Solution structure and refolding of the Mycobacterium tuberculosis pentapeptide repeat protein MfpA

The pentapeptide repeat is a recently discovered protein fold. Mycobacterium tuberculosis MfpA is a founding member of the pentapeptide repeat protein (PRP) family that confers resistance to the antibiotic fluoroquinolone by binding to DNA gyrase and inhibiting its activity. The size, shape, and surface potential of MfpA mimics duplex DNA. As an initial step in a comprehensive biophysical analysis of the role of PRPs in the regulation of cellular topoisomerase activity and conferring antibiotic resistance, we have explored the solution structure and refolding of MfpA by fluorescence spectroscopy, CD, and analytical centrifugation. A unique CD spectrum for the pentapeptide repeat fold is described. This spectrum reveals a native structure whose beta-strands and turns within the right-handed quadrilateral beta-helix that define the PRP fold differ from canonical secondary structure types. MfpA refolded from urea or guanidium by dialysis or dilution forms stable aggregates of monomers whose secondary and tertiary structure are not native. In contrast, MfpA refolded using a novel "time-dependent renaturation" protocol yields protein with native secondary, tertiary, and quaternary structure. The generality of "time-dependent renaturation" to other proteins and denaturation methods is discussed.     

Tritrichomonas foetus from domestic cats and cattle are genetically distinct

The genetic relationship amongst Tritrichomonas foetus isolated from domestic cats and cattle was investigated by DNA sequencing of the internal transcribed region of the ribosomal DNA unit and the TR7/TR8 variable-length repeat. The results reject the hypothesis that T. foetus from domestic cats is genetically identical to T. foetus in cattle. We suggest recognition of a ‘cat genotype’ and a ‘cattle genotype’ of T. foetus. Review of public nucleotide repositories revealed that the ‘cat genotype’ has not been isolated from cattle nor the ‘cattle genotype’ recovered from cats. However, at least one cat isolate has been shown to induce disease in experimentally infected cattle. We conclude that these genotypes fall within the single species T. foetus.     

Mycobacterium fluoroquinolone resistance protein B,a novel small GTPase,is involved in the regulation of DNA gyrase and drug resistance

DNA gyrase plays a vital role in resolving DNA topological problems and is the target of antibiotics such as fluoroquinolones. Mycobacterium fluoroquinolone resistance protein A (MfpA) from Mycobacterium smegmatis is a newly identified DNA gyrase inhibitor that is believed to confer intrinsic resistance to fluoroquinolones. However, MfpA does not prevent drug-induced inhibition of DNA gyrase in vitro, implying the involvement of other as yet unknown factors. Here, we have identified a new factor, named Mycobacterium fluoroquinolone resistance protein B (MfpB), which is involved in the protection of DNA gyrase against drugs both in vivo and in vitro. Genetic results suggest that MfpB is necessary for MfpA protection of DNA gyrase against drugs in vivo; an mfpB knockout mutant showed greater susceptibility to ciprofloxacin than the wild-type, whereas a strain overexpressing MfpA and MfpB showed higher loss of susceptibility. Further biochemical characterization indicated that MfpB is a small GTPase and its GTP bound form interacts directly with MfpA and influences its interaction with DNA gyrase. Mutations in MfpB that decrease its GTPase activity disrupt its protective efficacy. Our studies suggest that MfpB, a small GTPase, is required for MfpA-conferred protection of DNA gyrase.     

Pentapeptide repeat proteins

The pentapeptide repeat protein (PRP) family has more than 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S,T,A,V][D,N][L,F][S,T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Mycobacterium tuberculosis. The structure revealed that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral beta-helix. MfpA binds to DNA gyrase and inhibits its activity. The rod-shaped, dimeric protein exhibits remarkable similarity in size, shape, and electrostatics to DNA.     

