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1.
Crystallography of hemerythrin 总被引:1,自引:0,他引:1
X-ray crystallographic studies of hemerythrin from Golfingia gouldii show that the molecules pack in a tetragonal cell with two sets of molecules in an apparently face-centred array seen in projection along the 4-fold axis, but displaced relatively to each other by approximately in the axial direction. Both sets of molecules lie on 4-fold rotation axes, so that the subunits of each octameric molecule are related in pairs by a molecular 4-fold rotation axis. The two subunits of each pair are probably related by non-crystallographic 2-fold rotation axes perpendicular to the 4-fold axes and lying at 10 ° and 55 ° to the a and b axes. At low resolution the subunits are apparently arranged approximately in the form of a square prism. Along each 4-fold crystallographic axis there are two hemerythrin molecules, nearly equidistant and having similar but not identical orientations. 相似文献
2.
A characteristic of virus assembly is the use of symmetry to construct a complex capsid from a limited number of different proteins. Many spherical viruses display not only icosahedral symmetry, but also local symmetries, which further increase the redundancy of their structural proteins. We have developed a computational procedure for evaluating the quality of these local symmetries that allows us to probe the extent of local structural variations among subunits. This type of analysis can also provide orientation parameters for carrying out non-icosahedral averaging of quasi-equivalent subunits during three-dimensional structural determination. We have used this procedure to analyze the three types of hexon (P, E and C) in the 8.5 A resolution map of the herpes simplex virus type 1 (HSV-1) B capsid, determined by electron cryomicroscopy. The comparison of the three hexons showed that they have good overall 6-fold symmetry and are almost identical throughout most of their lengths. The largest difference among the three lies near the inner surface in a region of about 34 A in thickness. In this region, the P hexon displays slightly lower 6-fold symmetry than the C and E hexons. More detailed analysis showed that parts of two of the P hexon subunits are displaced counterclockwise with respect to their expected 6-fold positions. The most highly displaced subunit interacts with a subunit from an adjacent P hexon (P'). Using the local 6-fold symmetry axis of the P hexon as a rotation axis, we examined the geometrical relationships among the local symmetry axes of the surrounding capsomeres. Deviations from exact symmetry are also found among these local symmetry axes. The relevance of these findings to the process of capsid assembly is considered. 相似文献
3.
Low resolution crystal structure of muconolactone isomerase. A decamer with a 5-fold symmetry axis 总被引:1,自引:0,他引:1
Muconolactone isomerase from Pseudomonas putida crystallizes from sodium sulfate solution in space group P2(1) (a = 65.84 A, b = 105.70 A, c = 77.20 A, beta = 90.5 degrees) with ten 11,000 Mr subunits per asymmetric unit. The 7 A resolution crystal structure was solved by single isomorphous replacement followed by 10-fold symmetry averaging. The decameric enzyme has an uncommon non-crystallographic 5-fold symmetry axis and a large cavity in its center. 相似文献
4.
J K Stoops T S Baker J P Schroeter S J Kolodziej X D Niu L J Reed 《The Journal of biological chemistry》1992,267(34):24769-24775
Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components are attached. We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; M(r) = 2.7 x 10(6)) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. Using icosahedral particle reconstruction techniques, we determined its structure to 25 A resolution. Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence. The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of approximately 226 A (frozen-hydrated value), and 12 large openings (approximately 63 A in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (approximately 76-140 A diameter). The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center. The trimers are interconnected by 20 A thick "bridges" on the 2-fold axes. These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule. 相似文献
5.
V R Shatilov L P Loseva V L Tsuprun A S Kaftanova W L Kretovich 《Biochimica et biophysica acta》1989,995(1):17-20
The constitutive coenzyme non-specific glutamate dehydrogenase (GDH) from Chlorella pyrenoidosa 82T was purified to homogeneity by column immunoaffinity chromatography and examined by an electron microscope. The enzyme molecule was found to be a hexameric oligomer composed of monomers arranged in three 2-point group symmetry in two layers slightly twisted round the 3-fold axis. The molecule is 8 +/- 1 nm in diameter and 10 +/- 1 nm in height. The enzyme molecules appear both to dissociate into trimers and to associate along the 3-fold axis forming linear aggregates under certain conditions. A tentative model of the Chlorella GDH molecule is proposed, which is very similar to those described for bovine liver GDH and GDH from Clostridium symbiosum. 相似文献
6.
