Towards a complete description of the structural and dynamic properties of the denatured state of barnase and the role of residual structure in folding.

The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models using conventional techniques. Molecular dynamics simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here, we describe three independent 4 ns high-temperature molecular dynamics simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the beta(3-4) hairpin contained a particularly high degree of such side-chain interactions but it lacked beta-structure in two of the three denatured ensembles: beta(3-4) was the only portion of the beta-structure to contain significant residual structure in the denatured state. The two principal alpha-helices (alpha1 and alpha2) adopted dynamic helical structure. In addition, there were persistent contacts that pinched off core 2 from the body of the protein. The rest of the protein was unstructured, aside from transient and mostly local side-chain interactions. Overall, the simulated denatured state contains residual structure in the form of dynamic, fluctuating secondary structure in alpha1 and alpha2, as well as fluctuating tertiary contacts in the beta(3-4) region, and between alpha1 and beta(3-4), in agreement with previous NMR studies. Here, we also show that these regions containing residual structure display impaired mobility by both molecular dynamics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between beta(3-4) and alpha1 assisted in the refolding of alpha1. This contact-assisted helix formation was confirmed in fragment simulations of beta(3-4) and alpha1 alone and complexed, and, as such, alpha1 and beta(3-4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that earlier time-points from the simulations provide models of the major intermediate and transition states in quantitative agreement with data from both unfolding and refolding experiments. Both beta(3-4) and alpha1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further beta-strands pack. The beta-structure condenses about beta(3-4), while alpha1 aids in stabilizing beta(3-4) and maintaining its orientation. The resulting beta-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the beta-sheet twists, leading to the major transition state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensation of the structure then occurs to yield the native state.

The effects of disulfide bonds on the denatured state of barnase.

The effects of engineered disulfide bonds on protein stability are poorly understood because they can influence the structure, dynamics, and energetics of both the native and denatured states. To explore the effects of two engineered disulfide bonds on the stability of barnase, we have conducted a combined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain the denatured state. However, specific extended beta-sheet structure can also be detected in one of the mutant proteins. This mutant is also more stable than would be predicted. Our study suggests a possible cause of the very high stability conferred by this disulfide bond: the wild-type denatured ensemble is stabilized by a nonnative hydrophobic cluster, which is constrained from occurring in the mutant due to the formation of secondary structure.

Identification and distribution of a novel Malassezia species yeast on normal equine skin.

This study aimed to investigate the distribution of Malassezia species yeasts on the skin of healthy horses. Acetate tape samples were obtained from the lip, axilla, interbulbar region, groin and anus of 12 healthy horses. The samples were stained and examined microscopically and sites harbouring yeast-like organisms were identified. Contact plates were applied to the skin at these sites and cultured at 26 degrees C and 32 degrees C. No growth was obtained on horse blood, Sabouraud's dextrose or modified Dixon's agar. A pure growth of a Malassezia-type organism was obtained on Sabouraud's dextrose agar enriched with oleic acid when it was incubated at 30 degrees C. It was identified by 26S ribosomal DNA D1/D2 sequence analysis as a member of the genus Malassezia, and most closely related to Malassezia sympodialis. However, the level of sequence divergence indicated that it was a novel species.

Selective separation of microorganisms by lectins: yeast and concanavalin A as a model system.

Specific aggregation and separation of microorganisms was investigated using yeasts and concanavalin A as a model system. Cells of Saccharomyces cerevisiae were specifically aggregated and so separated from those of Schizosaccharomyces pombe. Optimum aggregation with over 99% of cells aggregated was achieved by adjustment to pH value and applied agitation. Dimeric lectin structure caused a far higher degree of aggregation than did tetrameric. Degree of aggregation was also strongly influenced by the ratio of lectin/cell concentrations, optimum aggregation occurring in the middle range of ratios. A high ratio of lectin to cells inhibited aggregation, occupation of most of the available receptors preventing intercellular bonding by divalent lectins. Detachment and reuse of concanavalin A was demonstrated using switching from moderate to low pH value. Potential uses for species-specific-separation of microorganisms are discussed.

Candida sorbosivorans sp. nov., a new member of the genus Candida Berkhout.

A yeast, strain NCYC 2938T, was isolated from contaminated industrial material. This material was involved in a cascade continuous process for oxidizing sorbitol (D-glucitol) to L-sorbose. The isolate is similar, although not identical, to Candida geochares and Candida magnoliae in its physiological characteristics. Sequence analysis of the 26S rDNA D1/D2 variable domain showed that it was similar to those of both Candida species, but differed sufficiently to be considered as a separate species. Both the physiological characteristics and the unique 26S rDNA D1/D2 sequence of NCYC 2938T are described here, and the yeast has been named Candida sorbosivorans sp. nov. The type strain is NCYC 2938T (= CBS 8768T).

Characterization of residual structure in the thermally denatured state of barnase by simulation and experiment: description of the folding pathway.

