Mapping the absolute value of M0 using dipolar field effects.

The ability to map the spatial variation of the absolute, rather than the relative value of the equilibrium magnetization could be advantageous in many areas of NMR. However, direct measurement of M(0) is usually difficult because of the multiparametric dependence of the NMR signal. Here we propose a technique for mapping the spatial variation of the absolute value of M(0), independent of relaxation weighting and flip angle calibration. This method, which works best at high field strengths, is based on the effect of the dipolar field due to the nuclear magnetization that is normally neglected in liquid-state NMR. The experimental implementation of this sequence at 3.0 T is described, and its initial application to the measurement of the water content of brain tissue is outlined.

Imaging the long-range dipolar field in structured liquid state samples.

We describe imaging experiments in which the pattern of the dipolar field generated by spatially modulated nuclear magnetization is directly visualized in simply structured phantoms. Two types of experiment have been carried out at 11.7 T using (1)H NMR signals. In the first, the field from a single spin species is imaged via its own NMR signal. In the second, the NMR signal from one spin species is used to image the field generated by a second species. The field patterns measured in these experiments correspond well with those calculated using simple theoretical expressions for the dipolar field. The results also directly demonstrate the spatial sensitivity of the signal generated using dipolar field effects, indicating that the range of the field depends upon the inverse of the spatial frequency with which the magnetization is modulated.

Plastome engineering of ribulose-1,5-bisphosphate carboxylase/oxygenase in tobacco to form a sunflower large subunit and tobacco small subunit hybrid.

Targeted gene replacement in plastids was used to explore whether the rbcL gene that codes for the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase, the key enzyme of photosynthetic CO2 fixation, might be replaced with altered forms of the gene. Tobacco (Nicotiana tabacum) plants were transformed with plastid DNA that contained the rbcL gene from either sunflower (Helianthus annuus) or the cyanobacterium Synechococcus PCC6301, along with a selectable marker. Three stable lines of transformants were regenerated that had altered rbcL genes. Those containing the rbcL gene for cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase produced mRNA but no large subunit protein or enzyme activity. Those tobacco plants expressing the sunflower large subunit synthesized a catalytically active hybrid form of the enzyme composed of sunflower large subunits and tobacco small subunits. A third line expressed a chimeric sunflower/tobacco large subunit arising from homologous recombination within the rbcL gene that had properties similar to the hybrid enzyme. This study demonstrated the feasibility of using a binary system in which different forms of the rbcL gene are constructed in a bacterial host and then introduced into a vector for homologous recombination in transformed chloroplasts to produce an active, chimeric enzyme in vivo.

Molecular mass determination for prostate-specific antigen and alpha 1-antichymotrypsin complexed in vitro.

The results from molecular mass determinations and amino acid analyses for prostate-specific antigen (PSA), alpha 1-antichymotrypsin (ACT) and PSA-ACT complexed in vitro are reported. Molecular masses for two separate PSA-ACT lots were determined by matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS) coupled to a time-of-flight (TOF) detector. Interestingly, both PSA-ACT lots contained two predominant protein species: 78,095 +/- 138 Da (approx. 67% of total protein) and 82,519 +/- 104 Da. Because a heterogneous population was observed and the masses were less than expected on the basis of less sensitive techniques, molecular masses for the individual PSA and ACT components were determined. As expected, PSA possessed a single molecular mass (27,755.8 Da). Each ACT raw material lot, however, also contained two predominant species: 55,106 +/- 111 and 51,414 +/- 32 Da. To assess the amino acid composition, each PSA, ACT and PSA-ACT lot was subjected to acid hydrolysis for 24, 48 and 72 h followed by amino acid analysis. Experimental results, expressed as mole percentages for each measurable residue and the number of residues per mole of protein, were compared with both the predicted and previously published values and were as expected. These more accurate molecular mass values for ACT and purified PSA-ACT complex should be considered in preparing and characterizing international standard preparations.

Aspartic proteinase genes in the Brassicaceae Arabidopsis thaliana and Brassica napus.

