Pair potentials for protein folding: choice of reference states and sensitivity of predicted native states to variations in the interaction schemes

We examine the similarities and differences between two widely used knowledge-based potentials, which are expressed as contact matrices (consisting of 210 elements) that gives a scale for interaction energies between the naturally occurring amino acid residues. These are the Miyazawa-Jernigan contact interaction matrix M and the potential matrix S derived by Skolnick J et al., 1997, Protein Sci 6:676-688. Although the correlation between the two matrices is good, there is a relatively large dispersion between the elements. We show that when Thr is chosen as a reference solvent within the Miyazawa and Jernigan scheme, the dispersion between the M and S matrices is reduced. The resulting interaction matrix B gives hydrophobicities that are in very good agreement with experiment. The small dispersion between the S and B matrices, which arises due to differing reference states, is shown to have dramatic effect on the predicted native states of lattice models of proteins. These findings and other arguments are used to suggest that for reliable predictions of protein structures, pairwise additive potentials are not sufficient. We also establish that optimized protein sequences can tolerate relatively large random errors in the pair potentials. We conjecture that three body interaction may be needed to predict the folds of proteins in a reliable manner.     

Mitochondrial Respiratory Chain Complex Patterns from Acanthamoeba castellanii and Lycopersicon esculentum: Comparative Analysis by BN-PAGE and Evidence of Protein–Protein Interaction Between Alternative Oxidase and Complex III

We have previously shown that a kinetic interplay exists between the cytochrome pathway and the alternative oxidase in mitochondria from amoeba Acanthamoeba castellanii . Native interaction analyses using blue native gel electrophoresis coupled to denaturating electrophoresis and immunodetection have indicated associations between alternative oxidase and oxidative phosphorylation complexes in both amoeba and tomato mitochondria. These associations are dependent on the expression level of alternative oxidase according to the physiological state in both organisms. Alternative oxidase associates broadly with large complexes of the respiratory chain when it is expressed in large amount, i.e., in ripe tomato and exponentially growing amoeba. On the contrary, alternative oxidase interacts specifically with complex III even if expression of the oxidase is low, i.e., in green tomato and stationary phase amoeba. This specific interaction represents a higher level of regulation driven by protein-protein interactions leading to a direct kinetic interplay between the cytochrome pathway and alternative oxidase in both plant and amoeba mitochondria.     

DNA-Hydrolyzing Antibodies from Sera of Autoimmune-Prone MRL/MpJ-lpr Mice

Catalytically active antibodies (abzymes) hydrolyzing proteins, polysaccharides, ATP, DNA, and RNA have been detected in the sera of patients with various autoimmune and some viral diseases, but abzymes from the sera of animals are practically unstudied. The development of lupus-like autoimmune disease of MRL/MpJ-lpr mice is an experimental model for study of autoimmune pathologies and immunopathogenesis. In this work, homogeneous IgG preparations were isolated from the sera of MRL/MpJ-lpr mice. These antibodies (Abs), their Fab-fragments, and isolated light chains were shown to possess catalytic activity in DNA hydrolysis, whereas Abs from the sera of control healthy mice did not hydrolyze DNA. The data demonstrate that DNA hydrolyzing activity is an intrinsic property of Abs from MRL/MpJ-lpr mice. It was shown that various markers of autoimmune pathologies (level of total protein concentration in urea (proteinuria), Abs titers to native and denatured DNA, and DNA-hydrolyzing activity of IgG) increased in animals with aging, but they noticeably increased (2-22 times) only after appearance of obvious indicators of pathology independently of age. The highest increase in proteinuria (25-fold), anti-DNA Abs titers (12-19-fold), and abzyme activity (120-fold) was found in mice after their immunization with DNA–protein complex.     

Chlorophyll b to chlorophyll a energy transfer kinetics in the CP29 antenna complex: a comparative femtosecond absorption study between native and reconstituted proteins

The energy transfer processes between Chls b and Chls a have been studied in the minor antenna complex CP29 by femtosecond transient absorption spectroscopy. Two samples were analyzed: the native CP29, purified from higher plants, and the recombinant one, reconstituted in vitro with the full pigment complement. The measurements indicate that the transfer kinetics in the two samples are virtually identical, confirming that the reconstituted CP29 has the same spectroscopic properties as the native one. In particular, three lifetimes (150 fs, 1.2 ps, and 5-6 ps) were identified for Chl b-652 nm to Chl a energy transfer and at least one for Chl b-640 nm (600-800 fs). Considering that the complexes bind two Chls b per polypeptide, the observation of more than two lifetimes for the Chl b to Chl a energy transfer, in both samples, clearly indicates the presence of the so-called mixed Chl binding sites--sites which are not selective for Chl a or Chl b, but can accommodate either species. The kinetic components and spectra are assigned to specific Chl binding sites in the complex, which provides further information on the structural organization.     

