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1.
The three-dimensional structure of bovine erythrocyte glutathione peroxidase, a tetrameric enzyme containing 4 gram atoms of selenium per mole (Mr = 84,000), has been determined at 2.8 Å resolution using the multiple isomorphous replacement method. By correlation calculations in Patterson space the tetramers were shown to exhibit molecular [222] symmetry, proving the monomers to be identical or at least very similar.The monomer consists of a single polypeptide chain of 178 amino acid residues. Its shape is nearly spherical with a radius of r ≈ 19 A?. A tentative sequence corresponding to a partially refined model (R = 0.38) is given. Each subunit is built up from a central core of two parallel and two anti-parallel strands of pleated sheet surrounded by four α-helices. One of the helices runs antiparallel to the neighbouring β-strands giving rise to a βαβ substructure, an architecture that has been found in several other proteins e.g. flavodoxin, thioredoxin, rhodanese and dehydrogenases. A comparison of the glutathione peroxidase subunit structure with thioredoxin-S2 revealed large regions of structural resemblance. The central four-stranded β structure together with two parallel α-helices resembles nearly 80% of the thioredoxin fold.The active sites of glutathione peroxidase are located in flat depressions on the molecular surface. Probably each active centre is built up by segments from two subunits. The catalytically active selenocysteines were found at the N-terminal ends of long α-helices and are surrounded by an accumulation of aromatic side-chains. A difference Fourier map between oxidized and substrate-reduced glutathione peroxidase as well as heavy-atom binding led to the conclusion that the two-electron redox-cycle involves a reversible transition of the active-site selenium from a selenenic acid (RSeOH) to a seleninic acid (RSeOOH).  相似文献   

2.
The packing of α-helices and β-sheets in six αβ proteins (e.g. flavodoxin) has been analysed. The results provide the basis for a computer algorithm to predict the tertiary structure of an αβ protein from its amino acid sequence and actual assignment of secondary structure.The packing of an individual α-helix against a β-sheet generally involves two adjacent ± 4 rows of non-polar residues on the α-helix at the positions i, i + 4, i + 8, i + 1, i + 5, i + 9. The pattern of interacting β-sheet residues results from the twisted nature of the sheet surface and the attendant rotation of the side-chains. At a more detailed level, four of the α-helical residues (i + 1, i + 4, i + 5 and i + 8) form a diamond that surrounds one particular β-sheet residue, generally isoleucine, leucine or valine. In general, the α-helix sits 10 Å above the sheet and lies parallel to the strand direction.The prediction follows a combinational approach. First, a list of possible β-sheet structures (106 to 1014) is constructed by the generation of all β-sheet topologies and β-strand alignments. This list is reduced by constraints on topology and the location of non-polar residues to mediate the sheet/helix packing, and then rank-ordered on the extent of hydrogen bonding. This algorithm was uniformly applied to 16 αβ domains in 13 proteins. For every structure, one member of the reduced list was close to the crystal structure; the root-mean-square deviation between equivalenced Cα atoms averaged 5.6 Å for 100 residues. For the αβ proteins with pure parallel β-sheets, the total number of structures comparable to or better than the native in terms of hydrogen bonds was between 1 and 148. For proteins with mixed β-sheets, the worst case is glyceraldehyde-3-phosphate dehydrogenase, where as many as 3800 structures would have to be sampled. The evolutionary significance of these results as well as the potential use of a combinatorial approach to the protein folding problem are discussed.  相似文献   

3.
13C n.m.r. CP/MAS spectra (50.3 and 75.4 MHz) of solid poly(l-lleucines) and poly(d-norvalines) measured with suitable acquisition parameters allow quantification of the composition of the secondary structure. The optimum acquisition parameters were found by systematic variation of the contact time by means of samples containing 5?0% α-helix structure. The polypeptides were prepared by primary or tertiary amine-initiated polymerizations of the corresponding amino acid NCAs and the average degrees of polymerization (DP) were determined by 1H n.m.r. endgroup analysis. The mole fraction of α-helices increases with increasing DP; it depends on the nature of the solvent and to a lesser degree on the polymerization temperature. When prepared under identical conditions, poly(d-norvaline) samples contain more β-sheet structure than poly(l-leucine. Reprecipitation increases the α-helix content, demonstrating that a part of the original β-sheet structure is thermodynamically unstable. The presence of oligomers of DP ?10 is mainly responsible for the thermodynamically stable part of the β-sheet structure. The chain growth mechanism is discussed.  相似文献   