Active aspartic proteinase is isolated from Brassica napus seeds and the peptide sequence is used to generate primers for PCR. We present here cDNA and genomic clones for aspartic proteinases from the closely related Brassicaceae Arabidopsis thaliana and Brassica napus. The Arabidopsis cDNA represents a single gene, while Brassica has at least 4 genes. Like other plant aspartic proteases, the two Brassicaceae enzymes contain an extra protein domain of about 100 amino acids relative to the mammalian forms. The intron/exon arrangement in the Brassica genomic clone is significantly different from that in mammalian genes. As the proteinase is isolated from seeds, the same tissue where 2S albumins are processed, this implies expression of one of the aspartic proteinase genes there.

Oligonucleotide sequence and composition determined by matrix-assisted laser desorption/ionization.

Molecular weight measurements of several oligonucleotides ranging in size from 12 to 60 bases were performed by matrix-assisted laser desorption/ionization with a time-of-flight mass spectrometer (MALDI-TOF). In each case, the mass accuracy was better than 0.1%. Sequences for two 12-base oligonucleotides and a 24-base oligonucleotide were determined using calf spleen phosphodiesterase to sequentially cleave from the 5' end. A MALDI-TOF spectrum of the digest mixture shortly after the addition of the enzyme produced a characteristic oligonucleotide ladder. Molecular ions in the mass spectrum corresponded to the products of enzymatic cleavage, and the mass differences between these peaks identified the individual nucleotides. The resolution and mass accuracy of MALDI-TOF were sufficient to unambiguously identify the individual nucleotides in the 12- and 24-base strands.

Structure of an effector-induced inactivated state of ribulose 1,5-bisphosphate carboxylase/oxygenase: the binary complex between enzyme and xylulose 1,5-bisphosphate.

BACKGROUND: Ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) catalyzes the addition of CO2 to ribulose 1,5-bisphosphate in all photosynthetic organisms. During catalysis, the bisphosphate is depleted by reactions other than carboxylation and some of the products are potent inhibitors of rubisco. We have used one of these, xylulose 1,5-bisphosphate as an analogue of the natural substrate and co-crystallized it with the enzyme. RESULTS: We have solved the crystal structure of Synechococcus rubisco with bound xylulose 1,5-bisphosphate to 2.3 A and compared it with the previously solved 2'-carboxylarabinitol 1,5-bisphosphate (2CABP) enzyme quaternary complex. Unlike 2CABP, xylulose 1,5-bisphosphate forms a binary complex with no activating CO2 or essential metal present. Five flexible elements that restrict access to the active site in the 2CABP complex also close off the active site in the xylulose 1,5-bisphosphate complex, stabilized by interactions with the hydrated form of the analogue. CONCLUSIONS: Xylulose 1,5-bisphosphate induces closure of critical loops of the protein without essential cofactors resident at the active site. In the case of rubisco in one species, catalysis is completely inhibited.

The X-ray structure of Synechococcus ribulose-bisphosphate carboxylase/oxygenase-activated quaternary complex at 2.2-A resolution.

The structure of the hexadecameric ribulose-bisphosphate carboxylase/oxygenase from Synechococcus PCC6301 has been solved to 2.2-A resolution. Crystallization was in the presence of CO2, Mg2+, and 2'-carboxyarabinitol bisphosphate to form a stable enzyme quaternary complex that mimics one of the intermediate states of the carboxylation reaction. The structure was solved by molecular replacement using the coordinates of spinach carboxylase. The deviations in C alpha positions of the L- and S-subunits are only 0.3 and 2.0 A, respectively, and localized at specific regions of the two polypeptides. One region that shows significant divergence of the peptide backbone is loop 6 of the beta barrel in the L-subunit. Two other elements, the C terminus, and a highly conserved loop of the N-terminal domain of a second L-subunit, interact with loop 6 in the quaternary complex. These three regions, plus two other flexible segments, completely enfold the bisphosphate inhibitor. Significant alteration in their spatial relationship must occur to allow substrates or products access to and from the active site. The active site residues, activating cofactors, and inhibitor are well resolved in the electron density map. The disposition of these groups around the essential metal provides some indication of their role at different stages of the catalytic cycle.