Direct unfolding of RuvA-HJ complex at the single-molecule level

The repair of double-stranded DNA breaks via homologous recombination involves a four-way cross-strand intermediate known as Holliday junction (HJ), which is recognized, processed, and resolved by a specific set of proteins. RuvA, a prokaryotic HJ-binding protein, is known to stabilize the square-planar conformation of the HJ, which is otherwise a short-lived intermediate. Despite much progress being made regarding the molecular mechanism of RuvA-HJ interactions, the mechanochemical aspect of this protein-HJ complex is yet to be investigated. Here, we employed an optical-tweezers-based, single-molecule manipulation assay to detect the formation of RuvA-HJ complex and determined its mechanical and thermodynamic properties in a manner that would be impossible with traditional ensemble techniques. We found that the binding of RuvA increases the unfolding force (F_unfold) of the HJ by ～2-fold. Compared with the ΔG_unfold of the HJ alone (54 ± 13 kcal/mol), the increased free energy of the RuvA-HJ complex (101 ± 20 kcal/mol) demonstrates that the RuvA protein stabilizes HJs. Interestingly, the protein remains bound to the mechanically melted HJ, facilitating its refolding at an unusually high force when the stretched DNA molecule is relaxed. These results suggest that the RuvA protein not only stabilizes the HJs but also induces refolding of the HJs. The single-molecule platform that we employed here for studying the RuvA-HJ interaction is broadly applicable to study other HJ-binding proteins involved in the critical DNA repair process.     

Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata

Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from ‘Wuzishatangju’ (self-incompatible, SI) and ‘Shatangju’ (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between ‘Wuzishatangju’ and ‘Shatangju’, respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of ‘Wuzishatangju’ and ‘Shatangju’. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of ‘Shatangju’ was approximately 10-fold higher than in anthers of ‘Wuzishatangju’. The highest expression level of CrUBE1 was detected in pistils at 7 days after self-pollination of ‘Wuzishatangju’, which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from ‘Wuzishatangju’ and ‘Shatangju’ were successfully expressed in Pichia pastoris. Pollen germination frequency of ‘Wuzishatangju’ was significantly inhibited with increasing of CrUBE1 protein concentrations from ‘Wuzishatangju’.     

Structural characterization of the fusion of two pentapeptide repeat proteins, Np275 and Np276, from Nostoc punctiforme: resurrection of an ancestral protein

The Nostoc punctiforme genes Np275 and Np276 are two adjacently encoded proteins of 98 and 75 amino acids in length and exhibit sequences composed of tandem pentapeptide repeats. The structures of Np275 and a fusion of Np275 and Np276 were determined to 2.1 and 1.5 A, respectively. The two Nostoc proteins fold as highly symmetric right-handed quadrilateral beta-helices similar to the mycobacterial protein MfpA implicated in fluoroquinolone resistance and DNA gyrase inhibition. The sequence composition of the intervening coding region and the ability to express a fused protein by removing the stop codon for Np275 suggests Np275 and Np276 were recently part of a larger ancestral pentapeptide repeat protein.     

Assimilate translocation and expression of sucrose transporter,OsSUT1, contribute to high-performance ripening under heat stress in the heat-tolerant rice cultivar Genkitsukushi

High temperature reduces the grain quality of rice, a situation likely to become more frequent because of global warming. We studied the effects of high-temperature stress on grain quality of heat-tolerant cultivar ‘Genkitsukushi’ and heat-sensitive cultivar ‘Tsukushiroman’. When day/night temperatures were 31/26 °C from heading until maturity, the grain quality of ‘Genkitsukushi’ was rated at the first inspection grade (high quality), whereas ‘Tsukushiroman’ showed a remarkable increase in the percentage of white immature kernels (low quality). Nonstructural carbohydrate content in the stem of ‘Genkitsukushi’ the early maturation was significantly higher than in ‘Tsukushiroman’ and greatly decreased under high temperature. From 14 to 21 days after heading, the expression of the sucrose transporter gene, OsSUT1, was higher in the stem of ‘Genkitsukushi’ grown under high temperature than in ‘Tsukushiroman’. In addition, the expression of OsSUT1 in the grains of ‘Genkitsukushi’ was significantly higher than in ‘Tsukushiroman’ during the ripening period. These results indicate that sugar transport functions more effectively in ‘Genkitsukushi’ than in ‘Tsukushiroman’, and that the effectiveness of sugar transport contributes to maintaining high grain quality in ‘Genkitsukushi’ under high-temperature conditions.     

