Metabolic fluxes in riboflavin-producing Bacillus subtilis.

The pentose phosphate pathway and the pyruvate shunt were identified as major pathways of glucose catabolism in a recombinant, riboflavin-producing Bacillus subtilis strain. Reactions connecting the tricarboxylic acid cycle and glycolysis, catalyzed by the malic enzyme and phosphoenolpyruvate carboxykinase, consume up to 23% of the metabolized glucose. These are examples of important fluxes that can be accessed explicitly using a novel analysis based on synergistic application of flux balancing and recently introduced techniques of fractional 13C-labeling and two-dimensional nuclear magnetic resonance spectroscopy. The overall flux distribution also suggests that B. subtilis metabolism has an unusually high capacity for the reoxidation of NADPH. Under the conditions investigated, riboflavin formation in B. subtilis is limited by the fluxes through the biosynthetic rather than the central carbon pathways, which suggests a focus for future metabolic engineering of this system.

Design of structurally distinct proteins using strategies inspired by evolution.

Natural recombination combines pieces of preexisting proteins to create new tertiary structures and functions. We describe a computational protocol, called SEWING, which is inspired by this process and builds new proteins from connected or disconnected pieces of existing structures. Helical proteins designed with SEWING contain structural features absent from other de novo designed proteins and, in some cases, remain folded at more than 100°C. High-resolution structures of the designed proteins CA01 and DA05R1 were solved by x-ray crystallography (2.2 angstrom resolution) and nuclear magnetic resonance, respectively, and there was excellent agreement with the design models. This method provides a new strategy to rapidly create large numbers of diverse and designable protein scaffolds.

Nuclear magnetic resonance solution structure of hirudin(1-51) and comparison with corresponding three-dimensional structures determined using the complete 65-residue hirudin polypeptide chain.

The three-dimensional structure of the N-terminal 51-residue domain of recombinant hirudin in aqueous solution was determined by 1H nuclear magnetic resonance (NMR) spectroscopy, and the resulting high-quality solution structure was compared with corresponding structures obtained from studies with the intact, 65-residue polypeptide chain of hirudin. On the basis of 580 distance constraints derived from nuclear Overhauser effects and 109 dihedral angle constraints, a group of 20 conformers representing the solution structure of hirudin(1-51) was computed with the program DIANA and energy-minimized with a modified version of the program AMBER. Residues 3 to 30 and 37 to 48 form a well-defined molecular core with two antiparallel beta-sheets composed of residues 14 to 16 and 20 to 22, and 27 to 31 and 36 to 40, and three reverse turns at residues 8 to 11 (type II), 17 to 20 (type II') and 23 to 26 (type II). The average root-mean-square deviation of the individual NMR conformers relative to their mean co-ordinates is 0.38 A for the backbone atoms and 0.77 A for all heavy atoms of these residues. Increased structural disorder was found for the N-terminal dipeptide segment, the loop at residues 31 to 36, and the C-terminal tripeptide segment. The solution structure of hirudin(1-51) has the same molecular architecture as the corresponding polypeptide segment in natural hirudin and recombinant desulfatohirudin. It is also closely similar to the crystal structure of the N-terminal 51-residue segment of hirudin in a hirudin-thrombin complex, with root-mean-square deviations of the crystal structure relative to the mean solution structure of 0.61 A for the backbone atoms and 0.91 A for all heavy atoms of residues 3 to 30 and 37 to 48. Further coincidence is found for the loop formed by residues 31 to 36, which shows increased structural disorder in all available solution structures of hirudin, and of which residues 32 to 35 are not observable in the electron density map of the thrombin complex. Significant local structural differences between hirudin(1-51) in solution and hirudin in the crystalline thrombin complex were identified mainly for the N-terminal tripeptide segment and residues 17 to 21. These are further analyzed in an accompanying paper.

NMR structure of the J-domain and the Gly/Phe-rich region of the Escherichia coli DnaJ chaperone.

The recombinant N-terminal 107-amino acid polypeptide fragment 2-108 of the DnaJ molecular chaperone of Escherichia coli, which contains the J-domain (residues 2 to 76) and the Gly/Phe-rich region (residues 77 to 108), was uniformly labeled with nitrogen-15 and carbon-13. The complete NMR solution structure of the J-domain was determined with the program DIANA on the basis of 682 nuclear Overhauser enhancement (NOE) upper distance limits and 180 dihedral angle constraints. It contains three well-defined helices comprising residues 6 to 10, 18 to 32 and 41 to 57, and a fourth helix, consisting of residues 61 to 68, which is well defined as a regular secondary structure but for which the location relative to the remainder of the molecule is not precisely determined. The helices II and III form an antiparallel helical coiled-coil. Helix I is approximately parallel to the plane defined by the helices II and III and runs from the carboxy-terminal end of the helix III to the center of helix II. Helix IV is positioned near the carboxy-terminal end of helix III and is on the same side of the coiled coil as helix I, but it is oriented approximately perpendicular to the plane of the helices II and III. This novel alpha-protein topology leads to formation of a hydrophobic core involving side-chains of all four helices. A strong correlation is seen between the extent of sequence-conservation of hydrophobic residues in the family of J-domain homologues, and the structural organization of the hydrophobic core in these proteins. The residues which have key roles for the specificity of the interaction of DnaJ-like proteins with their corresponding Hsp70 counterparts are located on the outer surfaces of the helices II and III, and in the loop connecting these two helices. Measurements of backbone amide proton exchange rates, 15N spin relaxation times and heteronuclear 15N {1H} NOEs provided additional insights into local conformational equilibria and internal rate processes in DnaJ(2-108). In the Gly/Phe-rich region, which is poorly ordered in the NMR solution structure and does not form a globular core, the polypeptide segment 90 to 103 differs from the segments 77 to 89 and 104 to 108 by reduced local flexibility. Considering that this same segment shows sequence conservation with corresponding segments in the Gly/Phe-rich regions of other DnaJ-like proteins, its reduced flexibility may be directly linked to the formation of the ternary DnaJ-DnaK-polypeptide complex.

