ambiente Extraterreno , Estereoisomerismo , Síncrotrons
15.
Chirality ; 26(8): 373-8, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24829143

RESUMO

The basic units that constitute essential biopolymers (proteins and nucleic acids) are enantiomerically biased. Proteins are constructed from L-amino acids and nucleic acids possess a backbone composed exclusively of D-sugars. Photochirogenesis has been postulated to be the source of this homochirality of biomolecules: Asymmetric photochemical reactions were catalyzed by circularly polarized light (cpl) in interstellar environments and generated the first chiral prebiotic precursors. Enantiomers absorb cpl differently and this difference can dictate the kinetics of asymmetric photochemical reactions. These differences in absorption can be studied using circular dichroism (CD) and anisotropy spectroscopy. Rather than measuring the CD spectrum alone, the anisotropy factor g is recorded (CD divided by absorption). This factor g is directly related to the maximum achievable enantiomeric excess. We now report on the substantial influence of solvent and molecular surroundings on CD and anisotropy spectroscopy. This shows for the first time that CD and anisotropy signals depend just as much on the molecular surroundings of a molecule as on the nature of the molecule itself. CD and g spectra of amino acids in different solvents and in the solid state are presented here and the influence of these different surroundings on the spectra is discussed.


Assuntos
Aminoácidos/química , Processos Fotoquímicos , Solventes/química , Anisotropia , Dicroísmo Circular , Modelos Moleculares , Conformação Molecular , Estereoisomerismo
16.
Astrobiology ; 14(2): 87-101, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24512475

RESUMO

We report results from the exposure of the metalloporphyrin iron tetraphenylporphyrin chloride (FeTPPCl) to the outer space environment, measured in situ aboard the Organism/Organic Exposure to Orbital Stresses nanosatellite. FeTPPCl was exposed for a period of 17 months (3700 h of direct solar exposure), which included broad-spectrum solar radiation (∼122 nm to the near infrared). Motivated by the potential role of metalloporphyrins as molecular biomarkers, the exposure of thin-film samples of FeTPPCl to the space environment in low-Earth orbit was monitored in situ via ultraviolet/visible spectroscopy and reported telemetrically. The space data were complemented by laboratory exposure experiments that used a high-fidelity solar simulator covering the spectral range of the spaceflight measurements. We found that thin-film samples of FeTPPCl that were in contact with a humid headspace gas (0.8-2.3% relative humidity) were particularly susceptible to destruction upon irradiation, degrading up to 10 times faster than identical thin films in contact with dry headspace gases; this degradation may also be related to the presence of oxides of nitrogen in those cells. In the companion terrestrial experiments, simulated solar exposure of FeTPPCl films in contact with either Ar or CO2:O2:Ar (10:0.01:1000) headspace gas resulted in growth of a band in the films' infrared spectra at 1961 cm(-1). We concluded that the most likely carriers of this band are allene (C3H4) and chloropropadiene (C3H3Cl), putative molecular fragments of the destruction of the porphyrin ring. The thin films studied in space and in solar simulator-based experiments show qualitatively similar spectral evolution as a function of contacting gaseous species but display significant differences in the time dependence of those changes. The relevance of our findings to planetary science, biomarker research, and the photostability of organic materials in astrobiologically relevant environments is discussed.


Assuntos
Metaloporfirinas/química , Radiação , Voo Espacial , Laboratórios , Nitrogênio/análise , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta , Fatores de Tempo
17.
Angew Chem Int Ed Engl ; 53(1): 210-4, 2014 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-24227543

RESUMO

Circularly polarized light (CPL) is known to be a true chiral entity capable of generating absolute molecular asymmetry. However, the degree of inducible optical activity depends on the λ of the incident CPL. Exposure of amorphous films of rac-alanine to tunable CPL led to enantiomeric excesses (ee) which not only follow the helicity but also the energy of driving electromagnetic radiation. Postirradiation analyses using enantioselective multidimensional GC revealed energy-controlled ee values of up to 4.2 %, which correlate with theoretical predictions based on newly recorded anisotropy spectra g(λ). The tunability of asymmetric photochemical induction implies that both magnitude and sign can be fully controlled by CPL. Such stereocontrol provides novel insights into the wavelength and polarization dependence of asymmetric photochemical reactions and are highly relevant for absolute asymmetric molecular synthesis and for understanding the origins of homochirality in living matter.

