RESUMEN
An LED based illumination device for mechanistic studies on photochemical reactions by means of NMR spectroscopy is presented. The LEDs are directly switched by the NMR spectrometer with the help of a one-stage electronic circuit. This allows for continuous or alternatively pulsed operation of the LEDs. Continuous operation provides direct comparability with conditions in synthetic chemistry, in pulsed operation the short time light power can be enhanced ninefold. The LEDs are efficiently coupled to a 1000 µm core optical fiber guiding the light into the spectrometer by simply bringing it in close contact to the fiber. The tip of the fiber is roughened by sandblasting and thus emits light in a uniform and efficient way over the full length of the receiver coil. The combination of these techniques tremendously increases the amount of light brought into the NMR sample and makes LEDs an easy, versatile and handy light source for the in situ illumination of NMR samples allowing even for single millisecond time resolved Photo-CIDNP spectroscopy.
RESUMEN
We present herein the first indications for dimeric structures in cometal-free asymmetric conjugate addition reactions of dialkylzinc reagents with aldehydes. These are revealed by nonlinear effect (NLE) studies. A monomer-dimer equilibrium can be assumed which explains the increase of the ee value in the product over time. Also, DOSY NMR spectroscopic measurements indicate the existence of the catalyst as [LZnEt](n) complexes in solution. Additionally, the first X-ray structure of a zinc complex with a [2.2]paracyclophane ligand was determined. The structures of the zinc complexes are supported by DFT calculations of monomeric and dimeric species.
RESUMEN
The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2-C-methyl-D-erythritol 4-phosphate pathway in Escherichia coli. In lytB-deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild-type E. coli, but is apparently absent in gcpE-deficient mutants. Here, this compound was purified from E. coli DeltalytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, (1)H, (13)C and (31)P NMR spectroscopy, and NOESY analysis as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is 10(4) times more potent in activating human Vgamma9/Vdelta2 T cells than isopentenyl pyrophosphate.
Asunto(s)
Difosfatos/farmacología , Enzimas , Eritritol/análogos & derivados , Proteínas de Escherichia coli , Escherichia coli/química , Activación de Linfocitos/efectos de los fármacos , Mitógenos/farmacología , Oxidorreductasas , Receptores de Antígenos de Linfocitos T gamma-delta , Subgrupos de Linfocitos T/efectos de los fármacos , Proteínas Bacterianas/genética , Difosfatos/química , Eritritol/biosíntesis , Humanos , Mitógenos/química , Modelos Biológicos , Resonancia Magnética Nuclear Biomolecular , Espectrometría de Masa por Ionización de Electrospray , Fosfatos de Azúcar/biosíntesis , Terpenos/metabolismoRESUMEN
H-Li distances and (1)H-(1)H dipolar interactions in Me(2)CuLiLiCN and Me(2)CuLi in diethyl ether (Et(2)O), obtained by NMR spectroscopy, were used to gain structural information about the contact ion pair of the salt-containing organocuprate Me(2)CuLiLiCN in this solvent. The H-Li distances of Me(2)CuLiLiCN and Me(2)CuLi in Et(2)O, resulting from the initial buildup rates in conjunction with the motional correlation times, are almost identical, indicating a similar homodimeric core structure [Me(2)CuLi](2) for both samples. However, the H-Li distances obtained for Me(2)CuLiLiCN do not rigorously exclude a heterodimeric structure [Me(2)CuLiLiCN] as proposed by ab initio calculations. Therefore, (1)H-(1)H dipolar interactions were investigated by SYM-BREAK-NOE/ROE-HSQC experiments, which allow for the observation of NOEs between equivalent protons. Since these experiments showed similar (1)H-(1)H dipolar interactions of Me(2)CuLiLiCN and Me(2)CuLi, we propose that for Me(2)CuLiLiCN a homodimeric core structure [Me(2)CuLi](2) indeed is predominant in Et(2)O.