Adenosine triphosphatase from the thermophilic bacterium PS3(TF1) has been studied by solution X-ray scattering. A structural change in TF1 caused by the binding of ADP was observed by examining the difference between the radii of gyration of the unligated and ligated forms. The radius of gyration of the unligated TF1 was found to be 49.5 +/- 0.3 A, and it decreased by approximately 3% after ligation with ADP. The positions and the amplitudes of a subsidiary maximum and a shoulder in the scattering profile showed subtle change on nucleotide binding. The lower limit of the maximum length of TF1 was determined to be 165 A for the unligated form and 150 A for the ligated form. The shape analysis of TF1 was performed by model calculations for simple triaxial bodies or their complexes. Among the various models tested, the one that gave the best fit with the experimental data consisted of seven ellipsoids of revolution; six identical ellipsoids with semi-axes: a = b = 18.5 A and c = 74 A. arranged hexagonally, and the other with a = b = 28 A and c = 45 A, located below the other six on the 6-fold axis. On the basis of this model it was suggested that there is a structural change on ligation with nucleotides, consisting of a shrinkage of the six long ellipsoids by 6% along their major axes. 相似文献
7.
The T = 3 capsid of southern bean mosaic virus is analyzed in detail. The beta-sheets of the beta-barrel folding motif that form the subunits show a high degree of twist, generated by several beta-bulges. Only 34 water molecules were identified in association with the three quasi-equivalent subunits, most of them on the external viral surface. Subunit contacts related by quasi-3-fold axes are similar, are dominated by polar interactions and have almost identical calcium binding sites. There is no metal ion on the quasi-3-fold axis, as previously reported. Subunits related by quasi-2-fold and icosahedral 2-fold axes have different contacts but nevertheless display almost identical interactions between the antiparallel helices alpha A. A dipole-dipole type interaction between these helices may produce an energetically stable hinge that allows two types of dimers in a T = 3 assembly. The temperature factor distribution, the hydrogen-bonding pattern, and the contacts across the icosahedral 2-fold axes suggest that one of the dimer types is present in the intact virion and probably also in solution; the other is produced only during capsid assembly. Interactions along the 5-fold axes are mainly polar and possibly form an ion channel. The beta-sheet structures of the three subunits can be superimposed with considerable precision. Significant relative distortions between quasi-equivalent subunits occur mainly in helices and loops. The two dimeric forms and the subunit distortions are the consequence of the non-equivalent subunit environments in the capsid. 相似文献
8.
James A. Lake 《Journal of molecular biology》1976,105(1):131-159
Unique, three-dimensional structures have been determined for Escherichia coli small subunits, large Subunits and monomeric ribosomes by electron microscopy of ribosomes and subunits and of antibody-labeled ribosomes and subunits.Small subunits orient on the carbon substrate with their long axes parallel to the plane of the carbon. In these projections the subunit is divided into a onethird and a two-thirds portion by a region of accumulated negative stain similar to that observed in eukaryotic small subunits. Four characteristic views, or projections, are readily recognized and correspond to orientations of approximately ?40 °, 0 °, +50 ° and +110 ° about the long axis of the subunit. Three of these have been described (Lake et al., 1974a; Lake & Kahan, 1975). The two most distinctive views are a quasi-symmetric view (0 °) that is characterized by an approximate line of mirror symmetry that coincides with the long axis of the subunit, and an asymmetric view (110 °) that is characterized by a concave and a convex subunit boundary. In the asymmetric projection, a platform or ledge is viewed “face-on”. The platform is attached to the lower two-thirds of the subunit just below the one-third/two-thirds partition. It is separated from the upper one-third of the subunit at the level of the partition and above the partition it forms a cleft approximately 30 to 40 Å wide, which has been suggested as the site of the codon-anticodon interaction (Lake & Kahan, 1975).Four characteristic views are presented for the large subunit. The most prominent of these, the quasi-symmetric view (θ = 90 °, φ = 0 °), is distinguished by a central protuberance located on a line of approximate mirror symmetry. The central protuberance is surrounded by projecting features inclined at about 50 ° on both sides of it. The smaller of these projections is rod-like, about 40 Å wide and approximately 100 Å long. The feature projecting from the other side of the central protuberance is shorter, more blunt and wider