Site-specific mutations in a loop region of the C-terminal domain of the large subunit of ribulose bisphosphate carboxylase/oxygenase that influence substrate partitioning.

Amino acids composing a flexible loop (loop 6) of the eight-stranded barrel domain of the L-subunit of Synechococcus ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) involved in reaction intermediate stabilization have been modified by site-specific mutagenesis. Changes at positions both distant and within the active site affect overall catalysis and substrate partitioning. Most significantly, replacement of the active site Lys (Lys-334) with Arg at the apex of the loop almost completely suppressed the carboxylase activity of the enzyme relative to oxygenation, with only a modest reduction in overall catalysis. Val-331 and Thr-342, more distant from the active site but with interacting side chains, were changed to larger and smaller residues with differential effects on both turnover and substrate partitioning. Substitution of the loop with the sequence found in more efficient carboxylases only increased partitioning marginally when accompanied by alterations in the C-terminal tail of the L-subunit that interacts with the loop. Generally, modifications to the loop composition also affected enediol formation, the first step of catalysis, suggesting that the geometry and hence flexibility of this segment affect more than just stabilization of the intermediates immediately following reaction with CO2 or O2.

Substitution of ASP193 to ASN at the active site of ribulose-1,5-bisphosphate carboxylase results in conformational changes.

The crystal structure of a mutant of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillium rubrum, where Asp193, one of the ligands of the magnesium ion at the activator site, is replaced by Asn, has been determined to a nominal resolution of 0.26 nm. The mutation of Asp to Asn induces both local and global conformation changes as follows. The side chain of Asn193 moves away from the active site and interacts with main-chain oxygen of residue 165, located in the neighbouring strand beta 1 of the alpha/beta barrel. The side chain of Lys166, which forms a salt bridge with Asp193 in the wild-type enzyme, interacts with Asn54 from the second subunit and creates a new subunit-subunit interaction. Another new subunit-subunit interaction is formed, more than 1.2 nm away from the site of the mutation. In the mutant enzyme, the side chain of Asp263 interacts with the side chain of Thr106 from the second subunit. Asp193 is not part of a subunit-subunit interface area or an allosteric regulatory site. Nevertheless, replacement of this residue by Asn results, unexpectedly, in a difference in the packing of the two subunits, which can be described as a slight rotation of one of the subunits relative to the second. The observed structural changes at the active site of the enzyme provide a molecular explanation for the differing behaviour of the Asp193----Asn mutant with respect to activation.

An intra-dimeric crosslink of large subunits of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase is formed by oxidation of cysteine 247.

Crystals of the hexadecameric form of ribulose-bisphosphate carboxylase used to solve the structure of the enzyme are composed of protein substantially crosslinked by a disulfide bond between pairs of large subunits. Conditions leading to the selective formation of dimers of the large subunits are described. The stability and specificity of the intra-dimeric crosslink was used to confirm that only one cysteine residue, Cys247 of neighboring large subunits, is involved in the bridge. The ability to generate this disulfide selectively, or alternatively replace the cysteine by site-directed mutagenesis, has led us to conclude that there is no effect of these changes on any of the critical kinetic parameters of the enzyme. The benign effect of the oxidation indicates that the crystal structures of the ribulose-bisphosphate carboxylase, particularly of the active site, are a true representation of the native enzyme.

The relative catalytic specificities of the large subunit core of Synechococcus ribulose bisphosphate carboxylase/oxygenase.

