19 research outputs found

    The Structure Of The D49 Phospholipase A2 Piratoxin Iii From Bothrops Pirajai Reveals Unprecedented Structural Displacement Of The Calcium-binding Loop: Possible Relationship To Cooperative Substrate Binding

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    Snake venoms are rich sources of phospholipase A2 homologues, both active calcium-binding Asp49 enzymes and essentially inactive Lys49 proteins. They are responsible for multiple pharmacological effects, some of which are dependent on catalytic activity and others of which are not. Here, the 2.4 Å X-ray crystal structure of an active Asp49 phospholipase A2 from the venom of the snake Bothrops pirajai, refined to conventional and free R values of 20.1 and 25.5%, respectively, is reported. Unusually for phospholipases A2, the dependence of the enzyme rate on the substrate concentration is sigmoidal, implying cooperativity of substrate binding. The unprecedented structural distortion seen for the calcium-binding loop in the present structure may therefore be indicative of a T-state enzyme. An explanation of the interaction between the substrate-binding sites based on the canonical phospholipase A2 dimer is difficult. 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    On the verge of Umdeutung in Minnesota: Van Vleck and the correspondence principle (Part One)

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    In October 1924, the Physical Review, a relatively minor journal at the time, published a remarkable two-part paper by John H. Van Vleck, working in virtual isolation at the University of Minnesota. Van Vleck combined advanced techniques of classical mechanics with Bohr's correspondence principle and Einstein's quantum theory of radiation to find quantum analogues of classical expressions for the emission, absorption, and dispersion of radiation. For modern readers Van Vleck's paper is much easier to follow than the famous paper by Kramers and Heisenberg on dispersion theory, which covers similar terrain and is widely credited to have led directly to Heisenberg's "Umdeutung" paper. This makes Van Vleck's paper extremely valuable for the reconstruction of the genesis of matrix mechanics. It also makes it tempting to ask why Van Vleck did not take the next step and develop matrix mechanics himself.Comment: 82 page

    Prediction of Biomolecular Complexes

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    Almost all processes in living organisms occur through specific interactions between biomolecules. Any dysfunction of those interactions can lead to pathological events. Understanding such interactions is therefore a crucial step in the investigation of biological systems and a starting point for drug design. In recent years, experimental studies have been devoted to unravel the principles of biomolecular interactions; however, due to experimental difficulties in solving the three-dimensional (3D) structure of biomolecular complexes, the number of available, high-resolution experimental 3D structures does not fulfill the current needs. Therefore, complementary computational approaches to model such interactions are necessary to assist experimentalists since a full understanding of how biomolecules interact (and consequently how they perform their function) only comes from 3D structures which provide crucial atomic details about binding and recognition processes. In this chapter we review approaches to predict biomolecular complexes, introducing the concept of molecular docking, a technique which uses a combination of geometric, steric and energetics considerations to predict the 3D structure of a biological complex starting from the individual structures of its constituent parts. We provide a mini-guide about docking concepts, its potential and challenges, along with post-docking analysis and a list of related software

    Molecular basis for the fold organization and sarcomeric targeting of the muscle atrogin MuRF1

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    Contains fulltext : 137461.pdf (publisher's version ) (Open Access)MuRF1 is an E3 ubiquitin ligase central to muscle catabolism. It belongs to the TRIM protein family characterized by a tripartite fold of RING, B-box and coiled-coil (CC) motifs, followed by variable C-terminal domains. The CC motif is hypothesized to be responsible for domain organization in the fold as well as for high-order assembly into functional entities. But data on CC from this family that can clarify the structural significance of this motif are scarce. We have characterized the helical region from MuRF1 and show that, contrary to expectations, its CC domain assembles unproductively, being the B2- and COS-boxes in the fold (respectively flanking the CC) that promote a native quaternary structure. In particular, the C-terminal COS-box seemingly forms an alpha-hairpin that packs against the CC, influencing its dimerization. This shows that a C-terminal variable domain can be tightly integrated within the conserved TRIM fold to modulate its structure and function. Furthermore, data from transfected muscle show that in MuRF1 the COS-box mediates the in vivo targeting of sarcoskeletal structures and points to the pharmacological relevance of the COS domain for treating MuRF1-mediated muscle atrophy

    Mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha-amylases.

