The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 A by multiwavelength anomalous dispersion phasing methods. The protein is in the shape of a cylinder, comprising 11 strands of beta-sheet with an alpha-helix inside and short helical segments on the ends of the cylinder. This motif, with beta-structure on the outside and alpha-helix on the inside, represents a new protein fold, which we have named the beta-can. Two protomers pack closely together to form a dimer in the crystal. The fluorophores are protected inside the cylinders, and their structures are consistent with the formation of aromatic systems made up of Tyr66 with reduction of its C alpha-C beta bond coupled with cyclization of the neighboring glycine and serine residues. The environment inside the cylinder explains the effects of many existing mutants of GFP and suggests specific side chains that could be modified to change the spectral properties of GFP. Furthermore, the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein.

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Recent studies demonstrate a pivotal role for bone morphogenic protein-6 (BMP6) and matriptase-2, a protein encoded by the TMPRSS6 gene, in the induction and suppression of hepatic hepcidin expression, respectively. We examined their expression profiles in the liver and showed a predominant localization of BMP6 mRNA in nonparenchymal cells and exclusive expression of TMPRSS6 mRNA in hepatocytes. In rats fed an iron-deficient (ID) diet for 24 hours, the rapid decrease of transferrin saturation from 71% to 24% (control vs ID diet) was associated with a 100-fold decrease in hepcidin mRNA compared with the corresponding controls. These results indicated a close correlation of low transferrin saturation with decreased hepcidin mRNA. The lower phosphorylated Smad1/5/8 detected in the ID rat livers suggests that the suppressed hepcidin expression results from the inhibition of BMP signaling. Quantitative realtime reverse transcription polymerase chain reaction analysis revealed no significant change in either BMP6 or TM-PRSS6 mRNA in the liver. However, an increase in matriptase-2 protein in the liver from ID rats was detected, suggesting that suppression of hepcidin expression in response to acute iron deprivation is mediated by an increase in matriptase-2 protein levels. (Blood. 2011;117(5): 1687-1699) IntroductionHepcidin is the key iron regulatory peptide hormone in the maintenance of iron homeostasis. It is secreted predominantly by hepatocytes. 1,2 Under physiologic conditions, its expression is regulated positively by body iron content through the bone morphogenic protein (BMP)-mediated signaling cascade. [3][4][5] In recent studies researchers have identified several proteins that can modulate BMP signaling and hepcidin expression directly or indirectly.BMP2, 4, 5, 6, 7, and 9 are cytokines of the BMP subfamily that belong to the transforming growth factor-␤ (TGF-␤) superfamily. 6 Each of these BMP ligands induces BMP signaling through receptor-activated Smad1, Smad5, and Smad8 (Smad1/5/8) and markedly increases hepcidin expression in hepatocytes. 7,8 BMP2,4,5,and 6 can also bind hemojuvelin (HJV), a BMP coreceptor, to enhance BMP signaling, resulting in an increase in hepcidin expression. 4,7 HJV is a glycosylphosphatidyl-inositol-linked membrane protein that is expressed in skeletal muscle, heart, and hepatocytes, and it plays a pivotal role in the induction of hepcidin expression. [9][10][11] Both homozygous or compound heterozygous mutations in the HJV gene, HFE2, in humans and disruption of both Hfe2 alleles in mice result in suppression of hepcidin expression and severe iron overload in the liver, pancreas, and heart. 10,12,13 In addition to BMPs, TGF-␤1 can also induce hepatic hepcidin expression. 5 BMP6 mRNA, but no other BMP mRNA, is downregulated by chronic iron depletion and up-regulated by iron loading. 3 Knockdown of the BMP6 gene in mice causes suppression of hepatic hepcidin expression. 3,14,15 These observations implicate BMP6 as a critical player in the iron-sensitive induction of hepcidin expr...

Amycolatopsis mediterranei is used for industry-scale production of rifamycin, which plays a vital role in antimycobacterial therapy. As the first sequenced genome of the genus Amycolatopsis, the chromosome of strain U32 comprising 10 236 715 base pairs, is one of the largest prokaryotic genomes ever sequenced so far. Unlike the linear topology found in streptomycetes, this chromosome is circular, particularly similar to that of Saccharopolyspora erythraea and Nocardia farcinica, representing their close relationship in phylogeny and taxonomy. Although the predicted 9 228 protein-coding genes in the A. mediterranei genome shared the greatest number of orthologs with those of S. erythraea, it was unexpectedly followed by Streptomyces coelicolor rather than N. farcinica, indicating the distinct metabolic characteristics evolved via adaptation to diverse ecological niches. Besides a core region analogous to that common in streptomycetes, a novel 'quasi-core' with typical core characteristics is defined within the non-core region, where 21 out of the total 26 gene clusters for secondary metabolite production are located. The rifamycin biosynthesis gene cluster located in the core encodes a cytochrome P450 enzyme essential for the conversion of rifamycin SV to B, revealed by comparing to the highly homologous cluster of the rifamycin B-producing strain S699 and further confirmed by genetic complementation. The genomic information of A. mediterranei demonstrates a metabolic network orchestrated not only for extensive utilization of various carbon sources and inorganic nitrogen compounds but also for effective funneling of metabolic intermediates into the secondary antibiotic synthesis process under the control of a seemingly complex regulatory mechanism.

The Retroviral integrase (IN)ʈ catalyzes the integration of reversetranscribed viral DNA into the host genome in two steps called processing and joining (1-3). These reactions are chemically similar, proceeding via nucleophilic attack on the DNA by a donor hydroxyl group (water or ribose 3Ј OH), activated by the catalytic site of the enzyme. In vitro, these reactions require only divalent cations and water as cofactors. The in vitro reactions are fastest with Mn 2ϩ as the metal cofactor, although Mg 2ϩ also can be used. Because of its greater abundance in living cells, Mg 2ϩ is assumed to be the cofactor in vivo. Previous structural studies using the catalytic core domain of avian sarcoma virus (ASV) IN showed the presence of one cation of Mn 2ϩ or Mg 2ϩ

Random encounters between proteins in crowded cells are by no means passive, but found to be under selective control. This control enables proteome solubility, helps to optimise the diffusive search for interaction partners, and allows for adaptation to environmental extremes. Interestingly, the residues that modulate the encounters act mesoscopically through protein surface hydrophobicity and net charge, meaning that their detailed signatures vary across organisms with different intracellular constraints. To examine such variations, we use in-cell NMR relaxation to compare the diffusive behaviour of bacterial and human proteins in both human and Escherichia coli cytosols. We find that proteins that ‘stick’ in E. coli are generally less restricted in mammalian cells. Furthermore, the rotational diffusion in the mammalian cytosol is less sensitive to surface-charge mutations. This implies that, in terms of protein motions, the mammalian cytosol is more forgiving to surface alterations than E. coli cells. The cellular differences seem not linked to the proteome properties per se , but rather to a 6-fold difference in protein concentrations. Our results outline a scenario in which the tolerant cytosol of mammalian cells, found in long-lived multicellular organisms, provides an enlarged evolutionary playground, where random protein-surface mutations are less deleterious than in short-generational bacteria.

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