Analysis of the stability of multimeric proteins by effective DeltaG and effective m-values

Analyzing the stability of a multimeric protein is challenging because of the intrinsic difficulty in handling the mathematical model for the folded multimer-unfolded monomer equilibrium. To circumvent this problem, we introduce the concept of effective stability, DeltaGeff (= -RTlnKeff), where Keff is the equilibrium constant expressed in monomer units. Analysis of the denaturant effect on DeltaGeff gives new insight into the stability of multimeric proteins. When a multimeric protein is mostly folded, the dependence of effective stability on denaturant concentration (effective m-value) is simply the m-value of its monomeric unit. However, when the protein is mostly unfolded, its stability depends on denaturant concentration with the m-value of its multimeric form. We also find that the effective m-value at the Cm is a good approximation of the apparent m-value determined by fitting the equilibrium unfolding data from multimeric proteins with a two-state monomer model. Moreover, when the m-value of a monomeric unit is estimated from its size, the effective stability of a multimeric protein can be determined simply from Cm and this estimated m-value. These simple and intuitive approaches will allow a facile analysis of the stability of multimeric proteins. These analyses are also applicable for high-throughput analysis of protein stability on a proteomic scale.     

Syntheses, structures and vibrational spectroscopy of some 1:1 and 1:2 adducts of silver(I) oxyanion salts with 2,2′-bis(pyridine) chelates

Syntheses and room-temperature single-crystal X-ray structure determinations are recorded for a number of adducts of 1:1 stoichiometry of silver(I) oxyanion salts (perchlorate, nitrate, trifluoroacetate (‘tfa’) (increasing basicity)) with 2,2′-bis(pyridine) ligands (2,2′-bipyridyl, ‘bpy’; 2,2′-biquinolyl, ‘bq’; 2,2′-dipyridylketone, ‘dpk’; 2,9-dimethylphenanthroline, ‘dmp’). The adducts take two forms: (a) neutral mononuclear molecules, in which the 2,2′-bis(pyridine) ligand behaves as a chelate, with the silver coordination number dependent on the denticity of the anion; these are Agtfa:bpy (1:1) and AgClO₄:bq (1:1) (and various (ionic) acetonitrile or pyridine solvates AgClO₄:bq/dmp:MeCN/py (1:1:1), in which the solvent molecules are coordinated); and (b) one-dimensional polymers. The latter are diverse: in AgClO₄:bpy, dpk (1:1), the anion is discrete, the polymer made up of an array of two-coordinate silver atoms linked by bpy ligands twisted about their central connecting element. In AgNO₃:bpy, bq (1:1), the bpy ligands are chelating with the oxyanions bridging, cf. previously reported AgNO₃:dpk (1:1), in which the nitrate chelates the metal, with the dpk bridging, chelating N,O to one silver, while the other nitrogen bridges to the next. With Agtfa, a novel binuclear adduct has been isolated in conjunction with the hydrated ligand, Agtfa:dpk:(dpk · H₂O) (1:1:2). The far-IR spectra of several of these complexes show bands that can be assigned to the ν(AgN) modes, the positions of these bands correlating well with the relative Ag-N bond lengths.Syntheses and single-crystal X-ray structural characterizations are also reported for various adducts of silver(I) perchlorate, nitrate and trifluoromethanesulfonate with bpy, bq, ‘phen’ (= 1,10-phenanthroline), and ‘dmp’, of stoichiometry AgX:L (1:2). In each case the complex is ionic [AgL₂]X; the silver atom is four-coordinate, but diverse and remarkable variations in stereochemistries associated with changes in the interligand N-Ag-N angles, presumably influenced by the different packing arrangements, are observed.     

