We have used intramolecular cross-linking, MS, and sequence threading to rapidly identify the fold of a model protein, bovine basic fibroblast growth factor (FGF)-2. Its tertiary structure was probed with a lysine-specific cross-linking agent, bis(sulfosuccinimidyl) suberate (BS 3 ). Sites of cross-linking were determined by tryptic peptide mapping by using time-of-flight MS. Eighteen unique intramolecular lysine (Lys-Lys) cross-links were identified. The assignments for eight cross-linked peptides were confirmed by using post source decay MS. The interatomic distance constraints were all consistent with the tertiary structure of FGF-2. These relatively few constraints, in conjunction with threading, correctly identified FGF-2 as a member of the ␤-trefoil fold family. To further demonstrate utility, we used the top-scoring homolog, IL-1␤, to build an FGF-2 homology model with a backbone error of 4.8 Å (rms deviation). This method is fast, is general, uses small amounts of material, and is amenable to automation. In recent years, the number of novel proteins identified by genomic (1, 2) and proteomic projects has dramatically increased, with a concomitant need for more rapid determination of their tertiary structures.Visualization of the three-dimensional structures of proteins has traditionally been realized by x-ray crystallography and NMR. These techniques produce high resolution atomic data but require relatively large amounts (10 to 100 mg) of pure analyte in a particular solution or crystalline state. Even if these conditions are met, it can take months or even years to generate a molecular structure by following these methodologies.To develop an alternative approach to structure determination that could keep pace with the rate of novel protein identification, we have re-examined cross-linking technology in the light of newer analytical protocols for the separation and identification of complex peptide mixtures. Previous investigators have shown that cross-linking experiments can provide low resolution interatomic distance information (3). In theory, given enough distance information, it is possible to solve the tertiary structure of a macromolecule (4, 5).The challenge we faced in trying to generate such information in a short time using cross-linking technology was to devise a rapid method for identifying cross-linked residues. MS affords high throughput but has rarely been used for the identification of cross-links. One study has been published where disuccinimidyl ester cross-linking, Edman sequencing, and MS were used to validate a model of human erythropoietin (6). Recent advances in MS (7, 8) gave us the means whereby we could determine the masses and sequences of large peptides with high accuracy and sensitivity (9, 10). These improvements make it feasible to analyze complex peptide mixtures from proteolytically digested, cross-linked proteins (11) very quickly. Specifically, we describe the use of chemical cross-linking and time-of-flight (TOF) MS to identify Lys-Lys cross-links. We also show how t...

One of the major goals of rational design of combinatorial libraries is to design libraries with maximum diversity to enhance the potential of finding active compounds in the initial rounds of high-throughput screening programs. We present strategies to visualize and optimize the structural diversity of sets of molecules, which can be either potential substituents to be attached at specific positions of the library scaffold, or entire molecules corresponding to enumerated libraries. The selection of highly diverse subsets of molecules from the library is based on the stochastic optimization of 'Diversity' functions using a single-point-mutation Monte Carlo technique. The Diversity functions are defined in terms of the distances among molecules in multidimensional property space resulting from the calculation of 2D and 3D molecular descriptors. Several Diversity functions, including an implementation of D-Optimal design, are applied to select diverse subsets and the results are compared. The diversity of the selected subsets of molecules is visualized by embedding the intermolecular distances, defined by the molecules in multidimensional property space, into a three-dimensional space.

No abstract

No abstract

High levels of β-carotene, lycopene, and the rare γ-carotene occur predominantly lipid-dissolved in the chromoplasts of peach palm fruits. First proof of their absorption from these fruits is reported. The structural diversity, the physical deposition state in planta, and the human bioavailability of carotenoids from the edible fruits of diverse orange and yellow-colored peach palm (Bactris gasipaes Kunth) varieties were investigated. HPLC-PDA-MS(n) revealed a broad range of carotenes, reaching total carotenoid levels from 0.7 to 13.9 mg/100 g FW. Besides the predominant (all-E)-β-carotene (0.4-5.4 mg/100 g FW), two (Z)-isomers of γ-carotene (0.1-3.9 mg/100 g FW), and one (Z)-lycopene isomer (0.04-0.83 mg/100 g FW) prevailed. Approximately 89-94 % of total carotenoid content pertained to provitamin A carotenoids with retinol activity equivalents ranging from 37 to 609 µg/100 g FW. The physical deposition state of these carotenoids in planta was investigated using light, transmission electron, and scanning electron microscopy. The plastids found in both orange and yellow-colored fruit mesocarps were amylo-chromoplasts of the globular type, containing carotenoids predominantly in a lipid-dissolved form. The hypothesis of lipid-dissolved carotenoids was supported by simple solubility estimations based on carotenoid and lipid contents of the fruit mesocarp. In our study, we report first results on the human bioavailability of γ-carotene, β-carotene, and lycopene from peach palm fruit, particularly proving the post-prandial absorption of the rarely occurring γ-carotene. Since the physical state of carotenoid deposition has been shown to be decisive for carotenoid bioavailability, lipid-dissolved carotenoids in peach palm fruits are expected to be highly bioavailable, however, further studies are required.

No abstract

No abstract

No abstract