An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21

We have measured the Raman spectrum of GDP bound to the elongation factor protein, EF-Tu, and the c-Harvey-ras protein, p21, two proteins of the guanine nucleotide binding family. In order to separate the Raman spectrum of the nucleotide from the much more intense protein spectrum, we investigate the feasibility of "tagging" the normal modes of the nucleotide by isotopic substitution, here by incoporating deuterium-labeled guanine at the C8 position into the active site. A difference spectrum between the labeled and unlabeled protein-nucleotide complex shows the changes in the Raman spectrum of the bound nucleotide that arise from the isotopic exchange. We find that surprisingly good Raman spectra of bound ligands can be obtained with this method and that the method can be easily generalized to other systems. The data show that the guanine amino group of the nucleotide interacts differently with both EF-Tu and p21 than it does with water, showing a change in hydrogen-bonding properties upon binding. On the other hand, no change in hydrogen bonding is observed at guanine's N7. The data strongly suggest that the conformation of the nucleotide when bound to EF-Tu and that p21 is the C2' endo pucker of the ribose ring and anti about the glycosidic bond. These results are compared to previous structural and chemical studies.     

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu have been derived from the atomic coordinates of the trypsin-modified form of EF-Tu-GDP and by comparison with the ras p21 structures. The significance of the differences in the guanine nucleotide binding sites of EF-Tu and ras p21 are discussed. Crystallization of the EF-Tu-GMPPNP complex is reported.     

Stabilization of the Escherichia coli elongation factor Tu-GTP-aminoacyl-tRNA complex

The effect of ammonium sulfate on the Escherichia coli elongation factor Tu-GTP-aminoacyl-tRNA complex has been studied. The half-lives of 12 E. coli aminoacyl-tRNA species were determined at 37 degrees C in the presence and absence of an equimolar amount of EF-Tu-GTP and in the presence and absence of 1.5 M ammonium sulfate. The results indicate that the addition of 1.5 M ammonium sulfate to the ternary complex increased the stability of all 12 complexes studied. In addition, the effects of various salts and crystallization agents on the stability of the E. coli EF-Tu-GTP-phenylalanyl-tRNA complex was studied in detail. Binding parameters were also measured under various conditions at 37 degrees C. The results indicate that the stability and the Kassoc of the ternary complex, using phenylalanyl-tRNA, can be increased by the presence of polyethylene glycol or ammonium sulfate.     

Preliminary crystallographic analysis of a complex between tetracycline and the trypsin-modified form of Escherichia coli elongation factor Tu

Crystals of a complex between the antibiotic tetracycline and the trypsin-modified form of the Escherichia coli protein elongation factor Tu have been grown in a form suitable for high-resolution X-ray diffraction analysis. The crystals belong to space group P2(1), with cell dimensions a = 69.7 A, b = 156.4 A, c = 135.4 A and beta = 95.3 degrees, and contain six molecules of the complex per asymmetric unit. The crystals are well ordered and diffract to a resolution of 2.3 A.     

Consumer Depletion Alters Seagrass Resistance to an Invasive Macroalga

Few field studies have investigated how changes at one trophic level can affect the invasibility of other trophic levels. We examined the hypothesis that the spread of an introduced alga in disturbed seagrass beds with degraded canopies depends on the depletion of large consumers. We mimicked the degradation of seagrass canopies by clipping shoot density and reducing leaf length, simulating natural and anthropogenic stressors such as fish overgrazing and water quality. Caulerpa racemosa was transplanted into each plot and large consumers were excluded from half of them using cages. Potential cage artifacts were assessed by measuring irradiance, scouring by leaf movement, water flow, and sedimentation. Algal invasion of the seagrass bed differed based on the size of consumers. The alga had higher cover and size under the cages, where the seagrass was characterized by reduced shoot density and canopy height. Furthermore, canopy height had a significant effect depending on canopy density. The alteration of seagrass canopies increased the spread of C. racemosa only when large consumers were absent. Our results suggest that protecting declining habitats and/or restoring fish populations will limit the expansion of C. racemosa. Because MPAs also enhance the abundance and size of fish consuming seagrass they can indirectly promote algal invasion. The effects of MPAs on invasive species are context dependent and require balancing opposing forces, such as the conservation of seagrass canopy structure and the protection of fish grazing the seagrass.     

Comparative evaluation of multiple protein extraction procedures from three species of the genus Caulerpa

Journal of Applied Phycology - The aim of this study was to define the simplest and least expensive protocol for total protein extraction for three different macroalgae of the genus Caulerpa (the...     

Preparation of Escherichia coli elongation factor Tu-guanosine 5'-triphosphate analogs

A simple procedure for the bulk preparation of 20 mg of Escherichia coli elongation factor (EF)-Tu-GTP analogs is described. The protocol is based upon the preparation and stabilization of nucleotide-free EF-Tu using an EF-Ts affinity chromatographic resin. The procedure is a general one for the preparation of any GTP analog of EF-Tu.     

A T-cell-selective interleukin 2 mutein exhibits potent antitumor activity and is well tolerated in vivo

Human interleukin 2 (IL-2; Proleukin) is an approved therapeutic for advanced-stage metastatic cancer; however, its use is restricted because of severe systemic toxicity. Its function as a central mediator of T-cell activation may contribute to its efficacy for cancer therapy. However, activation of natural killer (NK) cells by therapeutically administered IL-2 may mediate toxicity. Here we have used targeted mutagenesis of human IL-2 to generate a mutein with approximately 3,000-fold in vitro selectivity for T cells over NK cells relative to wild-type IL-2. We compared the variant, termed BAY 50-4798, with human IL-2 (Proleukin) in a therapeutic dosing regimen in chimpanzees, and found that although the T-cell mobilization and activation properties of BAY 50-4798 were comparable to human IL-2, BAY 50-4798 was better tolerated in the chimpanzee. BAY 50-4798 was also shown to inhibit metastasis in a mouse tumor model. These results indicate that BAY 50-4798 may exhibit a greater therapeutic index than IL-2 in humans in the treatment of cancer and AIDS.