Production of a self-assembled protein nanotube achieved through engineering of the 11mer ring protein trp RNA-binding attenuation protein is described. The produced mutant protein is able to stack in solution to produce an extremely narrow, uniform nanotube apparently stabilized by a mixture of disulfide bonds and hydrophobic interactions. Assembly is reversible and the length of tube can potentially be controlled. Large quantities of hollow tubes 8.5 nm in overall diameter with lengths varying from 7 nm to over 1 microm are produced. The structure is analyzed using transmission electron microscopy, atomic force microscopy, mass spectrometry, and single-particle analysis and it is found that component rings stack in a head-to-head fashion. The internal diameter of the tube is 2.5 nm, and the amino acid residues lining the central cavity can be mutated, raising the possibility that the tube can be filled with a variety of conducting or semiconducting materials.

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cyclic AMP-dependent protein kinase is a post-translational modification that increases its basal activity and facilitates its activation by the substrate L-Phe. So far there is no structural information on the flexible N-terminal tail (residues 1-18), including the phosphorylation site. To get further insight into the molecular basis for the effects of phosphorylation on the catalytic efficiency and enzyme stability, molecular modeling was performed using the crystal structure of the recombinant rat enzyme. The most probable conformation and orientation of the N-terminal tail thus obtained indicates that phosphorylation of Ser 16 induces a local conformational change as a result of an electrostatic interaction between the phosphate group and Phenylalanine hydroxylase (PAH 1 ; EC 1.14.16.1, phenylalanine 4-monooxygenase) belongs to the family of aromatic amino acid hydroxylases. PAH catalyzes the hydroxylation of L-Phe to L-Tyr, the rate-limiting step in the catabolism of L-Phe (1, 2), and it requires a nonheme iron, molecular oxygen, and a pterin cofactor for catalysis (3). Genetic defects in the human enzyme (hPAH) cause phenylketonuria, with a broad range of metabolic and clinical phenotypes (4) as well as enzymatic phenotypes (5, 6).hPAH is a tetrameric/dimeric enzyme, and crystal structure analyses (7-9) have shown that each of the chains folds into three domains, i.e. an N-terminal regulatory domain (residues 1-110) that includes the single phosphorylation site Ser 16 , a middle catalytic domain, and a C-terminal oligomerization domain. Mammalian PAH is activated severalfold by preincubation with its substrate L-Phe, which represents the most important mechanism for its regulation in hepatocytes (1). Phosphorylation of PAH at Ser 16 by cAMP-dependent protein kinase (PKA) represents an additional post-transcriptional regulation of the enzyme (10 -14). The two mechanisms of activation are interdependent, i.e. L-Phe enhances its rate of phosphorylation by PKA, and the phosphorylated enzyme requires a lower concentration of substrate for its activation (15,16). It appears that these two mechanisms act synergistically also in vivo and that L-Phe promotes the phosphorylation and activation of PAH in rat liver (13,17). However, the molecular mechanism of this interdependence is not well understood (18,19) and has not been explained by the crystal structure analyses of the phosphorylated (at Ser 16 ) ⌬C24-truncated dimeric form of rat PAH (rPAH) (9). Phosphorylation of the human enzyme results in a mobility shift on SDS-PAGE (20) that is also observed when hPAH is expressed in Escherichia coli (16,21) and in the in vitro transcription-translation system (5, 22). The enzyme expressed in the latter system is recovered as a double band on SDS-PAGE, corresponding to the phosphorylated (ϳ51 kDa) and nonphosphorylated (ϳ50 kDa) forms. Furthermore, we have previously shown by Fourier transform infrared spectroscopy that phosphorylation of the isolated recombi...

Vários derivados de 1,4-naftoquinonas contendo um grupo hidrazino como cadeia lateral foram sintetizados a partir do 3-diazo-naftaleno-1,2,4-triona e foram avaliados como potenciais agentes antimicrobianos. Os derivados naftoquinônicos 2--hidrazono]-malonato de etila mostraram maior atividade antibacteriana, ao nível de teste preliminar em disco, que o lapachol (1), uma 1,4-naftoquinona muito conhecida pelas suas variadas atividades biológicas. Estudo sobre a concentração mínima inibitória (MIC) para o Staphylococcus aureus mostrou que 2-[(3-hidroxi-1,4-dioxo-1,4-diidronaftaleno-2-il)-hidrazono]-malonato de etila tem uma atividade duas vezes maior que 1. Da mesma forma, o estudo da densidade ótica em cultura de S. aureus com esta substância mostrou uma atividade similar à da vancomicina na concentração de 2xMIC.Several 1,4-naphthoquinone derivatives having a hydrazino side chain were synthesized from 3-diazo-naphthalene-1,2,4-trione and tested as potential antimicrobial agents. These naphthoquinone derivatives 2-[N'-(1-acetyl-2-oxo-propylidene)-hydrazino]-3-hydroxy-[1,4]naphthoquinone, ethyl 2-[(3-hydroxy-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)-hydrazono]-3-oxo-butyrate, t-butyl 2-[(3-hydroxy-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)-hydrazono]-3-oxobutyrate, 3-hydroxy-2-[(di-O-isopropylidene-malonate)-hydrazino]-1,4-naphthoquinone, and diethyl 2-[(3-hydroxy-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)-hydrazono]-malonate showed greater antibacterial activity at the level of the preliminary susceptibility testing in disk than lapachol (1), a well known 1,4-naphthoquinone which has several biological activities. Studies on the minimal inhibitory concentration (MIC) for Staphylococcus aureus showed that diethyl 2-[(3-hydroxy-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)-hydrazono]-malonate has an activity twofold greater than 1. On the other hand, optical density measurement for S. aureus indicated that this compound has similar activity compared with vancomycin at 2xMIC.

Phosphorylation of phenylalanine hydroxylase (PAH) at Ser16 by cAMP-dependent protein kinase increases the basal activity of the enzyme and its resistance to tryptic proteolysis. The modeled structures of the full-length phosphorylated and unphosphorylated enzyme were subjected to molecular dynamics simulations, and we analyzed the energy of charge-charge interactions for individual ionizable residues in the final structures. These calculations showed that the conformational changes induced by incorporation of phosphate were localized and limited mostly to the region around the phosphoserine (Arg13-Asp17) and a region around the active site in the catalytic domain that includes residues involved in the binding of the iron and the substrate L-Phe (Arg270 and His285). The absence of a generalized conformational change was confirmed by differential scanning calorimetry, thermal-dependent circular dichroism, fluorescence spectroscopy, and limited chymotryptic proteolysis of the phosphorylated and unphosphorylated PAH. Our results explain the effect of phosphorylation of PAH on both the resistance to proteolysis specifically by trypsinlike enzymes and on the increase in catalytic efficiency.

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