RESUMEN
The basidiolipids of six mushroom species, i.e. the basidiomycetes Amanita virosa (engl., death cup), Calvatia exipuliformis (engl., puffball), Cantharellus cibarius (engl., chanterelle), Leccinum scabrum (engl., red birch boletus), Lentinus edodes (jap., Shiitake), and Pleurotus ostreatus (engl., oystermushroom), were isolated, and their chemical structures investigated. All glycolipids are structurally related to those of the Agaricales (engl., field mushroom). They are glycoinositolphosphosphingolipids, their ceramide moiety consisting of t18:0-trihydroxysphinganine and an alpha-hydroxy long-chain fatty acid. In contrast to a previous study [Jennemann, R., Bauer, B.L., Bertalanffy, H., Geyer, R., Gschwind, R.M., Selmer, T. & Wiegandt, H. (1999) Eur. J. Biochem. 259, 331--338], the glycoside anomery of the hexose (mannose) connected to the inositol of all investigated basidiomycete glycolipids, including the basidiolipids of Agaricus bisporus, was determined unequivocally to be alpha. Therefore, the root structure of all basidiolipids consists of alpha-DManp-2Ins1-[PO(4)]-Cer. In addition, for some mushroom species, the occurrence of an inositol substitution position variant, alpha-Manp-4Ins1-[PO(40]-Cer, is shown. The carbohydrate of chanterelle basidiolipids consists solely of mannose, i.e. Cc1, Man alpha-3 or -6Man alpha; Cc2, Man alpha-3(Man alpha-6)Man alpha-. All other species investigated show extension of the alpha-mannoside in the 6-position by beta-galactoside, which, in some instances, is alpha-fucosylated in 2-position (Fuc alpha-2)Gal beta-6Man alpha-. Further sugar chain elongation at the beta-galactoside may be in 3- and/or 6-position by alpha-galactoside, e.g. Ce4, Po2, Gal alpha-3-(Gal alpha-6)(Fuc alpha-2)Gal beta-6Man alpha-, whereas A. virosa, Av-3, has a more complex, highly alpha-fucosylated terminus, Gal alpha-3 (Fuc alpha-2)(Fuc alpha-6)Gal alpha-2(Gal alpha-3)Gal beta-6Man alpha-. L. edodes basidiolipids show further elongation by alpha-mannoside, e.g. Le3, Man alpha-2Man alpha-6Gal alpha-3(Fuc alpha-2)Gal beta-6Man alpha-, C. exipuliformis glycolipid by alpha-glucoside, i.e. Ce3, Glc alpha-6Gal beta-6Man alpha-. Basidiolipid Ls1 from L. scabrum, notably, has a 3-alpha-mannosylated alpha-fucose, i.e. Gal alpha-6(Man alpha-3Fuc alpha-2)Gal alpha-6Gal beta-6Man alpha-. In conclusion, basidiolipids, though identical in their ceramide constitution, display wide and systematic mushroom species dependent variabilities of their chemical structures.
Asunto(s)
Agaricales/química , Glucolípidos/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Ceramidas/química , Ceramidas/metabolismo , Cromatografía en Capa Delgada , Ácidos Grasos/análisis , Ácidos Grasos/química , Glucolípidos/metabolismo , Glicósido Hidrolasas/metabolismo , Hidrólisis , Inositol/análisis , Inositol/química , Espectroscopía de Resonancia Magnética , Manosa/análisis , Manosa/química , Metilación , Peso Molecular , Ácido Peryódico/metabolismo , Fósforo/análisis , Espectrometría de Masa por Ionización de Electrospray , Esfingosina/análisis , Esfingosina/químicaRESUMEN
A new multi-quantum version of the HBHA(CBCACO)NH experiment for partially deuterated protein samples is presented. The method is based on the significant reduction of the proton and carbon relaxation rates due to multi-quantum delays in highly deuterated proteins recently published by our group. The introduction of a multi-quantum period in the coherence transfer pathway of the HBHA(CBCACO)NH experiment yields a dramatic increase of sensitivity-on average 46% with a 75% deuterated sample of the homodimeric 31 kDa E. coli IIAMan domain. Additional resolution in the proton dimension can be achieved by a double time shared approach keeping the 1H single-quantum period at a minimum.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Aminoácidos/química , Óxido de Deuterio , Protones , Sensibilidad y Especificidad , Marcadores de SpinRESUMEN
From the edible mushroom, the basidiomycetes Agaricus bisporus and Agaricus campestris, a novel carbohydrate-homologous series of four glyco-inositol-phospho-sphingolipids, designated basidiolipids, was isolated and the constituents purified. The chemical structures of the basidiolipids were elucidated to be: Manpbeta1-2inositol1-phospho-ceramide, Galpalpha-6[Fucpalpha-2]Galpbeta-6Manpbeta-2i nositol1-phospho-ceramide, Galpalpha-6Galpalpha-6[Fucpalpha-2]Galpbeta- 6Manpbeta-2inositol1-phospho-ceramide and Galpalpha-6Galpalpha-6Galpalpha-6[Fucpalpha-2] Galpbeta-6Manpbeta-2ino sitol1-phospho-ceramide. All four glycolipids contained a ceramide which was composed of phytosphingosine and predominantly alpha-hydroxy-behenic and alpha-hydroxy-lignoceric acid.