Salt-stress induced changes in the leaf proteome of diploid and tetraploid mandarins with contrasting Na and Cl accumulation behaviour

To understand the genotypic variation of citrus to mild salt stress, a proteomic approach has been carried out in parallel on two citrus genotypes (‘Cleopatra’ and ‘Willow leaf’ mandarins), which differ for Na⁺ and Cl⁻ accumulation, and their cognate autotetraploids (4×). Using two-dimensional electrophoresis approximately 910 protein spots were reproducibly detected in control and salt-stressed leaves of all genotypes. Among them, 44 protein spots showing significant variations at least in one genotype were subjected to mass spectrometry analysis for identification. Salt-responsive proteins were involved in several functions, including photosynthetic processes, ROS scavenging, stress defence, and signalling. Genotype factors affect the salt-responsive pattern, especially that of carbon metabolism. The no ion accumulator ‘Cleopatra’ mandarin genotype showed the highest number of salt-responsive proteins, and up-regulation of Calvin cycle-related proteins. Conversely the ion accumulator ‘Willow leaf’ mandarin showed high levels of several photorespiration-related enzymes. A common set of proteins (twelve spots) displayed higher levels in salt-stressed leaves of 2× and 4× ‘Cleopatra’ and 4× ‘Willow leaf’ mandarin. Interestingly, antioxidant enzymes and heat shock proteins showed higher constitutive levels in 4× ‘Cleopatra’ mandarin and 4× ‘Willow leaf’ mandarin compared with the cognate 2× genotype. This work provides for the first time information on the effect of 8 weeks of salt stress on citrus genotypes contrasting for ion accumulation and their cognate autotetraploids. Results underline that genetic factors have a predominant effect on the salt response, although a common stress response independent from genotype was also found.     

Plasmid transformation in Bacillus subtilis: Fate of plasmid DNA

Summary Only multimeric, and not monomeric forms of B. subtilis plasmids can transform B. subtilis cells (Canosi et al. 1978). This finding prompted us to study the physico-chemical fate of plasmid DNA in transformation. Competent cells of B. subtilis were exposed to either unfractionated preparations or to preparations of multimeric plasmid DNA. Plasmid DNA was re-extracted from such cells and then analyzed by sedimentation and isopycnic centrifugation and also defined by its sensitivity to nuclease S1 degradation. No double-stranded plasmid DNA could be recovered from cells transformed with unfractionated plasmid preparations which contained predominantly monomeric covalently closed circular (CCC) DNA, Re-extracted plasmid DNA was single-stranded, had a molecular weight considerably smaller than monomer length DNA and had been subject to degradation to acid soluble products. However, when transformations were performed with multimeric DNA (constructed by in vitro ligation of linearized pC194 DNA), both double-stranded and partially double-stranded DNA could be recovered in addition to single-stranded DNA.We assume that plasmid DNA is converted to a single-stranded form in transformation, irrespective of its molecular structure. Double-stranded and partially double-stranded DNAs found in transformation with multimeric DNA would be the products of intramolecular annealing.Some of these results were presented at the 5th European Meeting on Bacterial Transformation and Transfection, September 1980, Florence     

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.     

Recent advances in the in vitro cultivation and genetic manipulation of Echinococcus multilocularis metacestodes and germinal cells

In order to elucidate host-parasite interactions and infection strategies of helminths at the molecular level, the availability of suitable in vitro cultivation systems for this group of parasites is of vital importance. One of the few helminth systems for which in vitro cultivation has been relatively successfully carried out in the past is the larval stage of the fox-tapeworm Echinococcus multilocularis, the causative agent of alveolar echinococcosis. Respective ‘first generation’ cultivation systems relied on the co-incubation of larval tissue, isolated from laboratory rodents, with host feeder cells. Although these techniques have been very successful in producing metacestode material for drug screening assays or the establishment of cDNA libraries, the continuous presence of host cells prevented detailed studies on the influence of defined host factors on larval growth. To facilitate such investigations, we have recently introduced the first truly axenic system for long-term in vitro maintenance of metacestode vesicles and used it to establish a technique for parasite cell cultivation. The resulting culture system, which allows the complete in vitro regeneration of metacestode vesicles from germinal cells, is a highly useful tool to study the cellular and molecular basis of a variety of developmental processes that occur during the infection of the mammalian host. Furthermore, it provides a solid basis for establishing transgenic techniques in cestodes for the first time. We consider it an appropriate time point to discuss the characteristics of these ‘second generation’ cultivation systems in comparison with former techniques, to present our first successful attempts to introduce foreign DNA into Echinococcus cells, and to share our ideas on how a fully transgenic Echinococcus strain can be generated in the near future.     

