Formation of Organized Protein Thin Films with External Electric Field.

The effect of an external electric field on the formation of protein GlnB-Hs films and on its buffer solution on siliconized glass slides has been analyzed by current versus electric field curves and atomic force microscopy (AFM). The Herbaspirillum seropedicae GlnB protein (GlnB-Hs) is a globular, soluble homotrimer (36 kDa) with its 3-D structure previously determined. Concentrations of 10 nM native denatured GlnB-Hs protein were deposited on siliconized glass slides under ambient conditions. Immediately after solution deposition a maximum electric field of 30 kV/m was applied with rates of 3 V/s. The measured currents were surface currents and were analyzed as transport current. Electric current started to flow only after a minimum electric field (critical value) for the systems analyzed. The AFM images showed films with a high degree of directional organization only when the proteins were present in the solution. These results showed that the applied electric field favored directional organization of the protein GlnB-Hs films and may contribute to understand the formation of protein films under applied electric fields.

Effect of protein solution components in the adsorption of Herbaspirillum seropedicae GlnB protein on mica.

The adsorption of proteins and its buffer solution on mica surfaces was investigated by atomic force microscopy (AFM). Different salt concentration of the Herbaspirillum seropedicae GlnB protein (GlnB-Hs) solution deposited on mica was investigated. This protein is a globular, soluble homotrimer (36kDa), member of PII-like proteins family involved in signal transducing in prokaryote. Supramolecular structures were formed when this protein was deposited onto bare mica surface. The topographic AFM images of the GlnB-Hs films showed that at high salt concentration the supramolecular structures are spherical-like, instead of the typical doughnut-like shape for low salt concentration. AFM images of NaCl and Tris from the buffer solution showed structures with the same pattern as those observed for high salt protein solution, misleading the image interpretation. XPS experiments showed that GlnB protein film covers the mica surface without chemical reaction.

Purification and characterization of the bifunctional uridylyltransferase and the signal transducing proteins GlnB and GlnK from Herbaspirillum seropedicae.

GlnD is a bifunctional uridylyltransferase/uridylyl-removing enzyme that has a central role in the general nitrogen regulatory system NTR. In enterobacteria, GlnD uridylylates the PII proteins GlnB and GlnK under low levels of fixed nitrogen or ammonium. Under high ammonium levels, GlnD removes UMP from these proteins (deuridylylation). The PII proteins are signal transduction elements that integrate the signals of nitrogen, carbon and energy, and transduce this information to proteins involved in nitrogen metabolism. In Herbaspirillum seropedicae, an endophytic diazotroph isolated from grasses, several genes coding for proteins involved in nitrogen metabolism have been identified and cloned, including glnB, glnK and glnD. In this work, the GlnB, GlnK and GlnD proteins of H. seropedicae were overexpressed in their native forms, purified and used to reconstitute the uridylylation system in vitro. The results show that H. seropedicae GlnD uridylylates GlnB and GlnK trimers producing the forms PII (UMP)(1), PII (UMP)(2) and PII (UMP)(3), in a reaction that requires 2-oxoglutarate and ATP, and is inhibited by glutamine. The quantification of these PII forms indicates that GlnB was more efficiently uridylylated than GlnK in the system used.

Effect of T- and C-loop mutations on the Herbaspirillum seropedicae GlnB protein in nitrogen signalling.

Proteins of the PII family are found in species of all kingdoms. Although these proteins usually share high identity, their functions are specific to the different organisms. Comparison of structural data from Escherichia coli GlnB and GlnK and Herbaspirillum seropedicae GlnB showed that the T-loop and C-terminus were variable regions. To evaluate the role of these regions in signal transduction by the H. seropedicae GlnB protein, four mutants were constructed: Y51F, G108A/P109a, G108W and Q3R/T5A. The activities of the native and mutated proteins were assayed in an E. coli background constitutively expressing the Klebsiella pneumoniae nifLA operon. The results suggested that the T-loop and C-terminus regions of H. seropedicae GlnB are involved in nitrogen signal transduction.

In vitro uridylylation of the Azospirillum brasilense N-signal transducing GlnZ protein.

Azospirillum brasilense is a diazotroph which associates with important agricultural crops. The nitrogen fixation process in this organism is highly regulated by ammonium and oxygen, and involves several proteins including the two PII-like proteins, GlnB and GlnZ. Although these proteins are structurally very similar, they play different roles in the control of nitrogen fixation. In this work, we describe the expression, purification, and uridylylation of the GlnZ protein of A. brasilense strain FP2. The amplified glnZ gene was sub-cloned and expressed as a His-tagged fusion protein. After purification, we obtained 30-40 mg of purified GlnZ per liter of culture. This protein was purified to 99% purity and assayed for in vitro uridylylation using a partially purified Escherichia coli GlnD as a source of uridylylyl-transferase activity. Analyses of the uridylylation reactions in non-denaturing and denaturing polyacrylamide gel electrophoresis showed that up to 74% of GlnZ monomers were modified after 30 min reaction. This covalent modification is strictly dependent on ATP and 2-ketoglutarate, while glutamine acts as an inhibitor and promotes deuridylylation.