Residual structure in the denatured state of a protein may contain clues about the early events in folding. We have simulated by molecular dynamics the denatured state of barnase, which has been studied by NMR spectroscopy. An ensemble of 10(4) structures was generated after 2 ns of unfolding and following for a further 2 ns. The ensemble was heterogeneous, but there was nonrandom, residual structure with persistent interactions. Helical structure in the C-terminal portion of helix alpha1 (residues 13-17) and in helix alpha2 as well as a turn and nonnative hydrophobic clustering between beta3 and beta4 were observed, consistent with NMR data. In addition, there were tertiary contacts between residues in alpha1 and the C-terminal portion of the beta-sheet. The simulated structures allow the rudimentary NMR data to be fleshed out. The consistency between simulation and experiment inspires confidence in the methods. A description of the folding pathway of barnase from the denatured to the native state can be constructed by combining the simulation with experimental data from phi value analysis and NMR.

Determinants of allosteric activation of yeast pyruvate kinase and identification of novel effectors using computational screening.

We have analyzed the structural determinants of the allosteric activation of yeast pyruvate kinase (YPK) by mutational and kinetic analysis and initiated a structure-based design project to identify novel effectors that modulate its allosteric response by binding to the allosteric site for fructose-1,6-bisphosphate (FBP). The wild-type enzyme is strongly activated by fructose-1,6-bisphosphate and weakly activated by both fructose-1-phosphate and fructose-6-phosphate; the strength of the activation response is proportional to the affinity of the allosteric effector. A point mutation within the 6'-phosphate binding loop of the allosteric site (T403E) abolishes activation of the enzyme by fructose-1, 6-bisphosphate. The mutant enzyme is also not activated by F1P or F6P. The mutation alone (which incorporates a glutamic acid that is strictly conserved in mammalian M1 isozymes) slightly reduces cooperativity of substrate binding. Three novel compounds were identified that effect the allosteric regulation of YPK by FBP and/or act as novel allosteric activators of the enzyme. One is a physiologically important diphospho sugar, while the other two are hydrophobic compounds that are dissimilar to the natural effector. These results demonstrate that novel allosteric effectors may be identified using structure-based screening and are indicative of the potential of this strategy for drug discovery. Regulatory sites are generally more divergent than catalytic sites and therefore offer excellent opportunities for discrimination and specificity between different organisms or between different tissue types.

Candida davenportii sp. nov., a potential soft-drinks spoilage yeast isolated from a wasp.

During a survey of yeast ecology in a soft-drinks production facility, a dead wasp was removed from the sampling tap of an external sugar-syrup storage tank. A yeast isolated from the dead wasp was found to be similar, although not identical, in its physiological characteristics to Candida lactis-condensi and Candida stellata. Sequence analysis of the 26S rDNA D1/D2 variable domain revealed that this isolate was most closely related to C stellata, but differed sufficiently in its D1/D2 sequence to indicate that it belonged to a separate species. The yeast species has been named Candida davenportii sp. nov.; the type strain is NCYC 3013T (= CBS 9069T). C davenportii sp. nov. was osmotolerant, moderately preservative-resistant and able to grow in very acidic conditions, i.e. pH 14. This yeast grew well in fruit-containing soft drinks, cola-type beverages and a synthetic soft drink and is therefore a potential cause of spoilage of soft drinks and other sugary food products. Other related yeast species in the same taxonomic clade as C davenportii sp. nov. are also osmotolerant, growing in < 50% (w/v) sugar. Many of these species are associated with insects, specifically bees, bumblebees and leafcutter bees, and many have been reported as the causative agent of spoilage of sugary foods, such as condensed milk, fruit juices and concentrates. It is proposed that C davenportii sp. nov. and other closely related yeasts are primarily associated with Aculeates (bees and wasps). In turn, bees and wasps are attracted by sugary residues in foods such as fruit juices and concentrates, forming the source of infection of these yeasts and thus instigating spoilage.

Cloning, sequence and crystallographic structure of recombinant iron superoxide dismutase from Pseudomonas ovalis.

The gene encoding the iron-dependent superoxide dismutase from Pseudomonas ovalis was cloned from a genomic library and sequenced. The ORF differs from the previously published protein sequence, which was used for the original structure determination, at 16 positions. The differences include three additional inserted residues, one deleted residue and 12 point substitutions. The gene was subcloned and the recombinant protein overexpressed, purified and crystallized in a trigonal space group. The structure was determined by molecular replacement and was refined to 2.1 A resolution.

Zygosaccharomyces lentus sp. nov., a new member of the yeast genus Zygosaccharomyces Barker.

Unusual growth characteristics of a spoilage yeast, originally isolated from spoiled whole-orange drink and previously identified as Zygosaccharomyces bailii, prompted careful re-examination of its taxonomic position. Small-subunit rRNA gene sequences were determined for this strain and for four other strains also originally described as Z. bailii but which, in contrast to other strains of this species, grew poorly or not at all under aerobic conditions with agitation, failed to grow in the presence of 1% acetic acid and failed to grow at 30 degrees C. Comparative sequence analysis revealed that these strains represented a phylogenetically distinct taxon closely related to, but distinct from, Z. bailii and Zygosaccharomyces bisporus. Furthermore, sequence analysis of the internal transcribed spacer (ITS) region showed that, while all five strains had identical ITS2 sequences, they could be subdivided into two groups based on ITS1 sequences. Despite such minor inter-strain sequence variation, these yeasts could readily be distinguished from all other currently described Zygosaccharomyces species by using ITS sequences. On the basis of the phylogenetic results presented, a new species comprising the five strains, Zygosaccharomyces lentus sp. nov., is described and supporting physiological data are discussed, including a demonstration that growth of this species is particularly sensitive to the presence of oxygen. The type strain of Z. lentus is NCYC D2627T.