The folding transition state of protein L is extensive with nonnative interactions (and not small and polarized)

Progress in understanding protein folding relies heavily upon an interplay between experiment and theory. In particular, readily interpretable experimental data that can be meaningfully compared to simulations are required. According to standard mutational ? analysis, the transition state for Protein L contains only a single hairpin. However, we demonstrate here using ψ analysis with engineered metal ion binding sites that the transition state is extensive, containing the entire four-stranded β sheet. Underreporting of the structural content of the transition state by ? analysis also occurs for acyl phosphatase [Pandit, A. D., Jha, A., Freed, K. F. &; Sosnick, T. R., (2006). Small proteins fold through transition states with native-like topologies. J. Mol. Biol. 361, 755–770], ubiquitin [Sosnick, T. R., Dothager, R. S. &; Krantz, B. A., (2004). Differences in the folding transition state of ubiquitin indicated by ? and ψ analyses. Proc. Natl Acad. Sci. USA 101, 17377–17382] and BdpA [Baxa, M., Freed, K. F. &; Sosnick, T. R., (2008). Quantifying the structural requirements of the folding transition state of protein A and other systems. J. Mol. Biol. 381, 1362–1381]. The carboxy-terminal hairpin in the transition state of Protein L is found to be nonnative, a significant result that agrees with our Protein Data Bank-based backbone sampling and all-atom simulations. The nonnative character partially explains the failure of accepted experimental and native-centric computational approaches to adequately describe the transition state. Hence, caution is required even when an apparent agreement exists between experiment and theory, thus highlighting the importance of having alternative methods for characterizing transition states.     

Proteins and surface proteins of Leishmania promastigotes and their possible relevance to the characterisation of strains

Ramasamy R., Jamnadas H. & Mutinga M.J. 1981. Proteins and surface proteins of Leishmania promastigotes and their possible relevance to the characterisation of strains. International Journal for Parasitology11: 387–390. Two strains of Leishmania isolated from phlebotomine flies and another one from a patient with kala-azar were grown in culture as promastigotes. They were analysed for protein composition and surface proteins by polyacrylamide gel electrophoresis after surface radiolabelling. Differences were observed in the characteristic patterns of proteins and surface proteins between the two strains that are likely to be Leishmania donovani and the other strain. Such differences may prove valuable in the classification of Leishmania strains.     

Polyacrylamide gel electrophoresis in the differentiation of Taenia (cestoda) by total protein

Bursey C. C., Mckenzie J. A. & Burt M. D. B. 1980. Polyacrylamide gel electrophoresis in the differentiation of Taenia (Cestoda) by total protein. International Journal for Parasitology10: 167–174, Step-gradient polyacrylamide gels were used to examine total protein in 3 taeniid species. Preliminary investigations revealed that band patterns of different parts of the taeniid strobila were basically identical; hence, individual worms were useful for analysis regardless of their state of development when collected. Using Hymenolepis diminula as a control, the band pattern for each of Taenia taeniaeformis, T. macrocystis and T. pisiformis was found to be species specific with marked differences. The procedure was adapted for use under the more adverse conditions present in field laboratories. Small quantities of tapeworm material used in analysis also allows specific differentiation of single proglottids of Taenia spontaneously expelled from live hosts.     

Method for isolation of intact titin (connectin) molecules from mammalian cardiac muscle