4.
Crystals of alkaline phosphatase (EC 3.1.3.1; Mr 94,000) grown at pH 9.5 from 2.25 m-(NH4)2SO4 with 5 × 10?5 m-Zn and 10?2 m-Mg present were analyzed by X-ray diffraction at pH 7.5 in 2.66 m-(NH4)2SO4 with 10?2 m-Zn and 10?2 m-Mg present. The crystals are orthorhombic with a = 195.5 A?, b = 168.3 A?and c = 76.33 A?, and the space group is I222. X-ray phases were determined by the multiple isomorphous replacement and anomalous dispersion method using K2PtCl4, KAu(CN)2 and K2OsO4 derivatives. The electron density maps and analysis of metal binding sites reveal one molecule per asymmetric unit with an internal, non-crystallographic, 2-fold rotation axis relating the subunits. Each subunit contains a major αβ domain with a seven-stranded β-sheet flanked by helices. The sheets are roughly coplanar but the general direction of the strands in each is at 20 ° to the rotation axis and thus 40 ° from each other. The helical content of the αβ domain is approximately 27% of the 459 residues in the monomer and the β content is approximately 7%. The chains in a smaller domain are more convoluted and less easily characterized than in the αβ domain. In both there is extensive monomer-monomer contact.Removal of the zinc and magnesium from the parent crystal produces a stable apoenzyme crystal and addition of cobalt at 10?2 m or cadmium at 10?2 or 5 × 10?2 m reveals seven metal binding sites per dimer. The active centers are 32 Å apart and each is shown by anomalous dispersion data to contain two metal binding sites, A and B. The cadmium derivative refinement determined the A-B separation to be 4.9 Å. Comparison of the parent and apo structures by means of difference maps reveals the double metal site with Zn at A and probably Mg at B. A prominent, partially resolved peak centered 7 Å away is interpreted as a stabilization of the backbone in this position by the metal ion co-ordination to a side-chain. Several negative peaks within 10 Å of the metals indicate local differences between apo and native structures but no significant differences are seen in the other parts of the molecule. At 5 × 10?2 m-Cd two metal sites (D and D′) are found 25.5 Å from the active center, on the surface of the minor domain. They are related to each other by the molecular 2-fold axis with a D-D′ distance of 25 Å. The seventh Cd site, E, is 20 Å from the active center, on the major domain, near a crystalline contact region, and devoid of any molecular symmetry mate.The apparent dissociation constants for cadmium at the A, B and D sites (and A′, B′, D′) are 3 × 10?3 m, 1.5 × 10?1 m and 1.3 × 10?2 m, respectively. Thus in these conditions cadmium is seen to distribute between A and B sites when the combined stoichiometry is two metal ions per dimer.  相似文献   

5.
6.
The regulation of the synthesis of trp operon enzymes was studied in streptomycin-resistant Escherichiacoli mutants temperature-sensitive for UGA suppression by normal tRNATrp. Our mutants carry a trpR+ allele that when transferred to a different genetic background causes repression of trp operon enzyme synthesis at both low (35°C) and high (42°C) temperatures; however, in our mutants with an excess of tryptophan and at increased temperatures trp enzyme synthesis is derepressed. Based on our results and the sequence data of the trpR gene [Singleton et al. (1980) Nucleic Acids Res., 8, 1551–1560], we offer a model for the involvement of the limited misreading of UGA codons by normal charged tRNATrp in the autogenous regulation of the trpR gene expression. The UGA readthrough process may be a regulatory amplifier of the effect of tryptophan starvation.  相似文献   

7.
The transducing phage λdarg14, carrying a portion of the E. coli chromosome including argECBH, is derived from the heat-inducible, lysis-defective strain λy199, which has the b519 and b515 deletions. Cleavage of λy199 DNA by EcoRI endonuclease, followed by agarose slab gel electrophoresis, results in bands corresponding to the known C, D, E, and F segments of λ, and a segment A′ (A plus B minus b519 minus b515, the cleavage site between A and B being eliminated). Cleavage of λdarg14 DNA by EcoRI yields the expected D, E, and F segments of λ and four other segments, termed 14-1 through 14-4, whose length is 17.5, 6.2, 3.0, and 2.0 kilobases, respectively, as determined by electron microscopy and corroborated by electrophoretic mobility. Heteroduplex analysis shows that the E. coli argECBH cluster is on the 14-1 segment.  相似文献   