than the rod-like appendage. In another view approximately orthogonal to the quasi-symmetric projection, the asymmetric projection (θ = 10 °, φ = 90 °), the subunit profile is distinguished by a convex lower edge and an upper boundary which is indented by a notch. The subunit is separated, in projection, by the notch into two unequal regions. The smaller region comprises about 20% of the total projected density and consists of the central protuberance and the rod-like appendage.The profiles observed in fields of monomeric 70 S ribosomes result from superpositions of the 30 S and 50 S profiles. Two major views are observed, an overlap and a non-overlap view, corresponding to whether or not the profile of the small subunit overlaps that of the large subunit in the 70 S profile. The small subunit is oriented in the monomeric ribosome so that the platform is in contact with the large subunit. The central protuberance of the large subunit overlaps part of the upper one-third of the small subunit in the overlap view of 70 S ribosomes, although in three dimensions they are probably separated by 30 to 50 Å. A region of the small subunit comprising the platform, the cleft and part of the upper one-third, suggested to be the approximate binding site of IF3 and IF2 (Lake & Kalian, 1975), is located at the interface between the large and small subunits, in a region of the small subunit that is close to, but probably not in physical contact with, the large subunit. 相似文献
9.
Rebière-Huët J Guérillon J Pimenta AL Di Martino P Orange N Hulen C 《FEMS microbiology letters》2002,215(1):121-126
Gene araA encoding an L-arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative L-arabinose isomerase of Thermotoga maritima. The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of L-arabinose isomerization and D-galactose isomerization at 85 degrees C, and required divalent cations such as Co(2+) and Mn(2+) for its activity and thermostability. The apparent K(m) values of the enzyme for L-arabinose and D-galactose were 116 mM (v(max), 119 micromol min(-1) mg(-1)) and 250 mM (v(max), 14.3 micromol min(-1) mg(-1)), respectively, that were determined in the presence of both 1 mM Co(2+) and 1 mM Mn(2+). A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80 degrees C. 相似文献
10.
E.J.M. van Schaick W.G. Schutter W.P.J. Gaykema A.M.H. Schepman W.G.J. Hol 《Journal of molecular biology》1982,158(3):457-485
The hemocyanin from the spiny lobster Panulirus interruptus, a hexamer with a molecular weight of approximately 540,000, was crystallized in space group P21 with two molecules in the unit cell and cell dimensions . With screened precession photographs a three-dimensional set of reflections was collected up to 10 Å resolution. Both the conventional and the fast rotation function programs were applied and gave results that were in excellent agreement with each other. The hemocyanin hexamer has 32 point group symmetry. Its 3-fold molecular axis runs approximately parallel to the crystallographic 2-fold screw axis.X-ray diffraction data to 5 Å resolution were collected by the oscillation method. Rotation function studies with data between 7 and 5 Å resolution confirmed the 10 Å studies and, furthermore, showed that the rotation axes relating subunits within one hexameric molecule can be distinguished from the rotation axes relating subunits belonging to different hexamers in the unit cell. The local 3-fold axis in the hexamer makes an angle of about 6 ° with the crystallographic 2-fold screw axis.For a mercury and a platinum derivative three-dimensional data sets were collected to 5 Å by the oscillation method. The difference Patterson of the platinum derivative could be solved. The eventual number of heavy-atom sites was 36 for the platinum derivative and 70 for the mercury derivative. From the well-occupied sites the point-group symmetry of the molecule could be established accurately. In addition, the centre of the hexamer could be located within 0.2 Å.Protein phases were obtained from isomorphous as well as anomalous differences. A “best” electron density map calculated with these phases showed the shape of the hexameric molecule as well as the boundaries of the six subunits. Correlation coefficients between the densities of the subunits showed little variation, suggesting a random distribution of the different subunit types (Van Eerd & Folkerts, 1981) over the six positions in the hexamer.The subunits are positioned at the corner of an antiprism. When viewed along the 3-fold axis the hexamer is roughly hexagonal in shape, with a diameter of approximately 120 Å. Viewed along one of the 2-fold axes the molecule is of rectangular shape with dimensions 95 Å × 120 Å. The subunit can be described as an ellipsoid of irregular shape with axes of 80 Å, 55 Å and 48 Å. Each subunit makes extensive contacts with three other subunits in the hexamer and, possibly, a much weaker contact with a fourth subunit. 相似文献
11.
