Isomerisation of 4-aryl-4-methylhex-5-en-2-ones to 5-aryl-4-methylhex-5-en-2-ones by an intramolecular ene-retro ene reaction sequence

Acid-catalysed thermal rearrangement of 4-aryl-4-methylhex-5-en-2-ones (products of the Claisen rearrangement of beta-methylcinnamyl alcohols and 2-methoxypropene) to isomeric 5-aryl-4-methylhex-5-en-2-ones via an intramolecular ene reaction of the enol tautomer followed by a retro ene reaction of the resultant acetylcyclopropane is described. Formation of the known diketone 13 via the ozonolysis of the rearrangement product 10, confirmed the structures of the rearranged enones, whereas formation of the enone 15 containing an extra methyl group on the styrene double bond confirmed the proposed mechanism. Finally, the rearrangement has been extended to the formal synthesis of beta-cuparenone 20 via the enones 22 and 23.

Primary structure of a cytotoxin-like basic protein from Naja naja naja (Indian Cobra) venom

The complete amino acid sequence of a cytotoxin-like basic protein (CLBP) from the venom of Naja naja naja (Indian Cobra) was determined by manual degradation using a 4-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method. Peptide fragments obtained by chemical cleavage with cyanogen bromide and enzymic cleavages with trypsin and Staphylococcus aureus proteases for sequence analysis were purified by reversed-phase chromatography. The total number of amino acid residues was 61, with leucine as the C-terminal residue. (C) Munksgaard 1995.

Purification and partial characterization of acyl carrier proteins from developing oil seeds of pisa (Actinodaphne hookeri) and ground nut (Arachis hypogaea)

Acyl carrier proteins (ACP) were purified to homogeneity in the active form from developing seeds of pisa (Actinodaphne hookeri) which synthesizes exclusively trilaurin and from ground nut (Arachis hypogaea) which synthesizes triacylglycerols containing long chain fatty acids. Two major isoforms of ACPs were purified from developing pisa seeds using DEAE-cellulose, Superose-6 FPLC and C-4 reversed phase HPLC chromatographic methods. In contrast, only a single form of ACP was present in ground nut seeds which was purified by anion-exchange and activated thiol-Sepharose 4B affinity chromatography. The two isoforms of ACPs from pisa showed nearly the same specific activity of 6,706 and 7,175 pmol per min per mg protein while ground nut ACP showed a specific activity of 3,893 pmol per min per mg protein when assayed using E. coli acyl-ACP synthetase and [1-C-14]palmitic acid. When compared with E. coli ACP, the purified ACPs from both the seeds showed considerable difference in their mobility in native PAGE, but showed similar mobility in SDS-PAGE under reducing conditions. In the absence of reducing agents formation of dimers was quite prominent. The ACPs from both the seed sources were acid- and heat-stable. The major isoform of pisa seed ACP and the ground nut ACP contain 91 amino acids with M(r) 11,616 and 1,228 respectively. However, there is significant variation in their amino acid composition. A comparision of the amino acid sequence in the N-terminal region of pisa and ground nut seed ACPs showed considerable homology between themselves and with other plant ACPs but not with E. coli ACP.

Primary Sequence That Determines the Functional Overlap between Mitochondrial Heat Shock Protein 70 Ssc1 and Ssc3 of Saccharomyces cerevisiae