The Single-Stranded DNA Binding Protein of Sulfolobus solfataricus Acts in the Presynaptic Step of Homologous Recombination

Homologous recombination is an important pathway in the repair of DNA double-strand breaks in all organisms. In mesophiles, single-stranded DNA binding proteins (SSBs) are believed to be involved in the removal of single-stranded DNA (ssDNA) secondary structure during the presynaptic step of homologous recombination, facilitating the formation of a contiguous Rad51/RecA nucleoprotein filament. Here we report a role for the thermophilic archaeal Sulfolobus solfataricus SSB (SsoSSB) in the presynaptic step of homologous recombination. We have identified multiple quaternary structural forms of this protein in vivo and examined the activity of SsoSSB with the strand-exchange protein S. solfataricus RadA (SsoRadA). Using gel-shift analysis, we found that the two major forms of SsoSSB have different DNA binding affinities and site sizes. Biochemical examination of the monomeric form of SsoSSB suggests that it has a minor role in presynapsis and may slightly inhibit the ssDNA-dependent ATPase activity of SsoRadA. The tetrameric form of SsoSSB, however, significantly inhibits SsoRadA ssDNA-dependent ATPase activity under both saturating and subsaturating conditions. Order-of-addition experiments indicate that preincubation of tetrameric SsoSSB and SsoRadA prior to reaction initiation with ssDNA relieves the inhibition observed when SsoSSB is added either before or after SsoRadA. In addition, we demonstrate a direct interaction between SsoRadA and SsoSSB using coimmunoprecipitation. Taken together, these results suggest that a direct interaction between SsoSSB and SsoRadA may occur in vivo prior to the formation of the SsoRadA nucleoprotein filament.     

A DNase from the trypanosomatid Crithidia fasciculata.

We have purified to homogeneity a DNase from a Crithidia fasciculata crude mitochondrial lysate. The enzyme is present in two forms, either as a 32 kDa polypeptide or as a multimer containing the 32 kDa polypeptide in association with a 56 kDa polypeptide. Native molecular weight measurements indicate that these forms are a monomer and possibly an alpha 2 beta 2 tetramer, respectively. The monomeric and multimeric forms of the enzyme are similar in their catalytic activities. Both digest double-stranded DNA about twice as efficiently as single-stranded DNA. They introduce single-strand breaks into a supercoiled plasmid but do not efficiently make double-strand breaks. They degrade a linearized plasmid more efficiently than a nickel plasmid. Both enzymes degrade a 5'-32P-labeled double-stranded oligonucleotide to completion, with the 5'-terminal nucleotide ultimately being released as a 5'-mononucleotide. One difference between the monomeric and multimeric forms of the enzyme, demonstrated by a band shift assay, is that the multimeric form binds tightly to double-stranded DNA, possibly aggregating it.     

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.     