The relative specificities of the carboxylase and oxygenase reactions catalyzed by the recombinant large subunit core (L8) of Synechococcus ribulose 1,5-bisphosphate carboxylase have been determined. The L8 core still retained the ability to catalyze both reactions but at a much reduced turnover rate, about 0.6% of the holoenzyme. The fate of ribulose 1,5-bisphosphate during carboxylation and oxygenation by L8 was compared with the Synechococcus holoenzyme (reconstituted from L8 and recombinant small subunits), the carboxylase from Rhodospirullum rubrum, and that of spinach. The absence of small subunits had no significant effect on the partitioning of the bisphosphate substrate between the two reactions. Thus the course of the two competing reactions is a characteristic of the structural elements that compose the L-subunits, whereas the S-subunits exert their effect on factors common to both reactions such as the specificity of the bisphosphate substrate.

The purification and preliminary X-ray diffraction studies of recombinant Synechococcus ribulose-1,5-bisphosphate carboxylase/oxygenase from Escherichia coli.

X-ray crystallographic diffraction data has been collected for recombinant hexadecameric ribulose-P2 carboxylase from the cyanobacterium Synechococcus PCC6301 expressed in Escherichia coli. The enzyme has been purified and then crystallized in a number of crystal forms from polyethylene glycol solutions. The best crystals were obtained with enzyme that was first activated with the cofactors CO2 and Mg2+ in the presence of the tight-binding intermediate analogue, 2'-carboxyarabinitol 1,5-bisphosphate. One crystal form with plate-like morphology diffracts beyond 2.5 A but has one axis greater than 350 A. A second crystal form that diffracts to similar resolution grows with space group P212121 and unit cell dimensions of a = 223.9 A, b = 111.9 A, and c = 199.7 A. The crystal forms used to collect the diffraction data have been redissolved to determine that the recombinant ribulose-P2 carboxylase L8S8 molecule is indeed composed of equal numbers of large and small subunits and also that a quaternary complex between activated ribulose-P2 carboxylase E.CO2.Mg2+, and the analogue was present in the crystals. Denaturation of the redissolved enzyme in the absence of thiol-reducing agents established that the L-subunits of the L8 core are substantially dimeric, cross-linked by a disulfide bridge. Crystals of spinach ribulose-P2 carboxylase were likewise analyzed to show that dimers of the L-subunit were also predominant. This report identifies a single cysteine residue in the L-subunit that forms a bridge between those L-monomers that compose the four putative functional dimers of the L8 core.

The orientation of substrate and reaction intermediates in the active site of ribulose-1,5-bisphosphate carboxylase.

There are four possible orientations of the substrate ribulose 1,5-bisphosphate in the active site of ribulose-1,5-bisphosphate carboxylase. Distinction between these four possible orientations has been made on the basis of 31P NMR and borohydride-trapping experiments. The orientation of the reaction-intermediate analog, 2'-carboxy-D-arabinitol 1,5-bisphosphate with respect to the divalent metal ion was determined by 31P NMR studies of the quaternary complex, enzyme.CO2.Ni2+.2'-carboxyarabinitol 1,5-bisphosphate. Assignment of the phosphorus resonances of this complex was made by labeling the phosphoryl group at either C-1 or C-5 with 17O. The phosphorus atom closer to the paramagnetic metal ion, Ni2+, to which the broader of the phosphorus resonances is attributed, has been identified as that attached to C-1. When bound to the active site of carbaminated enzyme, D-ribulose 1,5-bisphosphate was reduced by sodium borohydride with absolute stereospecificity to D-arabinitol 1,5-bisphosphate. The reduction of the enzyme-bound substrate thus occurred on the Si face of the C-2 carbonyl group. These two results together establish that ribulose 1,5-bisphosphate is oriented within the active site so that 1) the phosphoryl group at C-1 is closer to the divalent metal ion than that at C-5 and 2) the Si face of the carbonyl group points to the "outside world."

The synthesis and purification of 2'-carboxy-D-arabinitol 1-phosphate, a natural inhibitor of ribulose 1,5-bisphosphate carboxylase, investigated by 31P n.m.r.

