Biochemical, biophysical, and biological properties of densonucleosis virus. I. Structural proteins.

The polypeptide composition of highly purified densonucleosis virus was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The viral proteins showed a different behavior in sodium dodecyl sulfate-gels in comparison with the marker proteins. Therefore, the molecular weights were estimated by analyzing the retardation of the electrophoretic mobility of these proteins in gels with increasing polyacrylamide concentrations. Four structural proteins with molecular weights of 49,000, 58,500, 69,000, and 98,000 were found, ant they were designated p49, p59, p69, and p98, respectively. There are several indications that p98 is a dimer of p49. The relative quantity of the structural proteins in a virion suggests that at least p49 (accounting for +/-70% of total protein mass) is a capsid protein and that there will be 12 capsomers per virion.     

Biochemical, biophysical, and biological properties of densonucleosis virus (paravovirus). II. Two types of infectious virions.

Among the infectious densonucleosis virus (DNV) particles, two types of virions, DNV-I and DNV-II, have been identified. The density of DNV-I was about 1.40 g/ml and depended on the pH, whereas the density of DNV-II was independent of the pH and was 1.44 g/ml. The sedimentation rates of these particles were 111S and 89S, respectively. The specific extinction coefficients and the absorption spectra, corrected for light scattering, were also different for these two types. The electrophoretic mobility of both types of virions was identical under the experimental conditions used. Both types of particles contained single-stranded DNA with similar physicochemical properties. The difference between these types of virions seemed to be due to a different protein content, DNV-II containing about 600,000 daltons less protein, and to a different quaternary structure as indicated by the change in the density of DNV-II, in the presence of divalent cations, without any change in the density of DNV-I.     

Biochemical and biophysical properties of flacherie virus of the silkworm

Flacherie virus of the silkworm (FVS) was extracted from diseased silkworms, both larvae and pupae, and purified by 15 to 30% sucrose density gradient centrifugation. FVS III and FVS IV, in addition to the FVS I and FVS II described in the previous paper (Himeno et al., 1974), were found. The FVS I, FVS III, and FVS IV showed the same mobility in 2.4% polyacrylamide gel electrophoresis and could not be distinguished from each other in the gel. However, the purified FVS II was separated into two bands, FVS IIa and FVS IIb, in 2.4% gel. FVS III was a spherical particle with a diameter of 28 ± 1 nm and showed a sedimentation coefficient of about 90 S. FVS III was easily decomposed into FVS IV which sedimented at about 30 S in sucrose gradient centrifugation. FVS I and FVS II each contained a single molecule of RNA which showed the same molecular weight. FVS I consisted of three polypeptides with molecular weights of 67,000, 50,000, and 33,000. FVS II consisted of 10 polypeptides; among them 2 polypeptides with molecular weights of 50,000 and 33,000 were also found. Labeling experiments with [³²P]orthophosphate revealed that FVS II was found at an early stage of infection and FVS I at a late stage. FVS II was also isolated at an early stage from silkworms infected with FVS II, and FVS I was found at a late stage in these silkworms. The correlation among FVS I, FVS II, FVS III, and FVS IV was discussed and it was suggested that they might be closely related to one another and that few particles in them were immature. It is possible that FVS II changes to FVS I via FVS III by cleavage of large polypeptides.     

Screening methods to determine biophysical properties of proteins in structural genomics

We have developed and tested a simple and efficient protein purification method for biophysical screening of proteins and protein fragments by nuclear magnetic resonance (NMR) and optical methods, such as circular dichroism spectroscopy. The method constitutes an extension of previously described protocols for gene expression and protein solubility screening [M. Hammarstr?m et al., (2002), Protein Science 11, 313]. Using the present purification scheme it is possible to take several target proteins, produced as fusion proteins, from cell pellet to NMR spectrum and obtain a judgment on the suitability for further structural or biophysical studies in less than 1 day. The method is independent of individual protein properties as long as the target protein can be produced in soluble form with a fusion partner. Identical procedures for cell culturing, lysis, affinity chromatography, protease cleavage, and NMR sample preparation then initially require only optimization for different fusion partner and protease combinations. The purification method can be automated, scaled up or down, and extended to a traditional purification scheme. We have tested the method on several small human proteins produced in Escherichia coli and find that the method allows for detection of structured proteins and unfolded or molten globule-like proteins.     

Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides.