Cardiac titin was isolated from rabbit and ground squirrel ventricular muscles by a method that was used earlier to obtain myofibrils with intact minor proteins located in A-bands of sarcomeres (Podlubnaya, Z. A., et al. (1989) J. Mol. Biol., 210, 655–658). Small pieces of cardiac muscle were incubated for 2–3 weeks at 4°C in Ca²⁺-depleting solution before their homogenization to decrease activity of Ca²⁺-dependent proteases. Then the muscle was homogenized, and titin was isolated by the method of Soteriou, A., et al. (1993) J. Cell Sci., 14, 119–123. In control experiments, titin was isolated from cardiac muscle without its preincubation in Ca²⁺-depleting solution. Sometimes control titin preparations contained only T2-fragment, but generally they contained ～5–20% N2B-isoform of titin along with its T2-fragment. Preparations of titin obtained from rabbit cardiac muscle by our method contained ～30–50% of N2BA- and N2B-titin isoforms along with its T2-fragment. The content of α-structures in titin isolated by our method was increased. Actomyosin ATPase activity in vitro increased in the presence of titin preparations containing more intact molecules. This result confirms the significant role of titin in the regulation of actin-myosin interaction in muscles. The method used by us to preserve titin might be used for isolation of other proteins that are substrates of Ca²⁺-dependent proteases.     

The incorporation of radio-labelled nucleic acid precursors by Theileria parva in bovine blood and salivary glands of Rhipicephalus appendiculatus ticks

Irvin A.D., Boarer C.D.H., Kurtti T.J. and Ocama J.G.R. 1981. The incorporation of radio-labelled nucleic acid precursors by Theileria parva in bovine blood and salivary glands of Rhipicephalus appendiculatus ticks. International Journal for Parasitology11:451–456. The uptake of radio-labelled nucleic acid precursors by blood and tick salivary gland forms of Theileria pana was studied. Piroplasms took up tritiated purines, particularly hypoxanthine, but not pyrimidines. Similar uptake was recorded by T. parva, both in tick saliva and in salivary glands maintained in vitro. Intermediate parasite stages were those most readily labelled in glands; this reflected active nucleic acid synthesis associated with rapid parasite division. Radio-labelling of T. parva in tick salivary glands could be of value in procedures used for concentrating and purifying theilerial sporozoites.     

Quantitative assay for submicrogram amounts of protein

An ultrasensitive protein assay, linear between 0.01 and 0.2 μg protein, is described. In this assay, copper is complexed to protein, excess copper is removed by adsorption to a small Sephadex column, and the copper-protein complex is destroyed by digestion with hydroperoxide. Phenol and chloramine-T are added to the reaction mixture and the copper catalyzes the production of a color-producing reaction between the two compounds. The assay is not affected by low levels of phosphate, Tris, metals, or a tenfold excess of nucleic acid over protein. Reducing agents, sucrose, certain anions, high salt concentrations, and EDTA seriously interfere. The method is about 500 times as sensitive as that of Lowry et al. [Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951)J. Biol. Chem.193, 265]. Like the biuret procedure, the assay measures copper complexed to the peptide bonds but is probably influenced by other factors, since equivalent amounts of different proteins give similar but not identical amounts of color.     

Location of SH-1 and SH-2 in the heavy chain segment of heavy meromyosin.

The two essential thiol groups of myosin, SH-1 and SH-2, have been localized in an ~ 20K segment of the heavy chain by analysis of the distribution of radioactivity after tryptic digestion of tryptic heavy meromyosin (HMM) or papain-HMM subfragment-1, both labeled at SH-1 and SH-2 with [¹⁴C]iodoacetamide and [¹⁴C]N-ethyl maleimide, respectively. The results are discussed in the framework of earlier work (Bálint, M., Sréter, F. A., Wolf, I., Nagy, B., and Gergely, J. (1975) J. Biol. Chem. 250, 6168–6177) on the tryptic fragmentation of myosin heavy chain and in the light of more recent work on the location of a fragment that reacts with a photoaffinity analog of ATP (Szilágyi, L., Bálint, M., Sréter, F. A., and Gergely, J. (1978) Fed. Proc. 37, 1695) and of suggestions concerning the binding of ATP in the region containing the SH-1 and SH-2 (Elzinga, M., and Collins, J. H. (1977) Proc. Nat. Acad. Sci. USA74, 4281–4284).     