8.
In Vitro production of Immune Interferon (IF) in response to Herpes Simplex Virus (HSV) antigen by sensitized spleen cells from C57B1/6 (B6) mice could be detected as early as 3 and for at least 20 days after ip infection of HSV. Maximal levels of IF were produced after 10 hr of culture, but there was no decay of activity when supernatants were sampled during the subsequent 3 days. The IF produced shared certain known properties of immune IF and was not neutralized by an antiserum against viral-induced (type I) IF. DBA/2 (D2) mice which are considerably more sensitive in vivo to HSV infection than B6 mice produced significantly lower amounts of immune IF in the in vitro test system regardless whether high or low doses of virus were injected. The same pattern of results was observed when resistant B6D2F1 hybrid mice were compared with AJ and Balb/c mice which are about as sensible to ip infection with HSV as DBA/2 mice in our laboratory. These results demonstrate a remarkable defect of in vitro cellular immunity in mice susceptible to a virus infection when compared with resistant mice. Conceivably, a similar defect may be of in vivo relevance.  相似文献   

9.
10.
A β-sheet conformation is predicted at the N-terminal of β chains in sickle cell hemoglobin (Hb S) as a result of the β6 Glu → Val mutation. Since Glu is the weakest and Val is the strongest β-sheet former in the predictive method of Chou and Fasman [Biochemistry 13, 211, 222 (1974)], such a substitution greatly increases the β-sheet potential in the β 1–6 region. The similarity in the concentration and temperature dependence of Hb S gelation to β-sheet formation in polyamino acids suggest that a common aggregation mechanism may be involved. Conditions to cause a β → α trans-formation at the β 1–6 region of Hb S is discussed relative to the treatment of sickle cell disease.  相似文献   

11.
Chou-Fasman analysis of the secondary structure of F and Le interferons   总被引:1,自引:0,他引:1  
The Chou-Fasman method for calculating the secondary structure of F and Le interferons from their amino acid sequences predicts several regions of homologous structure in the two interferons. Those areas tend to be located in the middle and C- terminal ends of the molecules. Total predicted content of α helix is 55% for Le interferon and 36% for F interferon. Predicted β-pleated sheet residues total 33% for F and 16% for Le.  相似文献   

12.
Non-random segregation of DNA strands in Escherichia coli B-r   总被引:11,自引:0,他引:11  
The segregation of DNA strands during growth of Escherichia coliBr has been studied under conditions in which the chromosomal configuration and the ancestry of the cells during growth and division were known. Cells containing either one or two replicating chromosomes were pulse-labeled with [3H]thymidine, and the location of the radioactivity within chains of cells formed by growth in methylcellulose was determined by autoradiography. The locations of the radioactive cells within chains obtained after the second, third and fourth divisions were consistent with the co-segregation of only one of the replicating strands of each chromosome and a fixed region of the cell into daughter cells. The attachment of this strand to the region appeared to become permanent at the time the strand was used for the first time as a template. It is concluded that the segregation of DNA molecules into daughter cells is non-random in E. coli B/r.  相似文献   

13.
Sorting nexins are phox homology (PX) domain-containing proteins involved in diverse intracellular endosomal trafficking pathways. The PX domain binds to certain phosphatidylinositols and is recruited to vesicles rich in these lipids. The structure of the PX domain is highly conserved, containing a three-stranded β-sheet, followed by three α-helices. Here, we report the crystal structures of truncated human SNX11 (sorting nexin 11). The structures reveal that SNX11 contains a novel PX domain, hereby named the extended PX (PXe) domain, with two additional α-helices at the C terminus. We demonstrate that these α-helices are indispensible for the in vitro functions of SNX11. We propose that this PXe domain is present in SNX10 and is responsible for the vacuolation activity of SNX10. Thus, this novel PXe domain constitutes a structurally and functionally important PX domain subfamily.  相似文献   

14.
One-way mixed lymphocyte cultures were established between related cell donors A (haplotype designated ab) and B (ac). The cells from A, proliferating in response to stimulation by mitomycin treated cells from B, were eliminated from the culture by a hot pulse of 3H-thymidine. A marginal response was observed when the remaining cells from A reencountered additional stimulating cells from B, or cells from an HL-A identical sibling to B. In addition, the remaining responding cells were virtually incapable of responding to secondary stimulation by family member C (bc), who shared one haplotype (b) with individual A and the other haplotype (c) with the individual stimulating cell donor B. The MLC secondary stimulation response to family member D (cd), who differed from A by both haplotypes, but shared one haplotype with B, was reduced to approximately 50% of control values. In other experiments it was found possible to completely eliminate the response of A (ab) to D (cd) by using a combination of stimulating cells from related donors B (ab) and C (bc) in the initial hot pulse MLC.Separate populations of responding cells reactive to antigenic products of each major histocompatibility system haplotype is a likely explanation of these observations.  相似文献   

