Bioactive peptides: Conformational studies of [TYR4] cyclolinopeptide A

The solid state conformational analysis of [Tyr⁴] cyclolinopeptide A has been carried out by x-ray diffraction studies. The crystal structure of the monoclinic form, grown from a dioxane-water mixture [a = 9.849 (5) Å, b = 20.752 (4) Å, c = 16.728 (5) Å, β = 98.83 (3)°, space group P2₁, Z = 2], shows the presence of five intramolecular N-H? O?C hydrogen bonds, with formation of one C₁₇ ring structure, one α-turn (C₁₃), one inverse γ-turn (C₇), and two β-turns (C₁₀, one of type III and one of type 1). The Pro¹-Pro² peptide unit is cis (ω = 5°) all others are trans. The structure is almost superimposable with that of cyclolinopeptide A. The rms deviation for the atoms of the backbones is on the average 0.33 Å. © 1995 John Wiley & Sons, Inc.     

Lifetime distributions and anisotropy decays of indole fluorescence in cyclohexane/ethanol mixtures by frequency-domain fluorometry

We used frequency-domain fluorometry to measure intensity and anisotropy decay of indole fluorescence in cyclohexane/ethanol mixtures at 20 degrees C. In 100% cyclohexane or 100% ethanol the intensity decay of indole appears to be a single exponential with decay times of 7.66 and 4.10 ns, respectively. In cyclohexane containing a small percentage of ethanol (up to 10%), we observed increased heterogeneity in intensity decay, resulting in a 10-fold increase in chi 2R for the single-exponential fit, as compared with the double-exponential model. We obtained comparable or better fits using unimodal Lorentzian and Gaussian lifetime distributions (two floating parameters) than for the two-exponential model (three floating parameters). We believe that the distribution of decay times reflects a range of indole solvation states in the dominately nonpolar solutions. This result suggests that a variety of hydrogen-bonding configurations could be one origin of the distributions of decay times observed for tryptophan emission from proteins. We also measured rotational diffusion of indole in cyclohexane, ethanol and its mixtures at 20 degrees C. The picosecond correlation times required that the mean decay times be decreased by acrylamide quenching (in ethanol) or energy transfer (in cyclohexane). In ethanol we observed nearly isotropic rotation of indole; in cyclohexane we obtained two correlation times of 17 and 73 ps. The shorter correlation time in cyclohexane appears to be due to the slip boundary condition, which was found to be progressively eliminated by small percentages of ethanol. Hence, hydrogen-bonding interactions appear to have a substantial effect on the rotational dynamics of indole.     

Picosecond resolution of oxytocin tyrosyl fluorescence by 2 GHz frequency-domain fluorometry

The technique of frequency-domain fluorometry has been extended to 2000 MHz using the harmonic content of a picosecond laser source and a microchannel plate photomultiplier tube. This new instrument was used to resolve complex subnanosecond intensity and anisotropy decays of the tyrosyl emission of oxytocin. The intensity decay was found to contain at least three exponential components, 80, 359 and 927 ps. The anisotropy analysis revealed a 29 ps torsional motion of the tyrosine residue as well as a 454 ps overall rotational correlation time. The time resolution of this method should permit the comparison of experimental results with theoretical models for motions of proteins.     

波兰西南卢布林中部地区纵纹腹小鸮的繁殖期食性

2000、2001年4 -7月,我们对波兰东南部纵纹腹小( Athene noctua)繁殖期的食性进行了研究。通过对498个食丸的分析,检出了1 953类动物,其中昆虫占猎物总数量的60·5 % (生物量仅占2·7 %) ,且以鞘翅目(Coleoptera)昆虫居多。该地区纵纹腹小的主要食物是小哺乳动物(占总生物量的93·4 %和总数量的38·3 %) ,在4月出现了一个取食小哺乳动物的高峰。在所捕食的猎物中,个体最大的是欧鼹鼠(Talpa eu-ropaea) ( n=2)。研究还发现,纵纹腹小的猎物种类有季节性变化,以满足繁殖各阶段(如孵卵、育雏和饲喂离巢幼鸟)不同的能量需求。     

Lateral diffusion coefficients in membranes measured by resonance energy transfer and a new algorithm for diffusion in two dimensions

