Mustard seed (Brassica Juncea) Proteins

Quenched Molecular Dynamics (QMD) used to explore molecular conformations was developed to operate in Insight II platform for two simulation engines: CHARMm and Discover. Two scripts and procedures were written for molecular minimization, dynamics, minimization of each of several hundred conformers, and cut off. Experience with Insight II/Discover versus Quanta/CHARMm, and between Insight II/CHARMm versus Quanta/CHARMm has taught that the forcefield is the key factor in QMD studies. Protein A has been used for the purification of commercial antibodies, but it is expensive. Seven peptidomimetics of protein A were designed based on the hot-spots located at the helix-loop-helix region of protein A, and synthesized via solid phase using the Fmoc approach. These peptidomimetics were characterized by MS and NMR. The conformations of four peptidomimetics were studied by NMR and CD in water/hexafluoroisopropanol (pH 4). The CD and NMR data show that addition of hexafluoroisopropanol stabilizes their a-helical conformations. The structures of these peptidomimetics in solution were generated with Quanta/CHARMm using NMR data as limits for the QMD technique. Protein G has also been used to purify antibodies, but it is expensive too. A number of protein G mimics were designed as trivalent molecules. An efficient preparation of trivalent molecules having a useful primary amine arm has been developed through solid phase synthesis. The cheap, commercially available poly(propylene imine) dendrimers were used as scaffolds which allow multimerization of functionalized compounds. A small library of trivalent compounds were synthesized using this approach. A portion of compounds in this library were tested by Amersham Biosciences. The seven amino acid modified DAB-Am-4 exhibits strong binding to the IgG/Fab, and is a potential ligand for IgG purification. The interactions between neurotrophins (ie NGF and NT-3) and their receptors are typical drug targets. Fourteen second-generation peptidomimetics showing NGF-like or NT3-like activities in a preliminary bioassay, were resynthesized and tested again. Preliminary and retested data were compared. To access a direct binding assay, five fluorescently labeled peptidomimetics 41a-e were synthesized for a fluorescence activated cell sorting (FACScan) assay. Six monomeric precursors 42 and 43 were prepared on large scales for the library of bivalent turn analogs

Research has shown conflicting results involving the efficacy of carbohydrateprotein beverages on athletic performance. Purpose: To examine whether or not power output during the latter stages in a series of repeated maximal or near maximal effort anaerobic exercise bouts simulating a football game task was altered when consuming a carbohydrate-protein (CP) beverage versus either a carbohydrate-only (C) beverage or a placebo (P). Methods: Eighteen collegiate male football players participated in this investigation. The subjects' mean age, height, weight, and percent body fat were 20yr, 180.4cm, 92.4kg, and 12%, respectively. The experimental exercise sessions were completed by each athlete on three separate occasions, spaced one week apart. Subjects were asked to perform a series of maximal-effort weighted sled-pushes, which simulated a game-type activity over two halves of a football game separated by a 20-minute simulated halftime recovery period. Maximal muscle power was assessed through the use of a series of maximal jump-and-reach tests. The experimental beverages were administered during the first 5 minutes of halftime. Water was permitted ad libitum throughout each exercise session. The experimental beverages used included; 1) a commercially available flavored aspartame-sweetened P beverage, Crystal Light, (300 ml,5 kcal), 2) a commercially available C beverage, Gatorade Energy Drink®, (300 ml, 67.5 g CHO, 270 kcal), and 3) a commercially available CP beverage, Gatorade Nutrition Shake®, (243 ml diluted with water to 300 ml, 45 g CHO, 15 g Protein, 270 kcal). All beverages were randomly assigned and each player received all three beverages. An analysis of covariance (ANCOVA) was used to determine if differences existed in power output between the experimental beverages. Results: The Least Square Mean (LSM) for jump-power was significantly higher after C compared to CP (1587.36 watts vs. 1577.42 watts, respectively; p=0.0095). The LSM jump-power after the P beverage was also lower than after the C beverage (1582.52), but was not statistically significant. Conclusions: These data suggest that average power output over a series of high-intensity anaerobic exercise bouts, which simulate football game tasks, is greatest after consuming a C beverage during the halftime break compared with consuming a CP or P beverage.

