New Tools to Probe the Protein Surface: Ultrasmall Gold Nanoparticles Carry Amino Acid Binders.

A strategy toward epitope-selective functionalized nanoparticles is introduced in the following: ultrasmall gold nanoparticles (diameter of the metallic core about 2 nm) were functionalized with molecular tweezers that selectively attach lysine and arginine residues on protein surfaces. Between 11 and 30 tweezer molecules were covalently attached to the surface of each nanoparticle by copper-catalyzed azide alkyne cycloaddition (CuAAC), giving multiavid agents to target proteins. The nanoparticles were characterized by high-resolution transmission electron microscopy, differential centrifugal sedimentation, and 1H NMR spectroscopy (diffusion-ordered spectroscopy, DOSY, and surface composition). The interaction of these nanoparticles with the model proteins hPin1 (WW domain; hPin1-WW) and Survivin was probed by NMR titration and by isothermal titration calorimetry (ITC). The binding to the WW domain of hPin1 occurred with a KD of 41 ± 2 µM, as shown by ITC. The nanoparticle-conjugated tweezers targeted cationic amino acids on the surface of hPin1-WW in the following order: N-terminus (G) ≈ R17 > R14 ≈ R21 > K13 > R36 > K6, as shown by NMR spectroscopy. Nanoparticle recognition of the larger protein Survivin was even more efficient and occurred with a KD of 8 ± 1 µM, as shown by ITC. We conclude that ultrasmall nanoparticles can act as versatile carriers for artificial protein ligands and strengthen their interaction with the complementary patches on the protein surface.

The RxLR Motif of the Host Targeting Effector AVR3a of Phytophthora infestans Is Cleaved before Secretion.

When plant-pathogenic oomycetes infect their hosts, they employ a large arsenal of effector proteins to establish a successful infection. Some effector proteins are secreted and are destined to be translocated and function inside host cells. The largest group of translocated proteins from oomycetes is the RxLR effectors, defined by their conserved N-terminal Arg-Xaa-Leu-Arg (RxLR) motif. However, the precise role of this motif in the host cell translocation process is unclear. Here, detailed biochemical studies of the RxLR effector AVR3a from the potato pathogen Phytophthora infestans are presented. Mass spectrometric analysis revealed that the RxLR sequence of native AVR3a is cleaved off prior to secretion by the pathogen and the N terminus of the mature effector was found likely to be acetylated. High-resolution NMR structure analysis of AVR3a indicates that the RxLR motif is well accessible to potential processing enzymes. Processing and modification of AVR3a is to some extent similar to events occurring with the export element (PEXEL) found in malaria effector proteins from Plasmodium falciparum These findings imply a role for the RxLR motif in the secretion of AVR3a by the pathogen, rather than a direct role in the host cell entry process itself.

Supplementation with mixed fruit and vegetable juice concentrates increased serum antioxidants and folate in healthy adults.

OBJECTIVE: Epidemiological studies have shown that low plasma levels of antioxidant micronutrients, which are commonly found in fruit and vegetables, are associated with increased risk for diseases such as heart disease, cancer, metabolic disorders and the like. The aim of this study was to monitor the dietary habits of a group of healthy, middle-aged, men and women and to assess the effect of supplementation with a natural phytonutrient preparation from fruits and vegetables, on plasma levels of various antioxidant micronutrients and oxidative stress assessed by measuring 8-oxodGuo (8-oxo-7,8-dihydro-2'-deoxyguanosine) in urine. METHODS: The study followed a double-blind randomized cross-over design involving 59 healthy men and women (40-60 years of age). The supplement or a placebo was given to two groups for a total period of 14 weeks (crossover week 7). Blood levels of beta-carotene, vitamin C, vitamin E, selenium and folate were measured at 0, 7 and 14 weeks. Fruit and vegetable consumption was monitored by means of a retrospective food frequency questionnaire at week 0, 7 and 14. Urinary 8-oxodGuo was also determined at these time points. RESULTS: Significant increases in blood nutrient levels after active supplementation were observed for beta-carotene, vitamin C, vitamin E, selenium and folate. Ranges measured, after supplementation, often fell into those associated with a reduced risk for disease. Our data suggests that, although generally health conscious, participants still fell short of the recommended five portions of fruit and vegetables per day. No significant group changes were noted for 8-oxodGuo concentration in urine. CONCLUSION: Supplementation with mixed fruit and vegetable juice concentrates effectively increased plasma levels of important antioxidant nutrients and folate.

Expression, purification and crystallization of human 3'-phosphoadenosine-5'-phosphosulfate synthetase 1.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is used to incorporate sulfate into biomolecules. The human PAPS synthetase 1 catalyzes two steps leading from adenosine triphosphate (ATP) and sulfate to PAPS. The ATP sulfurylase domain catalyzes the formation of the intermediate adenosine-5'-phosphosulfate (APS). The APS kinase domain then adds a phosphate group to the 3'-ribose and releases PAPS. In this article, the recombinant expression, purification and crystallization of the full-length protein is described. In Escherichia coli the protein is only partly soluble and copurifies with GroEL. The pure protein migrates as a dimer in gel-filtration chromatography. It is moderately active, forming 25 nmol PAPS per minute per milligram. Crystals grow to 100 x 100 x 300 micro m and diffract to 1.75 A.