2'-Carboxy-D-arabinitol 1-phosphate (2CA1P), a natural inhibitor of ribulose 1,5-bisphosphate carboxylase was synthesized from 2'-carboxy-D-arabinitol 1,5-bisphosphate (2CABP). The selective dephosphorylation of 2CABP with either acid phosphatase or alkaline phosphatase was investigated by using 31P n.m.r. The n.m.r. spectra of the progress of the reactions indicated that both phosphatases preferentially removed the 5-phosphate from the bisphosphate. After the consumption of all of the bisphosphate, alkaline phosphatase generated a mixture of 2'-carboxy-D-arabinitol 1- and 5-monophosphates in the ratio of about 4:1, along with Pi. The enzyme also hydrolysed the monophosphates to 2'-carboxyarabinitol, thus decreasing the yield of 2CA1P further. In contrast, acid phosphatase catalysed almost quantitative conversion of 2CABP into 2CA1P, preferring to hydrolyse only the 5-phosphate. In either case, separation of the 2CA1P from Pi or other products of enzymic hydrolysis was readily accomplished by conventional ion-exchange chromatography or h.p.l.c.

The role of the N-terminus of the large subunit of ribulose-bisphosphate carboxylase investigated by construction and expression of chimaeric genes.

The genes for the large and small subunits of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Anacystis nidulans have been expressed in Escherichia coli under the control of the lac promoter to produce active enzyme. The enzyme can be purified from the cells to yield up to 200 mg Rubisco/l cultured bacteria, and is indistinguishable from the enzyme extracted from A. nidulans. In order to investigate the role of the N-terminus of the large subunit in catalysis, chimaeric genes were constructed where the DNA coding for the 12 N-terminal amino acids in A. nidulans was replaced by DNA encoding the equivalent, but poorly conserved, region of either the wheat or maize large subunit. These genes, in constructs also containing the gene for the A. nidulans small subunit, were expressed in E. coli and produced enzymes with similar catalytic properties to the wild-type Rubisco of A. nidulans. In contrast, when the N-terminal region of the large subunit was replaced by unrelated amino acids encoded by the pUC8 polylinker, enzyme activity of the expressed protein was reduced by 90% under standard assay conditions, due to an approximately tenfold rise in the Km for ribulose 1,5-bisphosphate. This confirms that the N-terminus of the large subunit has a function in catalysis, either directly in substrate binding or in maintaining the integrity of the active site.

Species variation in the predawn inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase.

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase was measured in extracts of leaves collected before dawn (predawn activity, pa) and at midday (midday activity, ma). Twenty-three of the 37 species examined showed a pa/ma ratio (

Large-scale preparation of ribulosebisphosphate carboxylase from a recombinant system in Escherichia coli characterized by extreme plasmid instability.

An ampicillin-resistant, RecA- strain of Escherichia coli (HB101) harboring the multicopy pBR322 plasmid containing the structural gene for ribulosebisphosphate carboxylase from Rhodospirillum rubrum was used to prepare large quantities of the carboxylase protein. This recombinant system was characterized by extreme plasmid instability, which resulted in part from the 1.7-fold faster growth rate of plasmid-free cells and in part from very rapid rates of plasmid segregation. The plasmid-containing organisms produced and excreted a large amount of beta-lactamase activity, with the result that ampicillin selection could only be maintained for a very short period of time, after which the plasmid-containing (carboxylase-producing) cells were overgrown by plasmid-free cells. The instability was so severe that even isolated colonies prepared on ampicillin-containing plates were impure and contained plasmid-free cells. Nevertheless, large quantities of carboxylase protein could be obtained from this system by using a highly dilute inoculum which allows selection of ampicillin-resistant (carboxylase-producing) organisms for a sufficient period of time so that the period of growth under nonselective conditions was minimized, and cells harvested at high cell densities contained large amounts of the carboxylase protein. In the present instance, 300-liter fermentations were initiated with a 0.3-microliter inoculum of freshly grown cells. After 20 h of growth in rich medium containing ampicillin, the harvested cells contained 74 g of ribulosebisphosphate carboxylase protein (average of two separate cultures). These results are discussed in terms of the general nature of plasmid instability and protocols available to minimize the effects of such instability.