18.
Eur Phys J E Soft Matter ; 36(9): 98, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24008406

RESUMO

The spectroscopic characterization of layer-by-layer (LbL) films containing liposomes is essential not only for determining the precise film architecture but also to guide the design of drug delivery systems. In this study we provide the first report of vacuum ultraviolet spectroscopy (VUV) characterization of LbL films made with liposomes from 1.2-dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt) (DPPG) alternated with poly(allylamine hydrochloride) (PAH). Measurements in the 6.0-9.5eV range allowed us to identify the electronic transitions responsible for the spectra, which were assigned to carboxyl, hydroxyl and phosphate groups in DPPG while the PAH spectra were governed by electronic transitions in the amino groups. The surface mass density of the LbL films could be determined, from which the formation of a DPPG bilayer was inferred. This rupture of the liposomes into bilayers was confirmed with atomic force microscopy measurements. In subsidiary experiments we ensured that the UV irradiation in vacuum had negligible damage in the DPPG liposomes during the course of the VUV measurements. In addition to demonstrating the usefulness of VUV spectroscopy, the results presented here may be exploited in biological applications of liposome-containing films.


Assuntos
Bicamadas Lipídicas/química , Lipossomos/química , Fosfatidilgliceróis/química , Espectroscopia Fotoeletrônica , Poliaminas/química , Vácuo
19.
Biochem Biophys Res Commun ; 435(2): 300-5, 2013 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-23618866

RESUMO

Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography.


Assuntos
Membrana Celular/química , Ouabaína/química , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/ultraestrutura , Cardiotônicos , Ativação Enzimática , Estabilidade Enzimática , Conformação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Temperatura Ambiente
20.
Biochim Biophys Acta ; 1834(2): 546-58, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23234758

RESUMO

Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a frequent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin (RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear phagocytes. The known receptor for LtxA on leukocytes is integrin α(L)ß(2) (LFA-1 or CD11a/CD18). However, the molecular mechanisms involved in LtxA-mediated cytotoxicity are poorly understood, partly because LtxA has proven difficult to prepare for experiments as free of contaminants and with its native structure. Here, we describe a protocol for the purification of LtxA from bacterial culture supernatant, which does not involve denaturing procedures. The purified LtxA was monodisperse, well folded as judged by the combined use of synchrotron radiation circular dichroism spectroscopy (SRCD) and in silico prediction of the secondary structure content, and free of bacterial lipopolysaccharide. The analysis by SRCD and similarity to a lipase from Pseudomonas with a known three dimensional structure supports the presence of a so-called beta-ladder domain in the C-terminal part of LtxA. LtxA rapidly killed K562 target cells transfected to express ß(2) integrin. Cells expressing α(M)ß(2) (CD11b/CD18) or α(X)ß(2) (CD11c/CD18) were killed as efficiently as cells expressing α(L)ß(2). Erythrocytes, which do not express ß(2) integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the ß(2) chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins α(M)ß(2) and α(X)ß(2) as novel receptors for LtxA.


Assuntos
Proteínas de Bactérias/química , Antígenos CD18/química , Eritrócitos/química , Exotoxinas/química , Pasteurellaceae/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antígenos CD18/genética , Antígenos CD18/metabolismo , Eritrócitos/metabolismo , Exotoxinas/genética , Exotoxinas/metabolismo , Humanos , Células K562 , Pasteurellaceae/genética , Pasteurellaceae/metabolismo , Infecções por Pasteurellaceae/genética , Infecções por Pasteurellaceae/metabolismo , Ligação Proteica
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