Caulobacter crescentus bacteriophage phiCbK: structure and in vitro self-assembly of the tail 总被引:3,自引:0,他引:3
Electron micrographs of negatively stained and platinum-shadowed bacteriophage φCbK have been analyzed by optical diffraction and computer Fourier transformation. The results show that the phage tail is a helical “stacked disc” structure with an annular repeat of about 38 Å and with 3-fold rotational symmetry about the helix axis. Phage tails exhibited lateral and rotational flexibility and were found to possess variable helical parameters. The smaller angle of rotation about the helix axis between equivalent asymmetric units on adjacent discs measured from a number of tail images was found to have an average value of 41.5±0.9 °. Cross-sectional views of short tail fragments were obtained after sonication at 0 °C. These views confirmed the 3-fold symmetry of the 38 Å annular unit, which most probably consists of three identical subunits of the major tail protein. Formation of extended tail polymers, both linear and circular, was found to take place spontaneously in vitro after sonication. On the basis of these results, a low-resolution model for the tail helix is presented. The questions of head-tail symmetry mismatch in the phage and of tail length regulation are discussed. 相似文献
12.
AIMS: Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. METHODS AND RESULTS: A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. CONCLUSIONS: The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme. 相似文献
13.
Transcarboxylase 12S crystal structure: hexamer assembly and substrate binding to a multienzyme core
Hall PR Wang YF Rivera-Hainaj RE Zheng X Pustai-Carey M Carey PR Yee VC 《The EMBO journal》2003,22(10):2334-2347
Transcarboxylase from Propionibacterium shermanii is a 1.2 MDa multienzyme complex that couples two carboxylation reactions, transferring CO(2)(-) from methylmalonyl-CoA to pyruvate, yielding propionyl-CoA and oxaloacetate. The 1.9 A resolution crystal structure of the central 12S hexameric core, which catalyzes the first carboxylation reaction, has been solved bound to its substrate methylmalonyl-CoA. Overall, the structure reveals two stacked trimers related by 2-fold symmetry, and a domain duplication in the monomer. In the active site, the labile carboxylate group of methylmalonyl-CoA is stabilized by interaction with the N-termini of two alpha-helices. The 12S domains are structurally similar to the crotonase/isomerase superfamily, although only domain 1 of each 12S monomer binds ligand. The 12S reaction is similar to that of human propionyl-CoA carboxylase, whose beta-subunit has 50% sequence identity with 12S. A homology model of the propionyl-CoA carboxylase beta-subunit, based on this 12S crystal structure, provides new insight into the propionyl-CoA carboxylase mechanism, its oligomeric structure and the molecular basis of mutations responsible for enzyme deficiency in propionic acidemia. 相似文献
14.
ABSTRACT: BACKGROUND: D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a beta-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. RESULTS: In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52[DEGREE SIGN]C; however, it exhibited over 60% of maximum activity at 30[DEGREE SIGN]C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50[DEGREE SIGN]C. In this study, a recombinant Pichia pastoris yeast strain secreting beta-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting beta-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the complete utilization of D-glucose and a 30% conversion of D-galactose to D-tagatose. CONCLUSIONS: The method developed for the simultaneous hydrolysis of lactose, utilization of D-glucose and isomerization of D-galactose using a P. pastoris strain secreting beta-D-galactosidase and recombinant L-arabinose isomerase seems to offer an interesting alternative for the production of D-tagatose from lactose-containing feedstock. 相似文献
15.