Short oligonucleotides that can bind to adjacent sites on target mRNA sequences are designed and evaluated for their binding affinity and biological activity. Sequence-specific binding of short tandem oligonucleotides is compared with a full-length single oligonucleotide (21mer) that binds to the same target sequence. Two short oligonucleotides that bind without a base separation between their binding sites on the target bind cooperatively, while oligonucleotides that have a one or two base separation between the binding oligonucleotides do not. The binding affinity of the tandem oligonucleotides is improved by extending the ends of the two oligonucleotides with complementary sequences. These extended sequences form a duplex stem when both oligonucleotides bind to the target, resulting in a stable ternary complex. RNase H studies reveal that the cooperative oligonucleotides bind to the target RNA with sequence specificity. A short oligonucleotide (9mer) with one or two mismatches does not bind at the intended site, while longer oligonucleotides (21mers) with one or two mismatches still bind to the same site, as does a perfectly matched 21mer, and evoke RNase H activity. HIV-1 inhibition studies reveal an increase in activity of the cooperative oligonucleotide combinations as the length of the dimerization domain increases.     

Stereo-enriched phosphorothioate oligodeoxynucleotides: synthesis, biophysical and biological properties

Stereo-enriched [Rp] and [Sp]-phosphorothioate oligodeoxynucleotides are synthesized using oxazaphospholidine derivatized monomers. Three different designs of phosphorothioate oligodeoxynucleotides (PS-oligos), (i) stereo-enriched all-[Rp] or all-[Sp] PS-linkages, (ii) stereo-random mixture of PS-linkages, and (iii) segments containing certain number of stereo-enriched [Rp] and [Sp] PS-linkages ([Sp-Rp-Sp] or [Rp-Sp-Rp]), have been studied. Thermal melting studies of these PS-oligos with RNA complementary strands showed that the binding affinities are in the order [Rp] > [Sp-Rp-Sp]-[Rp-Sp-Rp] > stereo-random > [Sp]. Circular dichroism (CD) studies suggest that the stereochemistry of the PS-oligo does not affect the global conformation of the duplex. The in vitro nuclease stability of these PS-oligos is in the order [Sp] > [Sp-Rp-Sp] > stereo-random > [Rp]. The RNase H activation is in the order [Rp] > stereo-random > [Rp-Sp-Rp] > [Sp] > [Sp-Rp-Sp]. Studies in a cancer cell line of PS-oligos targeted to MDM2 mRNA showed that all oligos had similar biological activity under the experimental conditions employed. Protein- and enzyme-binding studies showed insignificant stereo-dependent binding to proteins. The [Sp] and [Sp-Rp-Sp] chimeric and stereo-random PS-oligos that contained a CpG motif showed higher cell proliferation than [Rp] PS-oligo of the same sequence.     

Biochemical and biophysical properties of hyphomicrobium bacteriophage hyphi30

Hyphomicrobium bacteriophage Hy30 and its nucleic acid were studied to determine some of their biochemical and biophysical properties. The molecular weight of the phage is 55.4 × 10⁶, and its buoyant density is 1.508 g/ml. The nucleic acid of Hy30 is linear, double-stranded DNA with a molecular weight of 29.7 × 10⁶. The guanine-plus-cytosine content of the DNA was 62% as determined from its melting temperature and buoyant density.     

Molecular cloning, genomic analysis, and biological properties of rat leukemia virus and the onc sequences of Rasheed rat sarcoma virus.

Rasheed rat sarcoma virus (RaSV) has been shown to code for a protein of 29,000 Mr not present in replication-competent rat type C helper virus (RaLV)-infected cells. This protein is a fused gene product consisting of a portion of the RaLV p15 gag protein and the transformation-specific 21,000 Mr (p21) ras protein, which is also found in Harvey murine sarcoma virus. We now report the molecular cloning of both the SD-1 (Sprague-Dawley) strain of RaLV and the transforming ras sequences of RaSV. Heteroduplex analysis of these cloned DNAs demonstrated that the RaSV ras gene (v-Ra-ras) was inserted into the rat type C viral genome with a small deletion of RaLV genetic information in the 5' region of the gag gene and that the v-Ra-ras gene (0.72 kilobase pair) is homologous to and colinear with the p21 ras gene of Harvey murine sarcoma virus (v-Ha-ras). Restriction enzyme mapping confirmed the homology demonstrated by heteroduplex mapping, showing strong site conservation of restriction endonucleases known to cleave v-Ha-ras. Cloned v-Ra-ras DNA transformed NIH 3T3 cells, inducing the synthesis of the p29 RaSVgag-ras protein.     

