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.     

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.     

On the peptide-antipeptide interactions in interleukin-1 receptor system

Interleukin-1 receptor antagonist (IL-1Ra) and vaccinia virus protein C10L share a VTXFYF motif, with X being Lys or Arg residue, respectively. Peptides of such sequence compete successfully with IL-1 for the cellular receptor. A pair of complementary peptides, based on the Siemion's hypothesis on the periodicity of the genetic code (QWLNIN and QWANIN), and another pair, in which, following the Root- Bernstein theory, Lys was used as complementary amino acid to Phe (QWLKIK and QWAKIK), were investigated for the peptide-antipeptide interactions using mass spectrometry (ESI-MS) and circular dichroism (CD) methods. The CD measurements indicated some conformational changes, more pronounced in the Siemion's pairs, however, no heterodimer formation was found by MS. In the region of IL-1 receptor situated close to the position of IL-1Ra in the IL-1Ra-receptor complex, a KQKL motif is present, suggesting a possibility of complementary recognition of the Root-Bernstein type in the IL-1 receptor. The biological activity of the complementary peptides is similar to that of the original ones. They efficiently compete with IL-1 and show moderate immunosuppressory activity in humoral and cellular immune response. The inhibition of the IL-1-IL-1 receptor interaction may result from the complementary peptides acting as mini-receptors with affinity for IL-1.     

Radiative decay engineering 4. Experimental studies of surface plasmon-coupled directional emission

Fluorescence is typically isotropic in space and collected with low efficiency. In this paper we describe surface plasmon-coupled emission (SPCE), which displays unique optical properties and can be collected with an efficiency near 50%. SPCE occurs for fluorophores within about 200 nm of a thin metallic film, in our case a 50-nm-thick silver film on a glass substrate. We show that fluorophore proximity to this film converts the normally isotropic emission into highly directional emission through the glass substrate at a well-defined angle from the normal axis. Depending on the thickness of the polyvinyl alcohol (PVA) film on the silver, the coupling efficiency of sulforhodamine 101 in PVA ranged from 30 to 49%. Directional SPCE was observed whether the fluorophore was excited directly or by the evanescent field due to the surface plasmon resonance. The emission is always polarized perpendicular to the plane of incidence, irrespective of the polarization of the incident light. The lifetimes are not substantially changed, indicating a mechanism somewhat different from that observed previously for the effects of silver particles on fluorophores. Remarkably, the directional emission shows intrinsic spectral resolution because the coupling angles depend on wavelength. The distances over which SPCE occurs, 10 to 200 nm, are useful because a large number of fluorophores can be localized within this volume. The emission of more distant fluorophores does not couple into the glass, allowing background suppression from biological samples. SPCE can be expected to become rapidly useful in a variety of analytical and medical sensing applications.     

Fluorescence properties of labeled proteins near silver colloid surfaces

The fluorescence properties of a monolayer of labeled avidin molecules were studied near silver island films. We first adsorbed a monolayer of biotinylated-BSA as a base that was used to capture labeled avidin molecules. For labeled avidin on silver island films, we observed an increase of the fluorescence intensity of between 18 and 80 with one-photon excitation and up to several hundredfold or larger with two-photon excitation. The probes were moderately more photostable in the presence of silver islands. There was also a dramatic decrease in the lifetimes with the amplitude-weighted values decreasing from 7- to 35-fold. The data suggest that these spectral changes are due to both increased rates of excitation near the metallic particles and increases in the rates of radiative decay. Because these silver island surfaces are very heterogeneous, we are hopeful that larger increases in intensity and photostability can be obtained for probes situated at an optimal distance from the ideal island surfaces.     

Analogs of RGDVY and GRGD peptides inhibit Mycobacterium kansasii phagocytosis

Continuing our research on Mycobacteria kansasii phagocytosis inhibition, we have examined in that context three series of peptides derived from the RGDVY and GRGD sequences. It was found that the levels of the inhibitory activity depend on the amino acid composition as well as on the particular peptide sequence. Distinct inhibitory activity was found in the case of thymopentin (RKDVY), the active fragment of thymopoietin. In this case the Mycobacterium phagocytosis inhibition should be combined with general immunostimulatory activity of RKDVY peptide. Our examination of a series of GRGDV analogs with a successively prolonged oligo-Gly linker inserted into the peptide chain showed that the distance between the Arg and Asp residues required for such an activity should be about 9A.     

Antiadhesive peptides as the inhibitors of Mycobacterium kansasii phagocytosis

Initial entry of Mycobacteria into the cells depends upon the formation of a molecular complex between Antigen 85 (Ag85), located on the bacterial cell wall, and serum protein-fibronectin (FN) [Nat. Struct. Biol. 7 (2000) 141; Nat. Struct. Biol. 7 (2000) 94]. Therefore, a way of preventing a Mycobacteria invasion could be to inhibit the interaction between fibronectin and leucocyte cellular receptors of the integrin type. We found that some antiadhesive peptides (such as RGDVY and GRGD), derived of fibronectin and human leucocyte antigen DQ (HLA-DQ) sequences, are in fact very potent inhibitors of Mycobacterium kansasii phagocytosis. This observation may open new prospects in the search for tuberculosis therapy.