D B Wigley S J Gamblin J P Turkenburg E J Dodson K Piontek H Muirhead J J Holbrook 《Journal of molecular biology》1992,223(1):317-335
We report the refined structure of a ternary complex of an allosterically activated lactate dehydrogenase, including the important active site loop. Eightfold non-crystallographic symmetry averaging was utilized to improve the density maps. Interactions between the protein and bound coenzyme and oxamate are described in relation to other studies using site-specific mutagenesis. Fructose 1,6-bisphosphate (FruP2) is bound to the enzyme across one of the 2-fold axes of the tetramer, with the two phosphate moieties interacting with two anion binding sites, one on each of two subunits, across this interface. However, because FruP2 binds at this special site, yet does not possess an internal 2-fold symmetry axis, the ligand is statistically disordered and binds to each site in two different orientations. Binding of FruP2 to the tetramer is signalled to the active site principally through two interactions with His188 and Arg173. His188 is connected to His195 (which binds the carbonyl group of the substrate) and Arg173 is connected to Arg171 (the residue that binds the carboxylate group of the substrate). 相似文献
16.
Three-dimensional structure of infectious bursal disease virus determined by electron cryomicroscopy. 总被引:22,自引:8,他引:14
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B Bttcher N A Kiselev V Y Stel'Mashchuk N A Perevozchikova A V Borisov R A Crowther 《Journal of virology》1997,71(1):325-330
Infectious bursal disease virus (IBDV), a member of the Birnaviridae group, is a commercially important pathogen of chickens. From electron micrographs of frozen, hydrated, unstained specimens, we have computed a three-dimensional map of IBDV at about 2 nm resolution. The map shows that the structure of the virus is based on a T=13 lattice and that the subunits are predominantly trimer clustered. The subunits close to the fivefold symmetry axes are at a larger radius than those close to the two- or threefold axes, giving the capsid a markedly nonspherical shape. The trimer units on the outer surface protrude from a continuous shell of density. On the inner surface, the trimers appear as Y-shaped units, but the set of units surrounding the fivefold axes appears to be missing. It is likely that the outer trimers correspond to the protein VP2, carrying the dominant neutralizing epitope, and the inner trimers correspond to protein VP3, which has a basic carboxy-terminal tail expected to interact with the packaged RNA. 相似文献
17.
The Pseudomonas bacteriophage Pf1 is a long ( approximately 2000 nm) and thin ( approximately 6.5 nm) filament consisting of a covalently closed, single-stranded DNA genome of 7349 nucleotides coated by 7350 copies of a 46-residue alpha-helical subunit. The coat subunits are arranged as a superhelix of C(1)()S(5.4)() symmetry (class II). Polarized Raman and polarized FTIR spectroscopy of oriented Pf1 fibers show that the packaged single-stranded DNA genome is ordered specifically with respect to the capsid superhelix. Bases are nonrandomly arranged along the capsid interior, deoxynucleosides are uniformly in the C2'-endo/anti conformation, and the average DNA phosphodioxy group (PO(2)(-)) is oriented so that the line connecting the oxygen atoms (O.O) forms an angle of 71 degrees +/- 5 degrees with the virion axis. Raman and infrared amide band polarizations show that the subunit alpha-helix axis is inclined at an average angle of 16 degrees +/- 4 degrees with respect to the virion axis. The alpha-helical symmetry of the capsid subunit is remarkably rigorous, resulting in splitting of Raman-active helix vibrational modes at 351, 445 and 1026 cm(-)(1) into apparent A-type and E(2)()-type symmetry pairs. The subunit tyrosines (Tyr 25 and Tyr 40) are oriented with phenoxyl rings packed relatively close to parallel to the virion axis. The Tyr 25 and Tyr 40 orientations of Pf1 are surprisingly close to those observed for Tyr 21 and Tyr 24 of the Ff virion (C(5)()S(2)() symmetry, class I), suggesting a preferred tyrosyl side chain conformation in packed alpha-helical subunits, irrespective of capsid symmetry. The polarized Raman spectra also provide information on the orientations of subunit alanine, valine, leucine and isoleucine side chains of the Pf1 virion. 相似文献
18.