Preliminary study of genetic variation in Hawaiian isolates of Beauveria bassiana [Hypocreales, Cordycipitaceae

There have been no previous surveys documenting genetic diversity in Beauveria bassiana (Balsamo) Vuillemin in Hawaii. We used PCR primers and DNA sequencing to genetically characterize 14 isolates of B. bassiana collected from insects in east Hawaii island (the largest Hawaiian island, known as the ‘Big Island’) and compared these with the ‘GHA’ strain found in the commercial product BotaniGard®. Twelve of the 14 Hawaiian isolates were unique and the GHA strain was not among those isolated from the wild. Our data provides evidence that genetic diversity of B. bassiana in Hawaii is high over small spatial scales.     

Genetic diversity of a relict plant species, Ligularia sibirica (L.) Cass. (Asteraceae)

Rare plant species can be divided into naturally, ‘old rare’ species and anthropogenically, ‘new rare’ species. Many recent studies explored genetic diversity of ‘new rare’ species. Less is, however, known about genetic diversity of ‘old rare’ species. We examined isozyme genetic variability of 20 populations of an ‘old rare’ plant species, Ligularia sibirica (Asteraceae) in the Czech and Slovak Republic. It is a long-lived perennial herb with mixed-mating breeding system, widely distributed from East Asia to European Russia, with few isolated relict populations in the remaining part of Europe.The results showed high genetic diversity within populations (80.8%) and a low level of genetic differentiation (F_ST = 0.179). Genetic distance between populations correlated significantly with geographic distance. There was also a significant positive correlation between genetic diversity and population size. This is probably caused by destruction of habitats in last centuries and subsequent decrease of population size. Patterns of genetic diversity suggest that the recent distribution is a result of stepwise postglacial migration of the species and subsequent natural fragmentation.We conclude that L. sibirica populations preserve high levels of genetic diversity and are not yet threatened by genetic factors. However, this may change if changes in habitat conditions continue.     

Chimerical nature of the ribosomal RNA gene of a Nosema species

Taxonomic resolution of the Nosema/Vairimorpha clade has been augmented with DNA sequences of the small subunit (SSU) and large subunit (LSU) ribosomal RNA (rRNA) and the arrangement of SSU and LSU. Based on the two characteristics, the clade is largely divided into two, i.e. ‘true’ Nosema sub-group and non-‘true’ Nosema sub-group within the clade. Our study shows that a novel Nosema species isolated from Pieris rapae has mixed characteristics of the ‘true’ and non-‘true’ Nosema sub-group based on the topology of SSU and LSU sequences. To our knowledge, this may be the first case of the incongruent phylogenetic placement of SSU and LSU in the Nosema/Vairimorpha clade. Additionally, the length of internal transcribed spacer (ITS) can be a diagnostic tool to distinguish ‘true’ Nosema from non-’true’ Nosema in the Nosema/Vairimorpha clade based on its nucleotide length as reported before.     

Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength

DSC analysis has been used to quantify the reversibility of unfolding following thermal denaturation of lysozyme. Since the temperature at which protein unfolding occurs, T_m, varies with different solution conditions, the effect on the melting temperature and the degree of refolding after thermal denaturation in low ionic strength sodium phosphate buffers (5–1000 mM) over a range of pH (5–9) in the presence/absence of disaccharides is examined. This study compares the enthalpies of unfolding during successive heating cycles to quantify reversibility following thermal denaturation. The disaccharides, trehalose and maltose were used to assess if the disaccharide induced increase in T_m is reflected in the reversibility of thermally induced denaturation. There was extensive overlap between the T_m values where non-reversible and reversible thermal denaturation occurred. Indeed, for pH 6, at the highest and lowest T_m, no refolding was observed whereas refolding was observed for intermediate values, but with similar T_m values having different proportions of refolded protein. We established a method to measure the degree of reversible unfolding following thermal denaturation and hence indirectly, the degree to which protein is lost to irreversible aggregation, and show that solution conditions which increase melt transition temperatures do not automatically confer an increase in reversibility. This type of analysis may prove useful in assessing the stability of proteins in both the biopharmaceutical and food industries.     