The evolutionary diversity of the HSP70 gene family at the genetic level has generated complex structural variations leading to altered functional specificity and mode of regulation in different cellular compartments. By utilizing Saccharomyces cerevisiae as a model system for better understanding the global functional cooperativity between Hsp70 paralogs, we have dissected the differences in functional properties at the biochemical level between mitochondrial heat shock protein 70 (mtHsp70) Ssc1 and an uncharacterized Ssc3 paralog. Based on the evolutionary origin of Ssc3 and a high degree of sequence homology with Ssc1, it has been proposed that both have a close functional overlap in the mitochondrial matrix. Surprisingly, our results demonstrate that there is no functional cross-talk between Ssc1 and Ssc3 paralogs. The lack of in vivo functional overlap is due to altered conformation and significant lower stability associated with Ssc3. The substrate-binding domain of Ssc3 showed poor affinity toward mitochondrial client proteins and Tim44 due to the open conformation in ADP-bound state. In addition to that, the nucleotide-binding domain of Ssc3 showed an altered regulation by the Mge1 co-chaperone due to a high degree of conformational plasticity, which strongly promotes aggregation. Besides, Ssc3 possesses a dysfunctional inter-domain interface thus rendering it unable to perform functions similar to generic Hsp70s. Moreover, we have identified the critical amino acid sequence of Ssc1 and Ssc3 that can ``make or break'' mtHsp70 chaperone function. Together, our analysis provides the first evidence to show that the nucleotide-binding domain of mtHsp70s plays a critical role in determining the functional specificity among paralogs and orthologs across kingdoms.

Stochastic dynamics modeling of the protein sequence length distribution in genomes: implications for microbial evolution

In this paper, we report an analysis of the protein sequence length distribution for 13 bacteria, four archaea and one eukaryote whose genomes have been completely sequenced, The frequency distribution of protein sequence length for all the 18 organisms are remarkably similar, independent of genome size and can be described in terms of a lognormal probability distribution function. A simple stochastic model based on multiplicative processes has been proposed to explain the sequence length distribution. The stochastic model supports the random-origin hypothesis of protein sequences in genomes. Distributions of large proteins deviate from the overall lognormal behavior. Their cumulative distribution follows a power-law analogous to Pareto's law used to describe the income distribution of the wealthy. The protein sequence length distribution in genomes of organisms has important implications for microbial evolution and applications. (C) 1999 Elsevier Science B.V. All rights reserved.

Synthesis and structure of a novel terminal imido complex of titanium

The reaction of eta(5)-Cp*TiCl3 and (LiNHBu)-Bu-t in hexanes yields a novel [eta(5)-Cp*Ti(=(NBu)-Bu-t)((NHBu)-Bu-t)(2)]Li . (NH2Bu)-Bu-t complex with a terminal tert-butylimido moiety. The synthesis and X-ray structural characterization are described. (C) 1999 Elsevier Science S.A. All rights reserved.

Analysing Message Sequence Graph Specifications

We give a detailed construction of a finite-state transition system for a com-connected Message Sequence Graph. Though this result is well-known in the literature and forms the basis for the solution to several analysis and verification problems concerning MSG specifications, the constructions given in the literature are either not amenable to implementation, or imprecise, or simply incorrect. In contrast we give a detailed construction along with a proof of its correctness. Our transition system is amenable to implementation, and can also be used for a bounded analysis of general (not necessarily com-connected) MSG specifications.

iPBA: a tool for protein structure comparison using sequence alignment strategies

With the immense growth in the number of available protein structures, fast and accurate structure comparison has been essential. We propose an efficient method for structure comparison, based on a structural alphabet. Protein Blocks (PBs) is a widely used structural alphabet with 16 pentapeptide conformations that can fairly approximate a complete protein chain. Thus a 3D structure can be translated into a 1D sequence of PBs. With a simple Needleman-Wunsch approach and a raw PB substitution matrix, PB-based structural alignments were better than many popular methods. iPBA web server presents an improved alignment approach using (i) specialized PB Substitution Matrices (SM) and (ii) anchor-based alignment methodology. With these developments, the quality of similar to 88% of alignments was improved. iPBA alignments were also better than DALI, MUSTANG and GANGSTA(+) in > 80% of the cases. The webserver is designed to for both pairwise comparisons and database searches. Outputs are given as sequence alignment and superposed 3D structures displayed using PyMol and Jmol. A local alignment option for detecting subs-structural similarity is also embedded. As a fast and efficient `sequence-based' structure comparison tool, we believe that it will be quite useful to the scientific community. iPBA can be accessed at http://www.dsimb.inserm.fr/dsimb_tools/ipba/.