Probes for fluorescence energy transfer measurements were introduced into active sites of dodecameric glutamine synthetase from Escherichia coli by substituting appropriate ATP analogues for ATP in the autoinactivation reaction of this enzyme with L-Met-(S)-sulfoximine and Mn2+ [Maurizi, M. R., & Ginsburg, A. (1986) Biochemistry (preceding paper in this issue)]. Two fluorescent donors, 8-mercapto-ATP alkylated with either 5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid (AEDANS-ATP) or 1,N6-etheno-2-aza-ATP (aza-epsilon-ATP), and two acceptors, 6-mercaptopurine ribonucleotide triphosphate or 8-mercapto-ATP alkylated with the chromophore 4'-[[4-(dimethylamino)-phenyl]azo]-2-iodoacetanilide (6-Y- or 8-Y-ATP), were used. Fluorescence emissions of enzyme derivatives with 1 or 2 equiv of fluorescent donor per dodecamer and either an acceptor (Y) or ADP at the remaining active sites were compared at pH 7.0. The results, together with the known geometry of the enzyme, indicate that active site probes in the dodecamer are widely separated and that energy transfer occurs from a single donor to two or three acceptors on adjacent subunits. The calculated distance between equidistant active site probes on heterologously bonded subunits within the same hexagonal ring is 56-60 A. Probes on isologously bonded subunits are no closer than 60 A and may be as far apart as 78 A. Thus, active sites are away from the 6-fold axis of symmetry toward the outer edges of the dodecamer and are located greater than or equal to 30 A from the plane separating the hexagonal rings. During Zn2+-induced stacking of the same enzyme derivatives along the 6-fold axes of symmetry, additional quenches of fluorescent probes were dependent on the presence of acceptors on separate dodecamers. The Zn2+-induced face to face aggregation of dodecamers in the presence of 46 microM ZnCl2 and 9 mM MgCl2 at pH 7.0 had an Arrhenius activation energy of 22.3 +/- 0.2 kcal/mol and a second-order rate constant at 25 degrees C of approximately 10(5) M-1 s-1 at early stages. Time-dependent fluorescence quenches correlated well with the degree of linear polymer formation and reached maximum values of 47-70% quench when the average n-mer was six dodecamers. After correction for unquenched polymer ends, a fluorescent donor and an acceptor probe in layered dodecamers were estimated to be approximately 36 A apart--an average value if there is some twisting of single strands.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
19.
Symmetry, homology, and phrasing in the recognition of helical regulatory sequences in DNA.
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M C O''Neill 《Nucleic acids research》1977,4(12):4439-4463
Regulatory regions in DNA which have been sequenced have generally been found to contain one or more axes of two-fold rotational symmetry. If this symmetry is to be maintained in the helical sequence, the axis of rotation must be aligned with one of the two dyad axes of the helix. This is equivalent to saying that the rotational symmetry of the sequence can only be seen from certain viewing points in a circuit about the helix. More surprising is the fact that new symmetrical sequence arrangements can be seen at +/- 36 degrees, +/- 72 degrees, +/- 108 degrees, and +/- 144 degrees relative to the point at which the rotational symmetry is seen. This "amplification" of symmetry suggests a three-dimensional approach to sequence analysis. A specific reading frame, suggested by the geometry of the helix, is examined with regard to its elucidation of intra- and inter-sequence homologies. Two sequences are thus identified as being recurrent in a number of different regulatory sequences. 相似文献
20.
A Bacher S Weinkauf L Bachmann K Ritsert W Baumeister R Huber R Ladenstein 《Journal of molecular biology》1992,225(4):1065-1073
The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits enclosing a core of 3 alpha subunits. The preparation of reconstituted hollow capsids consisting of 60 beta subunits and their crystallization in a hexagonal (space group P6(3)22) and in a monoclinic (space group C2) modification have been described. The rotational and translational parameters of the protein molecules in both crystal forms were studied by electron microscopy of freeze-etch replicas and by Patterson correlation techniques. Decoration with silver and image processing provided images with the positions of the 3-fold and 5-fold molecular axes being labelled by metal clusters. This allowed the unequivocal determination of the orientation and translational position of the protein molecules with respect to the crystallographic axes in the hexagonal modification. From inspection of the decoration images it was immediately obvious that the hexagonal crystal forms of alpha 3 beta 60 and of beta 60 are isomorphous. In the monoclinic crystals, a local icosahedral 2-fold coincides with the crystallographic 2-fold axis. The exact solution of the particle orientation was determined by interpretation of Patterson self-rotation functions for the icosahedral symmetry axes. Rotational and translational parameters for the monoclinic modification are given. A rational procedure for the efficient application of freeze-etching techniques in order to elucidate the packing in crystals of large proteins is described. 相似文献