15.
The X-ray structure of cyanomet human hemoglobin C has been solved and refined, R ~27%. The molecular packing can be represented in two dimensions by two sets of parallel strands, one set in the b direction and the other in the c direction. Taken together the two sets of strands interconnect the molecules into square nets or layers where each molecule contacts its four nearest neighbors. Molecules in one layer are displaced in a and b so that they fit into the “holes” of the square arrays of the adjacent layers (normal to a) resulting in a pseudo body-centered cubic packing. This packing can account for the hemoglobin crystallization in and fragility of the erythrocytes. The aberrant β6A3 Lys residue is in a position to influence the crystal formation.  相似文献   

16.
The relationship between chromosome replication and the bacterial division cycle has been examined in three substrains of Escherichia coliBr obtained from different sources and designated Br A, Br F and Br K. At growth rates greater than 1.0 doubling per hour (μ > 1.0), the time for a round of chromosome replication (C) was 42 minutes in all three substrains, but the time between the end of a round and cell division (D) was 22 minutes in Br A, 16 minutes in Br F and 14 minutes in Br K. At slower growth rates C and D increased, but to significantly different extents in the three substrains. When μ = 0.5, C and D were approximately 80 and 40 minutes in Br A, 60 and 20 minutes in Br F, and 70 and 20 minutes in Br K.As a consequence of the lengths of the C and D periods in the three stocks of E. coliBr, the patterns of chromosome replication during the division cycle differed. The most obvious difference was that E. coliBr F and E. coliBr K possessed periods devoid of DNA synthesis at both the beginning and the end of the division cycle during slow growth, whereas E. coliBr A contained only one period devoid of DNA synthesis at the end of the cycle.  相似文献   

17.
Further details are given of crystals of glutamine synthetase prepared from Escherichia coli. Crystals of two kinds have been observed: (1) rhombic dodecahedra which correspond to the morphology of the crystals studied by Eisenberg et al. (1971) (and which were found by them to contain dodecamers), and (2) rhombohedra, reported here. Cell dimensions and packing considerations led to the consideration of two possible structures for the rhombohedral crystals. These we have called the “T = 7 structure” and the “B.C.C. structure”. The T = 7 structure would be related to that derived by Eisenberg and would contain dodecamers, but is inconsistent with our X-ray intensity data. The B.C.C. structure is considered more probable. It is built of cubic octomers or square tetramers. Electron micrographs of our glutamine synthetase preparations show a wide variety of aggregates, including dodecamers and tetramers. The unit cell dimensions of our crystals are a = 140 ± 2 Å, and c = 148 ± 2 Å. The Laue symmetry group is 3̄m P31.  相似文献   

18.
PERIOD proteins are central components of the Drosophila and mammalian circadian clocks. The crystal structure of a Drosophila PERIOD (dPER) fragment comprising two PER-ARNT-SIM (PAS) domains (PAS-A and PAS-B) and two additional C-terminal α-helices (αE and αF) has revealed a homodimer mediated by intermolecular interactions of PAS-A with tryptophane 482 in PAS-B and helix αF. Here we present the crystal structure of a monomeric PAS domain fragment of dPER lacking the αF helix. Moreover, we have solved the crystal structure of a PAS domain fragment of the mouse PERIOD homologue mPER2. The mPER2 structure shows a different dimer interface than dPER, which is stabilized by interactions of the PAS-B β-sheet surface including tryptophane 419 (equivalent to Trp482dPER). We have validated and quantitatively analysed the homodimer interactions of dPER and mPER2 by site-directed mutagenesis using analytical gel filtration, analytical ultracentrifugation, and co-immunoprecipitation experiments. Furthermore we show, by yeast-two-hybrid experiments, that the PAS-B β-sheet surface of dPER mediates interactions with TIMELESS (dTIM). Our study reveals quantitative and qualitative differences between the homodimeric PAS domain interactions of dPER and its mammalian homologue mPER2. In addition, we identify the PAS-B β-sheet surface as a versatile interaction site mediating mPER2 homodimerization in the mammalian system and dPER-dTIM heterodimer formation in the Drosophila system.  相似文献   

19.
Radiosensitivity and time of origin of Mauthner neuron in the zebra fish   总被引:1,自引:0,他引:1  
Regeneration was induced in the mature skeletal muscles of adult dystrophic mice (C57B16J dy/dy) by the im injection of hypertonic saline. Regenerating myogenic cells were isolated from the saline-treated muscles by cold trypsinization with the aid of Yaffe's selective plating procedure [Yaffe, D. (1968). Proc. Nat. Acad. Sci. USA61, 477–483]. The myogenic cells thus isolated readily developed into muscle fibers possessing cross striations when cultured in vitro.  相似文献   

20.
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