Putative superoxide dismutase activity of iron-EDTA: a reexamination

It has been reported that iron-EDTA complexes mimic the action of superoxide dismutase, displaying 0.01% of the activity of the enzyme (Halliwell, B., 1975, FEBS Lett., 56, 34–38). This was purportedly directly confirmed by J. G. McClune, J. A. Fee, G. A. McCluskey, and J. T. Groves, 1977, J. Amer. Chem. Soc., 99, 5220–5222. A reexamination of the behavior of this compound has demonstrated that it does not catalyze the dismutation of O₂^?, but rather inferferes with assays for superoxide dismutation activity, which are based on the reductions of nitroblue tetrazolium or of cytochrome c. The sources of this interference have been examined. Investigators engaged in searching for mimics of superoxide dismutase are urged to be wary of similar artifacts.     

Dose dependent responses of cattle to Theileria parva stabilate

Dolan T. T., Young A.S., Losos G.J., McMillan I., Minder Ch.E. and Soulsby K. 1984. Dose dependent responses of Theileria parva stabilate. International Journal for Parasitology14: 89–95. A tick derived stabilate of Theileria parva (Maguga) was titrated in a large group of Boran (Bos indicus) cattle of the same age, sex and origin. The infectivity data was analysed using the independent action model. The cattle were identified as heterogeneous in their response to infection with 75% showing one ID₅₀ (0.0014) and 25% showing another (0.01). The disease responses of the cattle given different dose levels were compared for a variety of parameters. The results obtained showed these parameters to be dose dependent including the time to onset of piroplasm parasitaemia. The stabilate is of large volume and can be used for controlled challenge in immunity studies and for comparison of susceptibility between cattle of different breeds and from different epidemiological backgrounds.     

Insect immunity: Early fate of bacteria injected in saturniid pupae

Saturniid pupae have previously been shown to synthesize a set of antibacterial proteins in response to an injection of viable nonpathogenic bacteria (Boman, H. G., Nilsson-Faye, I., Paul, K., and Rasmuson, T., Jr. 1974. Insect immunity. I. Characteristics of an inducible cell-free antibacterial reaction hemolymph of Samia cynthia pupae; Infec. Immun., 10, 136–145; Faye, I., Pye, A., Rasmuson, T., Boman, H. G., and Boman, I. A. 1975. Insect immunity. II. Simultaneous induction of antibacterial activity and selective synthesis of some hemolymph proteins in diapausing pupae of Hyalophora cecropia and Samia cynthia). Infec. Immun., 12, 1426–1438). It show here that two such injected bacteria, Enterobacter cloacae and Escherichia coli, were rapidly eliminated from the hemolymph. The distribution of the injected bacteria was studied by the use of radioactively labeled E. coli, which were traced by combustion of tissue samples and by radioautography. Both methods showed that the bacteria appeared most frequently in the upper distal ends of the pupae. In the radioautographic study this was expressed as a high number of silver grain-containing cells. These cells appeared singly or as two to five cells clumped together, preferentially attached to the fat body. No decisive effect was shown on either the elimination of bacteria from hemolymph or the appearance in the tissue when pupae were treated with actinomycin D or cycloheximide. Phagocytosis by adhesive hemocytes is discussed as an explanation of bacterial elimination from the hemolymph.     

Determination of proteins and sulfobetaine with the folin-phenol reagent

This paper describes a method for the quantitative analysis of solutions containing a mixture of proteins and sulfobetaine. In a preliminary step the proteins, which interfere with the detergent assay, are separated by precipitation with trichloroacetic acid (8%). The insoluble fraction, dissolved in NaOH (1.0 n), and the soluble fraction, containing the detergent, are treated with the Folin-Ciocalteu phenol reagent, essentially following the method of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951, J. Biol. Chem.193, 265–275). The absorbance of the protein fraction is read, as usual at 750 nm, while that of the detergent solution is read at 342 nm. At this wavelength, sulfobetaine, treated with the Folin reagent, absorbs strongly, the absorbance being proportional to its concentration up to 1.5 mg/ml.     