Biochemical and biophysical properties of cytochrome o of Azotobacter vinelandii

Cytochrome o, solubilized from the membrane of Azotobacter vinelandii, has been purified to homogeneity as judged by ultracentrifugation and polyacrylamide gel electrophoresis. The detergent-containing cytochrome o is composed of one polypeptide chain with a molecular weight of 28 000-29 000, associated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme exists as a dimer by gel filtration analysis. The amino analysis which reveals the majority of residues are of hydrophobic nature. The cytochrome o oxidase contains protoheme as its prosthetic group and about 20-40% of phospholipids. The phospholipids are identified as phosphatidylethanolamine and phosphatidylglycerol by radioautographic analysis using 2-dimensional thin-layer chromatography. No copper or nonheme iron can be detected in the purified oxidase preparation by atomic absorption and chemical analyses. Oxidation-reduction titration shows this membrane-bound cytochrome o to be a low-potential component, and Em was determined to be -18 mV in the purified form and -30 mV in the membrane-bound form. Both forms bind CO with a reduced absorption peak at 559 and 557-558 nm in the native and solubilized forms, respectively. A high-spin (g = 6.0) form is assigned to the oxidized cytochrome o by electron paramagnetic resonance analysis, and KCN abolishes this high-spin signal. CO titration of purified cytochrome o in the anaerobic conditions shows the enzyme binds one CO per four protohemes and a dissociation constant is estimated to be 3.2 microM for CO. Cyanide reacts with purified cytochrome o in both oxidized and CO-bound forms, identified by specific spectral compounds absorbed at the Soret region. Cytochrome c, often co-purified with cytochrome c from the membrane, cannot serve as a reductant for cytochrome o in vitro, due to the apparent potential difference of about 300 mV. Upon separation, both cytochrome o and cytochrome c4 show a great tendency of aggregation. Furthermore, the oxidase activity (measured by tetramethyl-p-phenylenediamine oxidation rate) decreases as the cytochrome c concentration is decreased by ammonium sulfate fractionation. All these suggest the structural and functional complex nature of cytochrome c4 and cytochrome o in the membrane of A. vinelandii.     

Biochemical and biological activities of N-ras proteins

Recombinant N-ras proteins, expressed and produced from synthetic genes cloned into E. coli, have been tested in vitro for GTPase and autophosphorylation activity. The genes corresponding to the assayed proteins were tested for their ability to transform NIH 3T3 cells. Mutations of glutamine to lysine at amino acid position 61 and glycine to valine at position 12 were both found to activate the ability of the N-ras gene to transform NIH 3T3 cells while significantly reducing the GTPase activity of the corresponding protein. N-ras proteins were also found to autophosphorylate in the presence of GTP when a threonine acceptor amino acid is provided at position 59.     

Three structural polypeptides coded for by minite virus of mice, a parvovirus.

Purified full and empty virions of minute virus of mice were separated on CsCl gradients, and their polypeptides were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The empty particle contains two polypeptides, A (83,300 daltons) and B (64,300 daltons), which are 15 to 18% and 82 to 85%, respectively, of the virion mass. The full particle contains the single-stranded DNA genome, proteins A and B, and a third polypeptide, C (61,400 daltons). Again A is 15 to 18% of the protein mass, but the amounts of B and C vary inversely in different preparations of full particles. These polypeptides comprise greater than 99.6% of the protein in either virion, and their molecular weights and molar ratios are independent of the species of host cell on which the virus is propagated, They are not found in uninfected cells, and no protein component of uninfected cells copurifies with either virion under our conditions. Pulse-chase experiments show that the three proteins are synthesized only after virus infection and are therefore probably virus coded. Sequential harvesting from the nuclei of cells infected under one cycle growth conditions shows an increase in the proportion of C in full particles as infection progresses, suggesting that C is derived from B in a late maturation step.     

