Inhibition of hyphal development and kill of Candida albicans blastospores by noxythiolin in vitro

The cidal activity of the antimicrobial agent, noxythiolin, was investigated against a laboratory strain and a fresh isolate of Candida albicans. The order of resistance to noxythiolin was hyphal form (isolate) ≥ 25°C-grown blastospores (isolate) ≥ 37°C-grown blastospores (isolate) > laboratory strain blastospores. Noxythiolin activity was superior to that of 'equivalent' formaldehyde concentrations. Mycelial transformation in C. albicans was examined by light and scanning electron microscopy and measured in terms of percentage germination and hyphal extension. Noxythiolin, 2·5%, in contact for 30 min prevented germination of the blastospore population whereas the decomposition products, formaldehyde and N-methylthiourea, showed no appreciable effect in the expected concentrations. The implications of these results are discussed in relation to the observed clinical efficacy of noxythiolin.     

Concanavalin A distorts the beta-GlcNAc-(1-->2)-Man linkage of beta- GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man- (1-- >6)]-Man upon binding

Carbohydrate recognition by proteins is a key event in many biological processes. Concanavalin A is known to specifically recognize the pentasaccharide core (beta-GlcNAc-(1-->2)-alpha- Man-(1-->3)-[beta- GlcNAc-(1-->2)-alpha-Man-(1-->6)]-Man) of N-linked oligosaccharides with a Ka of 1.41 x 10(6 )M-1. We have determined the structure of concanavalin A bound to beta-GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta- GlcNAc-(1-->2)-alpha-Man- (1-->6)]-Man to 2.7A. In six of eight subunits there is clear density for all five sugar residues and a well ordered binding site. The pentasaccharide adopts the same conformation in all eight subunits. The binding site is a continuous extended cleft on the surface of the protein. Van der Waals interactions and hydrogen bonds anchor the carbohydrate to the protein. Both GlcNAc residues contact the protein. The GlcNAc on the 1-->6 arm of the pentasaccharide makes particularly extensive contacts and including two hydrogen bonds. The binding site of the 1-->3 arm GlcNAc is much less extensive. Oligosaccharide recognition by Con A occurs through specific protein carbohydrate interactions and does not require recruitment of adventitious water molecules. The beta-GlcNAc-(1-->2)-Man glycosidic linkage PSI torsion angle on the 1-->6 arm is rotated by over 50 degrees from that observed in solution. This rotation is coupled to disruption of interactions at the monosaccharide site. We suggest destabilization of the monosaccharide site and the conformational strain reduces the free energy liberated by additional interactions at the 1-->6 arm GlcNAc site.      

The Evolution and Structure Prediction of Coiled Coils across All Genomes

Coiled coils are α-helical interactions found in many natural proteins. Various sequence-based coiled-coil predictors are available, but key issues remain: oligomeric state and protein-protein interface prediction and extension to all genomes. We present SpiriCoil (http://supfam.org/SUPERFAMILY/spiricoil), which is based on a novel approach to the coiled-coil prediction problem for coiled coils that fall into known superfamilies: hundreds of hidden Markov models representing coiled-coil-containing domain families. Using whole domains gives the advantage that sequences flanking the coiled coils help. SpiriCoil performs at least as well as existing methods at detecting coiled coils and significantly advances the state of the art for oligomer state prediction. SpiriCoil has been run on over 16 million sequences, including all completely sequenced genomes (more than 1200), and a resulting Web interface supplies data downloads, alignments, scores, oligomeric state classifications, three-dimensional homology models and visualisation. This has allowed, for the first time, a genomewide analysis of coiled-coil evolution. We found that coiled coils have arisen independently de novo well over a hundred times, and these are observed in 16 different oligomeric states. Coiled coils in almost all oligomeric states were present in the last universal common ancestor of life. The vast majority of occasions that individual coiled coils have arisen de novo were before the last universal common ancestor of life; we do, however, observe scattered instances throughout subsequent evolutionary history, mostly in the formation of the eukaryote superkingdom. Coiled coils do not change their oligomeric state over evolution and did not evolve from the rearrangement of existing helices in proteins; coiled coils were forged in unison with the fold of the whole protein.     

Primary interactions of taurulatam solutions with a urinary tract clinical isolate of Escherichia coli and mammalian erythrocytes

The primary interaction of taurultam with both Escherichia coli (isolated from a urinary tract infection) and mammalian erythrocytes was studied at 37C. Using E. coli as the adsorbate, the adsorption of taurultam was observed to follow a constant partitioning (C) isotherm. However, the interaction between taurultam and mammalian erythrocytes was shown to follow a Langmuirian (L) pattern. It is postulated that this difference may be due to the hydrolysis of taurultam by the bacterial cell after adsorption with the subsequent liberation of formaldehyde and the metabolite, taurinamide.     

Designed coiled coils promote folding of a recombinant bacterial collagen

Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ～37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ～37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat.     

A de novo peptide hexamer with a mutable channel

The design of new proteins that expand the repertoire of natural protein structures represents a formidable challenge. Success in this area would increase understanding of protein structure and present new scaffolds that could be exploited in biotechnology and synthetic biology. Here we describe the design, characterization and X-ray crystal structure of a new coiled-coil protein. The de novo sequence forms a stand-alone, parallel, six-helix bundle with a channel running through it. Although lined exclusively by hydrophobic leucine and isoleucine side chains, the 6-? channel is permeable to water. One layer of leucine residues within the channel is mutable, accepting polar aspartic acid and histidine side chains, which leads to subdivision and organization of solvent within the lumen. Moreover, these mutants can be combined to form a stable and unique (Asp-His)(3) heterohexamer. These new structures provide a basis for engineering de novo proteins with new functions.     

Structural Capacitance in Protein Evolution and Human Diseases

Canonical mechanisms of protein evolution include the duplication and diversification of pre-existing folds through genetic alterations that include point mutations, insertions, deletions, and copy number amplifications, as well as post-translational modifications that modify processes such as folding efficiency and cellular localization. Following a survey of the human mutation database, we have identified an additional mechanism that we term “structural capacitance,” which results in the de novo generation of microstructure in previously disordered regions. We suggest that the potential for structural capacitance confers select proteins with the capacity to evolve over rapid timescales, facilitating saltatory evolution as opposed to gradualistic canonical Darwinian mechanisms. Our results implicate the elements of protein microstructure generated by this distinct mechanism in the pathogenesis of a wide variety of human diseases. The benefits of rapidly furnishing the potential for evolutionary change conferred by structural capacitance are consequently counterbalanced by this accompanying risk. The phenomenon of structural capacitance has implications ranging from the ancestral diversification of protein folds to the engineering of synthetic proteins with enhanced evolvability.     

Chemical synthesis and cytotoxicity of dihydroxylated cyclopentenone analogues of neocarzinostatin chromophore

Compounds containing the naphthoate moiety of Neocarzinostatin chromophore or 2-hydroxynaphthoate have been synthesized and evaluated for cytotoxic activity against a leukemia cell line and a small panel of human-tumor cell lines. Those compounds containing a cyclopentenone moiety were active, with the carbonyl group being essential for biological activity.     

Design and applicability of DNA arrays and DNA barcodes in biodiversity monitoring

Background  

The rapid and accurate identification of species is a critical component of large-scale biodiversity monitoring programs. DNA arrays (micro and macro) and DNA barcodes are two molecular approaches that have recently garnered much attention. Here, we compare these two platforms for identification of an important group, the mammals.