Resistance of Adenoviral DNA Replication to Aphidicolin Is Dependent on the 72-Kilodalton DNA-Binding Protein

Aphidicolin is a highly specific inhibitor of DNA polymerase α and has been most useful for assessing the role of this enzyme in various replication processes (J. A. Huberman, Cell 23:647-648, 1981). Both nuclear DNA replication and simian virus 40 DNA replication are highly sensitive to this drug (Krokan et al., Biochemistry 18:4431-4443, 1979), whereas mitochondrial DNA synthesis is completely insensitive (Zimmerman et al., J. Biol. Chem. 255:11847-11852, 1980). Adenovirus DNA replication is sensitive to aphidicolin, but only at much higher concentrations. These patterns of sensitivity are seen both in vivo and in vitro (Krokan et al., Biochemistry 18:4431-4443, 1979). A temperature-sensitive mutant of adenovirus type 5 known as H5ts125 is able to complete but not initiate new rounds of replication at nonpermissive temperatures (P. C. van der Vliet and J. S. Sussenbach, Virology 67:415-426, 1975). When cells infected with H5ts125 were shifted from permissive (33°C) to nonpermissive (41°C) conditions, the residual DNA synthesis (elongation) showed a striking increase in sensitivity to aphidicolin. The temperature-sensitive mutation of H5ts125 is in the gene for the 72-kilodalton single-stranded DNA-binding protein. This demonstrated that the increased resistance to aphidicolin shown by adenovirus DNA replication was dependent on that protein. It also supports an elongation role for both DNA polymerase α and the 72-kilodalton single-stranded DNA-binding protein in adenovirus DNA replication. Further support for an elongation role of DNA polymerase α came from experiments with permissive temperature conditions and inhibiting levels of aphidicolin in which it was shown that newly initiated strands failed to elongate to completion.     

Gel permeation chromatography of asymmetric proteins

The gel permeation chromatographic behavior of three asymmetric proteins—collagen, fibrinogen, and the prolate ellipsoid lysozyme—was investigated using a variety of gel and high-performance liquid chromatographic media of various pore sizes and a wide range of flow rates. The time dependency of the elution patterns for columns and the partitioning of proteins between solvent and gel phases in batch experiments show that the “anomalous” behavior of asymmetric proteins is explicable by the mechanism proposed by Y. Nozaki, N. M. Schechter, J. A. Reynolds, and C. Tanford (1976, Biochemistry15, 3884); i.e., that these proteins penetrate pores of a size comparable to the minor semiaxis of the protein by end-on insertion. Thus, native type I collagen behaves as if it were a spherical protein of radius 8.2 Å, fibrinogen has an apparent radius of 32.4 Å, and lysozyme has an apparent radius of 14.6 Å. The rate at which asymmetric proteins penetrate the gel interior, however, is slow compared to the rate of gel penetration by globular proteins. The end-on insertion mechanism predicts that given infinite time, asymmetric proteins will be included into that portion of the internal volume of the gel which their smallest projectional cross sections allow them to penetrate. A method is presented for extrapolating the elution volume of asymmetric proteins to infinitely slow flow rate; from this extrapolation, one can calculate the minor semiaxis of the protein.     

Some improvements in two-dimensional gel electrophoresis of proteins. Protein mapping of eukaryotic tissue extracts.