Chicken avidin exhibits pseudo-catalytic properties. Biochemical, structural, and electrostatic consequences

Avidin and its bacterial analogue streptavidin exhibit similarly high affinities toward the vitamin biotin. The extremely high affinity of these two proteins has been utilized as a powerful tool in many biotechnological applications. Although avidin and streptavidin have similar tertiary and quaternary structures, they differ in many of their properties. Here we show that avidin enhances the alkaline hydrolysis of biotinyl p-nitrophenyl ester, whereas streptavidin protects this reaction even under extreme alkaline conditions (pH > 12). Unlike normal enzymatic catalysis, the hydrolysis reaction proceeds as a single cycle with no turnover because of the extremely high affinity of the protein for one of the reaction products (i.e. free biotin). The three-dimensional crystal structures of avidin (2 A) and streptavidin (2.4 A) complexed with the amide analogue, biotinyl p-nitroanilide, as a model for the p-nitrophenyl ester, revealed structural insights into the factors that enhance or protect the hydrolysis reaction. The data demonstrate that several molecular features of avidin are responsible for the enhanced hydrolysis of biotinyl p-nitrophenyl ester. These include the nature of a decisive flexible loop, the presence of an obtrusive arginine 114, and a newly formed critical interaction between lysine 111 and the nitro group of the substrate. The open conformation of the loop serves to expose the substrate to the solvent, and the arginine shifts the p-nitroanilide moiety toward the interacting lysine, which increases the electron withdrawing characteristics and consequent electrophilicity of the carbonyl group of the substrate. Streptavidin lacked such molecular properties, and analogous interactions with the substrate were consequently absent. The information derived from these structures may provide insight into the action of artificial protein catalysts and the evolution of catalytic sites in general.     

Structural proteins of densonucleosis virus isolated from the silkworm, Bombyx mori, infected with the flacherie virus

Four structural proteins were found in highly purified Bombyx densonucleosis virus particles which were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight was estimated from the relative mobility and the retardation coefficient. The major viral protein (VP1), accounting for 65% of the total virion protein, had a molecular weight of about 50,000, and the other three minor proteins (VP2, VP3, VP4) had molecular weights of about 57,000, 70,000, and 77,000, respectively. The Bombyx densonucleosis virus particle contains about 60 molecules of VP1, and VP1 is believed to be capsid protein.     

N-terminal amino acid sequences of the structural proteins of the tick-borne encephalitis virus

DNA-copies of the tick-borne encephalitis RNA fragments have been cloned in plasmid pBR322 in E. coli cells. The sequencing of the cloned DNA-copies revealed clones containing genes coding for viral structural proteins E and C. Strong homology between amino acid sequences of proteins E and C from two TBF strains is found.     

Recombinant-DNA-derived bovine growth hormone from Escherichia coli. 2. Biochemical, biophysical, immunological and biological comparison with the pituitary hormone

Bacterially synthesized, recombinant-DNA-derived bovine growth hormone (r-bGH), prepared as described in the preceding paper in this journal, has been characterized in comparison with pituitary bovine growth hormone (pit-bGH). The characterization criteria include sodium dodecyl sulfate/polyacrylamide gel electrophoresis, automated N-terminal sequence analysis, amino acid composition, isoelectric focusing, reverse-phase high-performance liquid chromatography, ultraviolet absorbance, analysis for free protein thiol, sizing by gel filtration, circular dichroism, radioimmunoassay and biological activity in the hypophysectomized rat weight-gain assay. In every respect the r-bGH appears to be virtually identical to pit-bGH.     

A common structural feature in promoter sequences of E. coli.

We have searched promoter regions of E. coli, structural genes of the same organism, and computer-generated random sequence DNA for the occurrence of common structural features. This is done by converting the base sequence to a series of numbers representing the sequence of helix twist angles and examining these numerical sequences statistically. Common structural features are shared by the promoter regions with a much higher frequency than are found in structural genes or in random sequences. These structures appear to be scattered randomly throughout the promoters, both in terms of the number of such structures per promoter and in terms of location within each promoter. One particular structure consisting of five successive helix twist angles is reported, along with a list of 60 different hexanucleotide sequences that share this structure. The locations of these structural elements in 61 E. coli promoters are also tabulated.     

Calponin: biological, chemical and structural properties

Calponin distribution in smooth muscle cells and its properties in experiments in vitro are described. A comparison of these properties with those of other regulatory proteins and of proteins presumed to play this role suggest that calponin has another, yet unknown function. On the basis of existing experimental and theoretical data, an attempt has been made to quantitatively estimate the content of structural elements of the polypeptide chain and their localization. With consideration of the structural calponin domains, a general model of the secondary structure of all known calponin sequences is suggested. Based on the known roentgenographic data of the CH-domain of other proteins a possible organization of the calponin molecule hydrophobic core has been proposed.