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    Despite the presence of a family of defense proteins, Phaseolus vulgaris can be attacked by bruchid insects resulting in serious damage to stored grains. The two distinct active forms of α-amylase inhibitors, α-AI1 and α-AI2, in P. vulgaris show different specificity toward α-amylases. Zabrotes subfasciatus α-amylase is inhibited by α-AI2 but not by α-AI1. In contrast, porcine α-amylase is inhibited by α-AI1 but not by α-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (α-AI1 and α-AI2) in relation to α-amylases. Mutants of α-AI2 were made and expressed in tobacco plants. The results showed that all the α-AI2 mutant inhibitors lost their activity against the insect α-amylases but none exhibited activity toward the mammalian α-amylase. The replacement of His33 of α-AI2 with the α-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus α-amylase. From structural modeling, the conclusion is that the size and complexity of the amylase-inhibitor interface explain why mutation of the N-terminal loop and resultant abolition of Z. subfasciatus α-amylase inhibition are not accompanied by gain of inhibitory activity against porcine α-amylase

    Glareosin: a novel sexually dimorphic urinary lipocalin in the bank vole, Myodes glareolus

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    The urine of bank voles (Myodes glareolus) contains substantial quantities of a small protein that is expressed at much higher levels in males than females, and at higher levels in males in the breeding season. This protein was purified and completely sequenced at the protein level by mass spectrometry. Leucine/isoleucine ambiguity was completely resolved by metabolic labelling, monitoring the incorporation of dietary deuterated leucine into specific sites in the protein. The predicted mass of the sequenced protein was exactly consonant with the mass of the protein measured in bank vole urine samples, correcting for the formation of two disulfide bonds. The sequence of the protein revealed that it was a lipocalin related to aphrodisin and other odorant-binding proteins (OBPs), but differed from all OBPs previously described. The pattern of secretion in urine used for scent marking by male bank voles, and the similarity to other lipocalins used as chemical signals in rodents, suggest that this protein plays a role in male sexual and/or competitive communication. We propose the name glareosin for this novel protein to reflect the origin of the protein and to emphasize the distinction from known OBPs

    Ferric Iron Uptake Genes Are Differentially Expressed In The Presence Of Copper Sulfides In Acidithiobacillus Ferrooxidans Strain Lr

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    Acidithiobacillus ferrooxidans is one of the most widely used microorganisms in bioleaching operations to recover copper from low-grade copper sulfide ores. This work aimed to investigate the relative expression of genes related to the iron uptake system when A. ferrooxidans LR was maintained in contact with chalcopyrite or bornite as the sole energy source. Real-time quantitative PCR analysis revealed that the presence of bornite had no effect on the expression of seven genes related to the siderophore-mediated Fe(III) uptake system, while in the presence of chalcopyrite the expression of the genes was up-regulated. Bioinformatic analysis of the genomic region where these genes were found revealed the existence of three new putative DNA-binding sequences for the ferric iron uptake transcriptional regulator (Fur). Electrophoretic mobility shift assays demonstrated that a purified A. ferrooxidans His-tagged Fur protein was able to bind in vitro to each of these putative Fur boxes, suggesting that Fur regulated the expression of these genes. The expression of fur and two known Fur-regulated genes, mntH and dsrK, was also investigated in the presence of chalcopyrite. While the expression of fur and mntH was up-regulated, the expression of dsrK was down-regulated. The low amount of ferrous iron in the medium was probably responsible for the up-regulation of fur and the genes related to the siderophore-mediated Fe(III) uptake system when A. ferrooxidans LR was kept in the presence of chalcopyrite. A homology model of the A. ferrooxidans Fur was constructed and revealed that the putative DNA-binding surface presents conserved positively charged residues, supporting a previously suggested mode of interaction with DNA. 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