Coupling of Domain Swapping to Kinetic Stability in a Thioredoxin Mutant

The thioredoxin (Trx) fold is a small monomeric domain that is ubiquitous in redox-active enzymes. Trxs are characterized by a typical WCGPC active-site sequence motif. A single active-site mutation of the tryptophan to an alanine in Staphylococcus aureus Trx converts the oxidized protein into a biologically inactive domain-swapped dimer. While the monomeric protein unfolds reversibly in a two-state manner, the oxidized dimeric form is kinetically stable and converts to the monomeric form upon refolding. After reduction, the half-life of the dimer decreases many orders of magnitude to ∼ 4.3 h, indicating that the active-site disulfide between Cys29 and Cys32 is an important determinant for the kinetics of unfolding. We propose kinetic stability as a possible evolutionary strategy in the evolution of multimeric proteins from their monomeric ancestors by domain swapping, which, for this biologically inactive Trx mutant, turned out to be an evolutionary dead end.     

Assessment of agroeconomic indices in polycultures of lettuce, rocket and carrot through uni- and multivariate approaches in semi-arid Brazil

The polyculture among vegetables is an activity that to have good results, needs a proper planning. Although it often requires more labor, has several advantages over monoculture, among them is that polycultures are generally are more productive, provide with productivity of various plant constituents and a more balanced human diet, contribute to economic return, economic and yield stability, social benefits and farmer's direct participation in decision-making. The objective of this study was to evaluate agroeconomic indices of polycultures derived from the combination of two cultivars of lettuce with two cultivars of rocket in two cultures strip-intercropped with carrot cultivar ‘Brasilia’ through uni-multivariate approaches in semi-arid Brazil. The experimental design used was of randomized complete blocks with five replications and the treatments arranged in a factorial scheme of 2 × 2. The treatments consisted of the combination of two lettuce cultivars (Babá de Verão and Tainá) with two rocket cultivars (Cultivada and Folha Larga) in two cultures associated with carrot cv. Brasília. In each block were grown plots with two lettuce cultivars and two rocket cultivars, and carrot in sole crop. In each system was determined the lettuce leaf yield, rocket green mass yield and carrot commercial yield. Agrieconomic indices such as operational cost, gross and net income, monetary advantage, rate of return, profit margin, land equivalent ratio and yield efficiency for DEA were used to measure the efficiency of intercropping systems. In the bicropping of lettuce and rocket associated with carrot cv. ‘Brasilia’, suggests the use of lettuce cultivar ‘Tainá’ combined with rocket cultivars ‘Cultivada’ or ‘Folha Larga’. It was observed significant effect of lettuce cultivars in the evaluation of polycultures of lettuce, carrot and rocket, with strong expression for the lettuce cultivar ‘Tainá’. Both uni- and multivariate approaches were effective in the discrimination of the best polycultures.     

alpha-Synuclein misfolding: single molecule AFM force spectroscopy study

Protein misfolding and aggregation are the very first and critical steps in development of various neurodegenerative disorders, including Parkinson’s disease, induced by misfolding of α-synuclein. Thus, elucidating properties of proteins in misfolded states and understanding the mechanisms of their assembly into the disease prone aggregates are critical for the development of rational approaches to prevent protein misfolding-mediated pathologies. To accomplish this goal and as a first step to elucidate the mechanism of α-synuclein misfolding, we applied single-molecule force spectroscopy capable of detecting protein misfolding. We immobilized α-synuclein molecules at their C-termini at the atomic force microscope tips and substrate surfaces, and measured the interaction between the proteins by probing the microscope tip at various locations on the surface. Using this approach, we detected α-synuclein misfolded states by enhanced interprotein interaction. We used a dynamics force spectroscopy approach to measure such an important characteristic of dimers of misfolded α-synuclein as their lifetimes. We found that the dimer lifetimes are in the range of seconds and these values are much higher than the characteristics for the dynamics of the protein in monomeric state. These data show that compared to highly dynamic monomeric forms, α-synuclein dimers are much more stable and thus can serve as stable nuclei for the formation of multimeric and aggregated forms of α-synuclein. Importantly, two different lifetimes were observed for the dimers, suggesting that aggregation can follow different pathways that may lead to different aggregated morphologies of α-synuclein.     