In the course of adapting O'Farrell's (1975, J. Biol. Chem.250, 4007–4021) two-dimensional separation technique for proteins to eukaryotic material, we have made some modifications. During sample preparation, sodium dodecyl sulfate (SDS) can be included, with a resulting enhancement in reproducibility of gel patterns. However, heating in the presence of SDS leads to artifactual spots in the gels, probably as a result of protein charge modifications. Ultracentrifugation reduces the clogging at the top of the isoelectric focussing gel. For electrophoresis, some modifications of apparatus and technique are suggested. For the analysis of gels, a simple high-efficiency method for the counting of radioactivity in spots from dried gel slabs is described. In addition, an inexpensive microdensitometer option is described for the analysis of the autoradiographs. Patterns of proteins obtained from superior cervical sympathetic ganglia of rats and from other eukaryotic tissues are illustrated. Finally, a few of the proteins commonly found in mammalian tissue are identified on the gels.     

Effect of selenium on determination of mercury in animal tissues.

Selenium in animal tissues was found to influence the reactivity of mercury in the tissues with stannous chloride or with stannous chloride plus cadmium chloride added as reducing agents for the determination of mercury by the method developed by L. Magos (1971, Analyst, 96, 847–853) and L. Magos and T. W. Clarkson (1972, J. Assoc. Offic. Anal. Chem., 55, 966–971). The recovery of mercury in the tissues of animals to which inorganic mercury and selenite were simultaneously administered was low compared to the case in which inorganic mercury alone was administered. Of the in vitro interactions of inorganic mercury and selenite examined in tissue homogenates and blood samples, only those interactions in blood samples caused the difficulty in mercury analysis mentioned above, i.e., there was a marked decrease in recovery of mercury when an equimolar amount of each compound was added to the blood. These facts suggest that selenium and inorganic mercury in the animal tissues are likely to interact with each other and might form a chemically stable state of inorganic mercury which resists reduction with stannous chloride in the procedure for mercury determination.     

Isoelectric focusing of interacting systems III. Carrier ampholyte-induced macromolecular association or dissociation into subunits.

A phenomenological theory of isoelectric focusing of interacting systems (Cann, J. R., and Stimpson, D. I. Biophys. Chem., 7, 103, (1977) has been extended to include carrier ampholyte-induced dimerization or dissociation of a subunit macromolecule. Equilibrium as well as transient focusing patterns can show two well-resolved peaks similar to the patterns for a mixture of two noninteracting macromolecules. Characteristically, one of the peaks in the transient pattern grows at the expense of the other, which under certain conditions may disappear completely as equilibrium is approached. The implications of these findings for conventional applications of isoelectric focusing and for the detection and characterization of macromolecular interactions are discussed.     

Glycolipids of cell surfaces: Isolation of specific sugar sequences using carbohydrate-binding proteins

Proteins that bind carbohydrates can be used to isolate specific sugar sequences from complex mixtures. Free sialyloligosaccharides or sialyloligosaccharides released from gangliosides by ozonolysis and alkaline fragmentation are labeled at their reducing ends by reduction with NaB[³H]₄. After partial separation by column chromatography, oligosaccharide fractions are tested for binding to anti-sialyloligosaccharide antibodies [Smith, D. F., and Ginsburg, V. (1980) J. Biol. Chem.255, 55–59] and cholera toxin by a nitrocellulose filter assay. Oligosaccharides bound by the proteins can be eluted from the filters and further characterized. The method can be used to isolate and identify carbohydrate ligands of cell surfaces.     

Formyl peptide receptor polymorphisms: 27 most possible ways for phagocyte dysfunction

Formyl peptide receptors (FPRs) expressed by mammalian myeloid cells are the important part of innate immunity. They belong to the seven-transmembrane domain class of receptors coupled to heterotrimeric GTP-binding proteins. Binding of the receptor with a wide spectrum of exogenous and endogenous ligands triggers such defensive phagocyte reactions as chemotaxis, secretory degranulation, and respiratory burst, keeping a balance of inflammatory and antiinflammatory processes in the organism. The association between single nucleotide polymorphisms in the gene of FPR1 receptor resulting in disruption of the receptor structure and the development of certain pathologies accompanied with inflammation, such as aggressive periodontitis, macular degeneration, and even gastric cancer (Maney, P., and Walters, J. D. (2009) J. Periodontol., 80, 1498-1505; Liang, X. Y., et al. (2014) Eye, 28, 1502-1510; Otani, T., et al. (2011) Biochem. Biophys. Res. Commun., 405, 356-361) has been shown. In this review, we matched the missense mutation of formyl-peptide receptors with their known functional domains and classified them according to their potential significance in pathology.     