Thesis (Ph. D.)--California Institute of Technology, 2001. PQ #3011579. / Advisor names found in the Acknowledgements pages of the thesis. Title from home page. Viewed 01/13/2010. Includes bibliographical references.

Thesis (Ph. D.)--California Institute of Technology, 1993. UM #93-25,374. / Advisor names found in the Acknowledgements pages of the thesis. Title from home page. Viewed 01/15/2010. Includes bibliographical references.

Thesis (Ph. D.)--University of Queensland, 2003. / Includes bibliographical references.

Presented in this dissertation are studies of protein dynamics and protein/protein interactions using solution phase hydrogen/deuterium exchange in combination with mass spectrometry (HXMS). In addition, gas phase fragmentation behaviors of deuterated peptides are investigated, with the purpose of increasing resolution of the HXMS. In the area of single protein dynamics, two protein systems are studied. Studies on the cytochrome c2 from Rhodobacter capsulatus indicate its domain stability to be similar to that of the horse heart cytochrome c. Further comparison of the exchange kinetics of the cytochrome c2 in its reduced and oxidized state reveals that the so-called hinge region is destabilized upon oxidation. We also applied a similar approach to investigate the conformational changes of photoactive yellow protein when it is transiently converted from the resting state to the signaling state. The central β-sheet of the protein is shown to be destabilized upon photoisomerization of the double bond in the chromophore. Another equally important question when it comes to understanding how proteins work is the interactions between proteins. To this end, two protein complexes are subjected to studies by solution phase hydrogen deuterium exchange and mass spectrometry. In the case of LexA/RecA interaction, both proteins show decreases in their extents of exchange upon complex formation. The potential binding site in LexA was further mapped to the same region that the protein uses to cleave itself upon interacting with RecA. In the sHSP/MDH system, hydrogen/deuterium exchange experiments revealed regions within sHSP-bound MDH that were significantly protected against exchange under heat denaturing condition, indicative of a partially unfolded state. Hydrogen/deuterium exchange therefore provides a way of probing low resolution protein structure within protein complexes that have a high level of heterogeneity. Finally, the feasibility of increasing resolution of HXMS by gas phase peptide fragmentation is investigated by using a peptide with three prolines near the C-terminus. Our data show that deuterium migration indeed occurs during the collision activated dissociation process. Caution is required when interpreting the MS/MS spectra as a way of pinpointing the exact deuterium distribution within peptides.

Directed evolution studies were done with PTE for the enhancement of hydrolysis of both sarin and soman analogs. Particular attention was focused on the toxic SpRc and SpSc isomers of the soman analog, and the Sp isomer of the sarin analog. Double substitution libraries yielded several mutants that had enhanced activity for the substrates. Among them was the double mutant, H257Y-L303T, which displayed a 462-fold increase in activity for hydrolysis of the most toxic SpSc isomer of the GD analog in comparison to the wild type. Several other mutants such as the triple mutants H254R-H257A-L303T and H254R-H257S-L303T had enhancements of between 150- and 200-fold, and had also displayed a different order of stereoselectivity relative to the wild type. For these mutants, the order of preferential hydrolysis was such that the SpRc isomer was preferentially hydrolyzed first. In contrast, the order of preferential hydrolysis for the wild type was that the RpRc was hydrolyzed first, followed by the RpSc, SpRc, and then the SpSc isomer. The reversal of stereoselective preference was also seen with the double substitution library members for hydrolysis of the sarin analog isomers. However, there were no significant improvements for sarin analog hydrolysis in these libraries. Among the best mutants obtained were H254G-H257W, H254G-H257R, and H257Y, all of which had catalytic efficiencies on the order of 106 M-1 s-1 for hydrolysis of the Sp isomer. The toxicity for analogs of sarin, soman, and VX was evaluated using Hydra attenuata as a model organism. The toxicity of each compound was assessed quantitatively by measuring the minimal effective concentration within 92 h in H. attenuata. There was a positive correlation between the molecular hydrophobicity of the compound and its ability to cause toxicity. Results from this study indicate the potential for application of this assay in the field of organophosphate nerve agent detection, as well as for the prediction of toxicity of structurally similar organophosphate compounds. The minimal effective concentration for two of the VX analogs was 2 orders of magnitude more toxic than the analog for soman and four orders of magnitude more toxic than the analog for sarin.

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