Crystal structure of Rv2118c: an AdoMet-dependent methyltransferase from Mycobacterium tuberculosis H37Rv

Rv2118c belongs to the class of conserved hypothetical proteins from Mycobacterium tuberculosis H37Rv. The crystal structure of Rv2118c in complex with S-adenosyl-Image -methionine (AdoMet) has been determined at 1.98 Å resolution. The crystallographic asymmetric unit consists of a monomer, but symmetry-related subunits interact extensively, leading to a tetrameric structure. The structure of the monomer can be divided functionally into two domains: the larger catalytic C-terminal domain that binds the cofactor AdoMet and is involved in the transfer of methyl group from AdoMet to the substrate and a smaller N-terminal domain. The structure of the catalytic domain is very similar to that of other AdoMet-dependent methyltransferases. The N-terminal domain is primarily a β-structure with a fold not found in other methyltransferases of known structure. Database searches reveal a conserved family of Rv2118c-like proteins from various organisms. Multiple sequence alignments show several regions of high sequence similarity (motifs) in this family of proteins. Structure analysis and homology to yeast Gcd14p suggest that Rv2118c could be an RNA methyltransferase, but further studies are required to establish its functional role conclusively.

The destabilization of human GCAP1 by a proline to leucine mutation might cause cone-rod dystrophy

Guanylate cyclase activating protein-1 (GCAP1) is required for activation of retinal guanylate cyclase-1 (RetGC1), which is essential for recovery of photoreceptor cells to the dark state. In this paper, experimentally derived observations are reported that help in explaining why a proline→leucine mutation at position 50 of human GCAP1 results in cone–rod dystrophy in a family carrying this mutation. The primary amino acid sequence of wild-type GCAP1 was mutated using site-directed mutagenesis to give a leucine at position 50. In addition, serine replaced a glutamic acid residue at position 6 to promote N‐terminal myristoylation, yielding the construct GCAP1 E6S/P50L. The enzyme was over-expressed in Escherichia coli cells, isolated and purified before being used in assays with RetGC1, characterized by circular dichroism (CD) spectroscopy, and investigated for protease resistance and thermal stability. Assays of cyclic guanosine monophosphate (cGMP) synthesis from guanosine triphosphate by RetGC1 in the presence of E6S/P50L showed that E6S/P50L could activate RetGC1 and displayed similar calcium sensitivity to wild-type GCAP1. In addition, E6S/P50L and wild-type GCAP1 possess similar CD spectra. However, there was a marked increase in the susceptibility to protease degradation and also a reduction in the thermal stability of E6S/P50L as observed by both the cGMP assay and CD spectroscopy. It is therefore suggested that although GCAP1 E6S/P50L has a similar activity and calcium dependency profile to the wild-type GCAP1, its lower stability could reduce its cellular concentration, which would in turn alter [Ca2+] and result in death of cells.

Cloning, expression, purification and preliminary X-ray diffraction studies of a putative Mycobacterium smegmatis thiolase

Thiolases are important in fatty-acid degradation and biosynthetic pathways. Analysis of the genomic sequence of Mycobacterium smegmatis suggests the presence of several putative thiolase genes. One of these genes appears to code for an SCP-x protein. Human SCP-x consists of an N-terminal domain (referred to as SCP2 thiolase) and a C-terminal domain (referred as sterol carrier protein 2). Here, the cloning, expression, purification and crystallization of this putative SCP-x protein from M. smegmatis are reported. The crystals diffracted X-rays to 2.5 angstrom resolution and belonged to the triclinic space group P1. Calculation of rotation functions using X-ray diffraction data suggests that the protein is likely to possess a hexameric oligomerization with 32 symmetry which has not been observed in the other six known classes of this enzyme.

Enantiospecific synthesis of functionalised bicyclo[3.3.1] nonanes from 10-bromocarvones via tandem intermolecular alkylation-intramolecular Michael addition sequence

A simple and convenient tandem methodology for the enantiospecific generation of functionalised bicyclo[3.3.1] nonanes 9,14-18, via intermolecular alkylation of Michael donors with 10-bromocarvones 7, 10 and 11, followed by intramolcular Michael addition, is achieved. An unsuccessful attempt for the extension of the methodology for a possible short enantiospecific approach to AB-ring system 22 of taxanes via the allyl bromide 21, is also described.