Chasing coccidia--new tools enter the race

The 8th International Coccidiosis Conference, held on 9--13 July 2001 in Palm Cove, Australia, was a showcase of the latest studies on widely known coccidia, including Eimeria and Toxoplasma in addition to the emerging or re-emerging parasites such as Neospora, Cryptosporidium and Cyclospora. This meeting was staged in conjunction with the Annual Scientific Meeting of the Australian Society for Parasitology.     

Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation

Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By comparison with analytical ultracentrifugation, we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EII(Mtl). For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented.Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dye is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EII(Mtl) and four other membrane (transport) proteins could be deduced within 10 % error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric.     

Plant regeneration from callus cultures of <Emphasis Type="Italic">Maclura tinctoria</Emphasis>, an endangered woody species

Summary Some native species produce seeds with a low frequency of germination accompanied with a period of dormancy. These features make it difficult to produce new phenotypes through sexual propagation. Maclura tinctoria has been considered an endangered species due to extensive use of its wood and low frequency of seed germination. The objective of the present study is to establish an in vitro propagation system for this species. Organogenic friable callus formation from nodal segments has been obtained using woody plant medium (WPM) supplemented with 10.74 μM 1-naphthaleneacetic acid (NAA)+4.43 μM 6-benzylaminopurine (BA). Results indicate that the highest frequency of shoot formation is observed when WPM supplemented with 4.03 μM NAA+4.43 BA is used. For root formation, the use of WPM medium (pH adjusted to 7.0) supplemented with 23.62 μM indole-3-butyric acid (IBA) and 4.7gl⁻¹ activated charcoal is recommended. For acelimatization, subjecting rooted plantlets to 70%, 50%, and 30% mesh screen, each successively for a period of 7 d, has resulted in 97% plantlet survival.     

Chickens fed with biomass of the red microalga Porphyridium sp. have reduced blood cholesterol level and modified fatty acid composition in egg yolk

The biomass of the red alga Porphyridium sp.constitutes a unique combination of soluble sulfatedpolysaccharide that accounts for about 70% of thealgal dry weight, and various polyunsaturatedfatty acids (PUFA) such as arachidonic andeicosapentaenoic acid (AA, 20:4 6 and EPA,20:5 3). In view of earlier results in ourlaboratory showing a reduction in serum cholesteroland triglyceride levels in rodents fed with red algalbiomass, we set out to examine the influence of algalbiomass as a feed additive on the metabolism ofchickens, with an emphasis on blood and eggcholesterol levels. For that purpose, lyophilizedalgal biomass was fed to 12–13, 30-week-old, WhiteLeghorn chickens for 10 days at a proportion of 5% or10% of the standard chicken diet. Twelve chickensfed with unsupplemented diet served as the control. No differences in body weight, egg number, and eggweight were found between the algal-fed chickens (atboth concentrations) and the control. However,chickens fed with algal biomass consumed 10% lessfood for both groups, and their serum cholesterollevels were significantly lower (by 11% and 28% forthe groups fed with 5% and 10% supplement,respectively) as compared with the respective valuesof the control group. Egg yolk of chickens fed withalgae tended to have reduced cholesterol levels (by10%) and increased linoleic acid and arachidonic acidlevels (by 29% and 24%, respectively). In addition,the color of the egg yolk was darker as a result ofthe higher carotenoid levels (2.4 fold higher) forchickens that fed with 5% supplement. Theseresults encourage the development of an improvedchicken feed having dietary fibers and polyunsaturatedfatty acids.     

Crystallization and initial X-ray diffraction characterization of complexes of FxFG nucleoporin repeats with nuclear transport factors

NTF2 and importin-beta are transport factors that mediate nuclear protein import and which interact with nuclear pore proteins (nucleoporins) during translocation from the cytoplasm to the nucleus through nuclear pore complexes. We employed a native gel electrophoresis method to assess the interaction of nucleoporin constructs that contain FxFG sequence repeats with NTF2 and truncation mutants of importin-beta to determine suitable fragments for crystallization. Based on these data, we obtained crystals of complexes between yeast NTF2 and a construct containing five FxFG nucleoporin repeats from the yeast nucleoporin Nsp1p and between a construct containing residues 1-442 of human importin-beta and the same nucleoporin construct. The yeast NTF2-nucleoporin crystals have trigonal symmetry and diffract past 2.8 A resolution using synchrotron radiation, whereas the importin-beta-nucleoporin complex crystals have P2(1)2(1)2 orthorhombic symmetry and diffract past 3.2 A resolution.