DFT conformation and energies of amylose fragments at atomic resolution. Part 1: syn forms of α-maltotetraose

DFT optimization studies of 90 syn α-maltotetraose (DP-4) amylose fragments have been carried out at the B3LYP/6-311++G∗∗ level of theory. The DP-4 fragments studied include V-helix, tightly bent conformations, a boat, and a ¹C₄ conformer. The standard hydroxymethyl rotamers (gg, gt, tg) were examined at different locations in the residue sequence, and their influence on the bridge conformations ?/ψ values and conformer energy is described. Hydroxyl groups were considered to be homodromic, that is, they are either in the all clockwise, ‘c’, or all counterclockwise, ‘r’. Energy differences between conformations are examined in order to assess the stability of the different conformations and to identify the sources of energy that dictate amylose polymer formation. A small nearly cyclic compact structure is of low energy as one would expect when these flexible molecules are studied in vacuo. Many conformations in which the only differences are a single hydroxymethyl variation in the residue sequence show similar energies and bridge conformations, with trends being a result of the hydroxymethyl as well as hydroxyl orientation. In general the ‘c’ structures are of lower energy than the ‘r’ structures, although this is only true for the in vacuo state. The solvent dependence on conformational preference of several low-energy DP-4 structures was investigated via the continuum solvation method COSMO. These results suggest that the ‘r’ structures may be favored for fully solvated molecules.     

Characterization of the complete mitochondrial genome of Tryporyza incertulas, in comparison with seven other Pyraloidea moths

The complete 15,223-bp mitochondrial genome (mitogenome) of Tryporyza incertulas (Walker) (Lepidoptera: Pyraloidea: Crambidae) was determined, characterized and compared with seven other species of superfamily Pyraloidea. The order of 37 genes was typical of insect mitochondrial DNA sequences described to date. Compared with other moths of Pyraloidea, the A + T biased (77.0%) of T. incertulas was the lowest. Eleven protein-coding genes (PCGs) utilized the standard ATN, but cox1 used CGA and nad4 used AAT as the initiation codons. Ten protein-coding genes had the common stop codon TAA, except nad3 having TAG as the stop codon, and cox2, nad4 using T, TA as the incomplete stop codons, respectively. All of the tRNA genes had typical cloverleaf secondary structures except trnS1(AGN), in which the dihydrouridine (DHU) arm did not form a stable stem-loop structure. There was a spacer between trnQ and nad2, which was common in Lepidoptera moths. A 6-bp motif ‘ATACTA’ between trnS2(UCN) and nad1, a 7-bp motif “AGC(T)CTTA” between trnW and trnC and a 6-bp motif “ATGATA” of overlapping region between atp8 and atp6 were found in Pyraloidea moths. The A + T-rich region contained an ‘ATAGT(A)’-like motif followed by a poly-T stretch. In addition, two potential stem-loop structures, a duplicated 19-bp repeat element, and two microsatellites ‘(TA)₁₂’ and ‘(TA)₉’ were observed in the A + T-rich region of T. incertulas mitogenome. Finally, the phylogenetic relationships of Pyraloidea species were constructed based on amino acid sequences of 13 PCGs of mitogenomes using Bayesian inference (BI) and maximum likelihood (ML) methods. These molecular-based phylogenies supported the morphological classification on relationships within Pyraloidea species.     

Mating,insemination, and ovulation in the tsetse fly, Glossina morsitans

Virgin females of the tsetse fly, Glossina morsitans orientalis, retain their first egg within the right ovary whereas mated females ovulate. The component of the mating act which causes ovulation and thereby initiates the ovarian cycles which follow, is not insemination, the construction of a spermatophore in the uterus, or a humoral factor associated with the testes, accessory glands or ejaculatory ducts of donor males, or ‘full’ spermathecae of donor females. The ovulation rate increases with copulation time and females are shown to ‘add up’ their ‘sexual experience’ independently of that of the males. Inter-specific matings between G. morsitans and G. austeni also result in ovulation, but an unidentified factor associated with the completion of the mating act was also apparent. It is suggested that a ‘mechanical’ factor probably with nervous and endocrine components is responsible for the release of the egg from the ovary. Observations on the construction of empty spermatophores by aspermic males of G. morsitans are included.     