Asunto(s)
Agaricus/química , Ceramidas/química , Fosfatos de Inositol/aislamiento & purificación , Fosfolípidos/química , Esfingosina/análogos & derivados , Adyuvantes Inmunológicos/química , Secuencia de Carbohidratos , Ácidos Grasos/química , Hidroxiácidos/química , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Esfingosina/aislamiento & purificaciónRESUMEN
The sequential assignment of backbone resonances is the first step in the structure determination of proteins by heteronuclear NMR. For larger proteins, an assignment strategy based on proton side-chain information is no longer suitable for the use in an automated procedure. Our program PASTA (Protein ASsignment by Threshold Accepting) is therefore designed to partially or fully automate the sequential assignment of proteins, based on the analysis of NMR backbone resonances plus C beta information. In order to overcome the problems caused by peak overlap and missing signals in an automated assignment process, PASTA uses threshold accepting, a combinatorial optimization strategy, which is superior to simulated annealing due to generally faster convergence and better solutions. The reliability of this algorithm is shown by reproducing the complete sequential backbone assignment of several proteins from published NMR data. The robustness of the algorithm against misassigned signals, noise, spectral overlap and missing peaks is shown by repeating the assignment with reduced sequential information and increased chemical shift tolerances. The performance of the program on real data is finally demonstrated with automatically picked peak lists of human nonpancreatic synovial phospholipase A2, a protein with 124 residues.
Asunto(s)
Algoritmos , Espectroscopía de Resonancia Magnética/métodos , Proteínas/química , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Fosfolipasas A/química , Fosfolipasas A2 , Programas InformáticosRESUMEN
The introduction of deuterated and partially deuterated protein samples has greatly facilitated the 13C assignment of larger proteins. Here we present a new version of the HC(CO)NH-TOCSY experiment, the ed-H(CCO)NH-TOCSY experiment for partially deuterated samples, introducing a multi-quantum proton evolution period. This approach removes the main relaxation source (the dipolar coupling to the directly bound 13C spin) and leads to a significant reduction of the proton and carbon relaxation rates. Thus, the indirect proton dimension can be acquired with high resolution, combined with a phase labeling of the proton resonances according to the C-C spin system topology. This editing scheme, independent of the CHn multiplicity, allows to distinguish between proton side-chain positions occurring within a narrow chemical shift range. Therefore this new experiment facilitates the assignment of the proton chemical shifts of partially deuterated samples even of high molecular weights, as demonstrated on a 31 kDa protein.
RESUMEN
The product of the nusB gene of Escherichia coli modulates the efficiency of transcription termination at nut (N utilization) sites of various bacterial and bacteriophage lambda genes. Similar control mechanisms operate in eukaryotic viruses (e.g. human immunodeficiency virus). A recombinant strain of E. coli producing relatively large amounts of NusB protein (about 10% of cell protein) was constructed. The protein could be purified with high yield by anion-exchange chromatography followed by gel-permeation chromatography. The protein is a monomer of 15.6 kDa as shown by analytical ultracentrifugation. Structural studies were performed using protein samples labelled with 15N, 13C and 2H in various combinations. Heteronuclear three-dimensional triple-resonance NMR experiments combined with a semi-automatic assignment procedure yielded the sequential assignment of the 1H, 13C and 15N backbone resonances. Based on experimentally derived scalar couplings, chemical-shift values, amide-exchange data, and a semiquantitative interpretation of NOE data, the secondary structure of NusB has classified as alpha helical, comprising seven alpha helices.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , División Celular , Cromatografía por Intercambio Iónico/métodos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Factores de Transcripción/metabolismoRESUMEN
The mannose transporter of the Escherichia coli bacterial phosphotransferase system consists of three subunits: IIAB, IIC and IID. IIABMan transfers phosphoryl groups to the transported substrate via phosphohistidine intermediates. Its IIB domain was overexpressed and isotopically labelled with 13C, 15N and 2H. Heteronuclear 3D triple-resonance NMR experiments combined with a semi-automatic assignment procedure yielded the sequential assignment of the 1H, 13C and 15N backbone resonances. Based on the evaluation of conformationally sensitive parameters, the secondary structure of the IIBMan domain has been determined as an alpha/beta twisted open-sheet structure consisting of a six-stranded parallel beta-sheet with the novel strand order 3-2-4-1-5-6, six helices and a short two-stranded antiparallel beta-sheet.