Taxonomic and nomenclatural rearrangement in the Hookeriales with notes on West Indian taxa

The Hookeriales are evaluated to discern familial limits. Five families are recognized in the order: Hookeriaceae, Leucomiaceae, Daltoniaceae, Callicostaceae, and Adelotheciaceae fam. nov. Chaetomitrium Dozy & Molk., Chaetomitriopsis. Fleisch., Dimorphocladon Dix., and Elharveya Crum are transferred to the Hypnaceae. Hookeriopsis (Besch.) Jaeg. is split into four genera: Hookeriopsis s. str., Brymela Crosby & Allen, Thamniopsis (Mitt.) Fleisch., and Trachyxiphium Buck, gen. nov. The following new combinations are proposed to coincide with the recognition of these four genera rather than Hookeriopsis s. lat.: Brymela acuminata, B. callicostelloides, B. cuspidata, B. fissidentoides, B. fluminensis, B. obtusifolia, B. parkeriana, B. rugulosa, B. websteri, Thamniopsis cheiloneura, T. cruegeriana, T. diffusa, T. incurva, T. langsdorffii, T. pappeana, T. purpureophylla, T. secunda, T. sinuata, T. terrestris, T. undata, T. utacamundiana, T. versicolor, Trachyxiphium aduncum, T. guadalupense, T. heteroicum, T. hypnaceum, T. pernutans, T. subfalcatum, T. tenue, T. vagum, and T. variable. Excluded species are treated as Isopterygium plumicaule, Schizomitrium cirrhosum, and S. subsecundum. Schizomitrium belangerianum (Besch.) Buck, comb. nov. is considered distinct from S. depressum (Hedw.) Buck & Steere, and S. pallidum (Hornsch.) Crum & Anders. is shown to have a smooth seta sometimes. The taxonomy of the aquatic species of Cyclodictyon Mitt. and Lepidopilum (Brid.) Brid. is clarified, resulting in the recognition of three species, Cyclodictyon subtortifolium (Bartr.) Buck, comb. nov., C. roridum (Hampe) Kuntze and L. tortifolium Mitt. Two new combinations are proposed in Calyptrochaeta Desv., C. setigera and C. albescens.     

Statistical evaluation of ways to analyze nonideal systems by sedimentation equilibrim

Three equations describing sedimentation equilibrium are examined and tested for their ability to analyze data. The testing procedure using simulated data is similar to that described previously (Holladay, L. A., and Sophianopoulos, A. J. (1972) J. Biol. Chem.247, 427–439) and used with another equation. The equations examined here are found to be of much less statistical reliability and of a more restricted range of application than the previously examined equation. The equation described previously, (Holladay, L. A., and Sophianopoulos, A. J. (1972) J. Biol. Chem.247, 427–439) is also used here to examine the conditions necessary to detect isodesmic systems of more than four components. The self-association of lysozyme reported previously (Sophianopoulos, A. J., and Van Holde, K. E. (1964) J. Biol. Chem.239, 2516–2524) is reexamined at pH 8.2, 0.15 ionic strength, and 13°C. The tentative conclusion is that the system is mainly a monomer-dimer, with a small, uncertain amount of tetramer possibly present. Under the above conditions the second virial coefficient, B, is estimated to lie in the range 0–4.4 × 10^?6 mole·dl·g^?2, the dimerization constant. K₂₁, lies in the range 2.3–2.7 × 10^?3m, and the tetramerdimer constant, K₄₂, is in the range 1.5–15 × 10^?3m.