Genetic Relationships of Ornamental Cultivars of Ginkgo biloba Analyzed by AFLP Techniques

Eight primer combinations that produced clear and a large number of polymorphic bands were screened from 64 EcoR I/Mse I primer combinations (Mse I fluorescent labeled). The genetic relationships of 21 ornamental cultivars of Ginkgo biloba L. from the United States of America, Holland, Japan, France, and China were analyzed. These primer combinations produced a total of 1 119 bands, 229 specific loci (including 54 absent bands, and 175 monomorphic bands). Among them, 983 polymorphic bands (PPB), accounting for 88%, were detected. The percentage of identification per primer combination was as high as 100%. The average PPB of 14 foreign cultivars was 35.86% and the average PPB of seven domestic cultivars was 31.51%. Genetic similarity coefficient (SC) among all cultivars varied from 0.4899 to 0.8499, and all cultivars were divided into the four clusters when SC was set at 0.7300. The cultivars from the same origin did not fall into the same group. The cultivars from France and China were classified into three groups. According to the comprehensive analyses based on specific loci, similarity coefficient, and clustering results, eight cultivars ‘Fastigiata’, ‘Tit’, ‘Tubifolia’, ‘Daeryinxing’, ‘Variegata’, ‘Horizontalis, ‘Pendula’, and ‘Yiyuanyeziyinxing’ were considered to be important germplasms of ornamental cultivars of Ginkgo biloba.     

DFT conformation and energies of amylose fragments at atomic resolution. Part 2: ‘band-flip’ and ‘kink’ forms of α-maltotetraose

In Part 2 of this series of DFT optimization studies of α-maltotetraose, we present results at the B3LYP/6-311++G∗∗ level of theory for conformations denoted ‘band-flips’ and ‘kinks’. Recent experimental X-ray studies have found examples of amylose fragments with conformations distorted from the usual syn forms, and it was of interest to examine these novel structural motifs by the same high-level DFT methods used in Part 1. As in Part 1, we have examined numerous hydroxymethyl rotamers (gg, gt, and tg) at different locations in the residue sequence, and include the two hydroxyl rotamers, the clockwise ‘c’ and counterclockwise ‘r’ forms. A total of fifty conformations were calculated and energy differences were found to attempt to identify those sources of electronic energy that dictate stressed amylose conformations. Most stressed conformations were found to have relative energies considerably greater (i.e., ∼4 to 12 kcal/mol) than the lowest energy syn forms. Relative energy differences between ‘c’ and ‘r’ forms are somewhat mixed with some stressed conformations being ‘c’ favored and some ‘r’ favored, with the lowest energy ‘kink’ form being an all-gg-r conformation with the ‘kink’ in the bc glycosidic dihedral angles. Comparison of our calculated structures with experimental results shows very close correspondence in dihedral angles.     

Characterization of two potentially universal turn motifs that shape the repeated five-residues fold--crystal structure of a lumenal pentapeptide repeat protein from Cyanothece 51142

The genome of the diurnal cyanobacterium Cyanothece sp. PCC 51142 has recently been sequenced and observed to contain 35 pentapeptide repeat proteins (PRPs). These proteins, while present throughout the prokaryotic and eukaryotic kingdoms, are most abundant in cyanobacteria. The sheer number of PRPs in cyanobacteria coupled with their predicted location in every cellular compartment argues for important, yet unknown, physiological and biochemical functions. To gain biochemical insights, the crystal structure for Rfr32, a 167-residue PRP with an N-terminal 29-residue signal peptide, was determined at 2.1 A resolution. The structure is dominated by 21 tandem pentapeptide repeats that fold into a right-handed quadrilateral beta-helix, or Rfr-fold, as observed for the tandem pentapeptide repeats in the only other PRP structure, the mycobacterial fluoroquinoline resistance protein MfpA from Mycobacterium tuberculosis. Sitting on top of the Rfr-fold are two short, antiparallel alpha-helices, bridged with a disulfide bond, that perhaps prevent edge-to-edge aggregation at the C terminus. Analysis of the main-chain (Phi,Psi) dihedral orientations for the pentapeptide repeats in Rfr32 and MfpA makes it possible to recognize the structural details for the two distinct types of four-residue turns adopted by the pentapeptide repeats in the Rfr-fold. These turns, labeled type II and type IV beta-turns, may be universal motifs that shape the Rfr-fold in all PRPs.