We describe measurements of lateral diffusion in membranes using resonance energy transfer. The donor was a rhenium (Re) metal-ligand complex lipid, which displays a donor decay time near 3 micros. The long donor lifetime resulted in an ability to measure lateral diffusion coefficient below 10(-8) cm(2)/s. The donor decay data were analyzed using a new numerical algorithm for calculation of resonance energy transfer for donors and acceptors randomly distributed in two dimensions. An analytical solution to the diffusion equation in two dimensions is not known, so the equation was solved by the relaxation method in Laplace space. This algorithm allows the donor decay in the absence of energy transfer to be multiexponential. The simulations show that mutual lateral diffusion coefficients of the donor and acceptor on the order of 10(-8) cm(2)/s are readily recovered from the frequency-domain data with donor decay times on the microsecond timescale. Importantly, the lateral diffusion coefficients and acceptor concentrations can be recovered independently despite correlation between these parameters. This algorithm was tested and verified using the donor decays of a long lifetime rhenium lipid donor and a Texas red-lipid acceptor. Lateral diffusion coefficients ranged from 4.4 x 10(-9) cm(2)/s in 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG) at 10 degrees C to 1.7 x 10(-7) cm(2)/s in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) at 35 degrees C. These results demonstrated the possibility of direct measurements of lateral diffusion coefficients using microsecond decay time luminophores.     

To the problem of biologically active conformation of enkephalin

Summary A semi-rigid structural analog of [Leu⁵] enkephalin, possessing the azo-bridge between Tyr¹ and Phe⁴ residues, was synthesized, along with two other linear enkephalin analogs: [4′-amino Phe⁴] enkephalin and [4′hydroxyphenyl/-azo Phe⁴] enkephalin. The results of the determination of the analgesic activity of the synthesized compounds suggest that the biologically active conformation of the enkephalin molecule should be such that both aromatic rings, Tyr¹ and Phe⁴, are situated in close proximity.     

Effect of temperature on oxygen affinity and anion binding of bovine hemoglobin

Measurements of oxygen binding to bovine hemoglobin have been carried out over the temperature range 15-37 degrees C at pH 7.33. The standard enthalpy of oxygenation after correction for the heat of oxygen solution and of the Bohr protons is found to be -7.1 or -7.2 kcal/mol in the presence of 0.1 M chloride or bromide, respectively. This value is well below the -14.4 kcal/mol determined for human hemoglobin under identical experimental conditions. As reported by Fronticelli et al. (C. Fronticelli, E. Bucci and A. Razynska, J. Mol. Biol. 202 (1988) 343), the preferential binding of anions by bovine hemoglobin recognizes the various halides. Measurements at various temperatures reveal that this is true only above 25 degrees C. The halide recognition and the less exothermic enthalpy of oxygenation of bovine hemoglobin are probable due to oxygen-linked hydrophobic effects that are larger in bovine than in human hemoglobin.     

Combined effects of nitrogen enrichment, sulphur pollution and climate change on fen meadow vegetation N:P stoichiometry and biomass

Nitrogen (N) and sulphur (S) deposition, as well as altered soil moisture dynamics due to climate change can have large effects on fen meadow biogeochemistry and vegetation. Their combined effects may differ strongly from their separate effects, since each process affects different nutrients through different mechanisms. However, the impacts of these environmental problems are rarely studied in combination. We therefore investigated the separate and interactive effects of current levels of N- and S-deposition and changes in soil moisture dynamics on fen meadow vegetation. We focused on vegetation biomass and N:P stoichiometry, including access to soil P through root surface phosphatase activity, in a 3-year factorial addition experiment in an N-limited rich fen meadow in the Biebrza valley in Poland. We applied 29.5 kg N ha^?1 year^?1 and 32.1 kg S ha^?1 year^?1, which correspond to current deposition levels in Western Europe. Changes in soil moisture dynamics due to climate change were mimicked by amplified drying of the soil in summer. This level of N-deposition had limited effects on plant biomass production in this rich fen, despite low foliar N:P ratios that suggest N limitation. This level of S-deposition, however, resulted in decreased vegetation P-uptake and biomass. We also showed that increased summer drought resulted in considerable increases in vegetation biomass. We found no interactive effects on vegetation biomass or N:P stoichiometry, possibly as a result of the limited main effects of the separate processes.     

Anisotropy-based sensing with reference fluorophores

We describe a new approach to fluorescence sensing based on measurements of steady-state anisotropies in the presence of reference fluorophores with known anisotropies. The basic concept is that the anisotropy of a mixture reflects a weighted average of the anisotropies of the emitting species. By use of reference fluorophores the starting anisotropy can be near zero, or near 0.9 for oriented films which contain the reference fluorophore. Changing intensities of the analyte result in changes in anisotropy. A wide dynamic range of anisotropies is available because of the freedom to select high or low starting values. Anisotropy-based sensing was demonstrated for pH using 6-carboxyfluorescein and for protein affinity or immunoassay using an oriented film with high anisotropy and a protein labeled with a metal-ligand complex. The latter measurements were performed with a simple light-emitting diode excitation source without an excitation polarizer. The sensitive range of the assay can be adjusted by changing the intensity of the reference fluorophore. Anisotropy-based sensing can have numerous applications in clinical and analytical chemistry.