Stability, denaturation and refolding of Mycobacterium tuberculosis MfpA, a DNA mimicking protein that confers antibiotic resistance

MfpA from Mycobacterium tuberculosis is a founding member of the pentapeptide repeat class of proteins (PRP) that is believed to confer bacterial resistance to the drug fluoroquinolone by mimicking the size, shape and surface charge of duplex DNA. We show that phenylalanine side chain stacking stabilizes the N-terminus of MfpA's pentapeptide thus extending the DNA mimicry analogy. The Lumry-Eyring model was applied to multiple spectral measures of MfpA denaturation revealing that the MfpA dimer dissociates to monomers which undergo a structural transition that leads to aggregation. MfpA retains high secondary and tertiary structure content under denaturing conditions. Dimerization stabilizes MfpA's pentapeptide repeat fold. The high Arrhenius activation energy of the barrier to aggregate formation rationalizes its stability. The mechanism of MfpA denaturation and refolding is a ‘double funnel’ energy landscape where the ‘native’ and ‘aggregate’ funnels are separated by the high barrier that is not overcome during in vitro refolding.     

Study of the inclusion of the (R)- and (S)-camphor enantiomers in α-cyclodextrin by X-ray crystallography and molecular dynamics

The inclusion of (R)- and (S)-camphor compounds in α-cyclodextrin has been studied by X-ray crystallography. The crystal structures of the complexes reveal that one guest molecule is accommodated inside the cavity formed by a head-to-head cyclodextrin dimer. In the crystal lattice, the dimers form layers which are successively shifted by half a dimer. In both (R)- and (S)-cases, the camphor molecule exhibits disorder and occupies three major sites with orientations that can be described as either ‘polar’ or ‘equatorial’. Molecular dynamics simulations performed for the observed complexes indicate that although the carbonyl oxygen of both (R)- and (S)-camphor switches between different hydrogen bonding partners, it maintains the observed mode of ‘polar’ or ‘equatorial’ alignment.     

Computational methods for yeast prion curing curves

If the chemical guanidine hydrochloride is added to a dividing culture of yeast cells in which some of the protein Sup35p is in its prion form, the proportion of cells that carry replicating units of the prion, termed propagons, decreases gradually over time. Stochastic models to describe this process of ‘curing’ have been developed in earlier work. The present paper investigates the use of numerical methods of Laplace transform inversion to calculate curing curves and contrasts this with an alternative, more direct, approach that involves numerical integration. Transform inversion is found to provide a much more efficient computational approach that allows different models to be investigated with minimal programming effort. The method is used to investigate the robustness of the curing curve to changes in the assumed distribution of cell generation times. Matlab code is available for carrying out the calculations.     

Predator-induced changes in fluted giant clam (Tridacna squamosa) shell morphology

Predator-induced defences have been demonstrated in numerous marine molluscs, but never in giant clams (Bivalvia: Tridacnidae). Water-borne cues from the predatory crab Myomenippe hardwickii were tested for their ability to induce changes in the shell morphology, strength and growth of juvenile fluted giant clams, Tridacna squamosa. Specimens were maintained in effluent from tanks holding ‘fed crabs’, ‘starved crabs’ and ‘no crabs’. To ensure changes were due to predator cues only, food for the crabs was provided ex situ and measures were taken to minimise the prey signal. After 182 days, MANOVA identified differences in various shell parameters relating to shape and strength. CDA showed a clear change in overall morphology, with strong separation among the three treatments. Effluent from ‘fed crabs’ had a greater influence than effluent from ‘starved crabs’, possibly because the starved crabs were perceived as weaker, and thus less risk. Significantly more clams died in ‘fed crabs’ treatments (25%) and ‘starved crabs’ treatments (30%) compared to the ‘no crabs’ control (0%). This degree of mortality has not been observed in similar experiments elsewhere and represents a new challenge to interpret. We suggest that the cause is unlikely to be related to the plastic responses, but rather a result of some form of crab-associated contaminant.     

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.