Impact of protein-protein contacts on the conformation of thrombin-bound hirudin studied by comparison with the nuclear magnetic resonance solution structure of hirudin(1-51).

The impact of protein-protein interactions on the conformation of the N-terminal hirudin domain consisting of residues 1 to 51 in the X-ray crystal structure of a hirudin-thrombin complex was investigated through comparisons with the nuclear magnetic resonance solution structure of hirudin(1-51). The close overall similarity observed between these two structures contrasts with the behavior of the C-terminal 17-residue polypeptide segment of hirudin, which is flexibly disordered in solution but exhibits a defined conformation in the complex with thrombin. Localized structural differences in the N-terminal domain include that residues 1 to 3 of hirudin in the crystalline complex form a hydrogen-bonding network with thrombin that is reminiscent of a parallel beta-sheet. Moreover, the backbone conformation of residues 17 to 20 in the complex does not contain the characteristic hydrogen bond observed for the type II' reverse turn in the solution structure, and the side-chains of Ser19 and Val21 have significantly different orientations in the two structures. Most of these structural changes can be related directly to thrombin-hirudin contacts, which may also be an important factor in the mechanism of hirudin action. In this context, it is of special interest that other residues that also make numerous contacts with thrombin, e.g. Thr4, Asp5 and Asn20, have identical conformations in free hirudin and in the complex.

Internal mobility of the basic pancreatic trypsin inhibitor in solution: a comparison of NMR spin relaxation measurements and molecular dynamics simulations.

Order parameters as well as longitudinal and transverse relaxation rates are calculated for the backbone 15N and 13C alpha nuclei of the basic pancreatic trypsin inhibitor (BPTI) from a 1000 ps molecular dynamics trajectory in explicit water at 277 K using the "model free" approach of Lipari and Szabo. New NMR relaxation data at 277 K are presented, and a comparison is made between NMR relaxation measurements and molecular dynamics relaxation data. It is found that the relaxation processes determining the longitudinal (T1) relaxation rates are inadequately sampled even during this length of simulation. In effect, the calculated relaxation rates are determined almost solely by the order parameters and the overall rotational correlation time of the protein, which appears to be in clear contrast to experimental relaxation rates.

NMR structure of the sterol carrier protein-2: implications for the biological role.

The determination of the NMR structure of the sterol carrier protein-2 (SCP2), analysis of backbone (15)N spin relaxation parameters and NMR studies of nitroxide spin-labeled substrate binding are presented as a new basis for investigations of the mode of action of SCP2. The SCP2 fold is formed by a five-stranded beta-sheet and four alpha-helices. Fatty acid binding to a hydrophobic surface area formed by amino acid residues of the first and third helices, and the beta-sheet, which are all located in the polypeptide segment 8-102, was identified with the use of the spin-labeled substrate 16-doxylstearic acid. In the free protein, the lipid-binding site is covered by the C-terminal segment 105-123, suggesting that this polypeptide segment, which carries the peroxisomal targeting signal (PTS1), might be involved in the regulation of ligand binding.

Conformational changes of the BS2 operator DNA upon complex formation with the Antennapedia homeodomain studied by NMR with 13C/15N-labeled DNA.

The NMR structures have been determined for a 13C/15N doubly labeled 14 base-pair DNA duplex comprising the BS2 operator sequence both free in solution and in the complex with the Antennapedia homeodomain. The impact of the DNA labeling is assessed from comparison with a previous structure of the same complex that was determined using isotope labeling only for the protein. Differences between the two structure determinations are nearly completely limited to the DNA, which retains the global B -conformation of the free DNA also in the complex. Local protein-induced conformational changes are a narrowing of the minor groove due to the interaction with the N-terminal arm of the homeodomain, and changes of the sugar puckers of the deoxyriboses G5 and C6, which are apparently induced by van der Waals interactions with Tyr25, and with Gln50 and Arg53, respectively. The high conservation of these amino acid residues in homeodomains suggests that protein-induced shifts in some sugar puckers contribute to the affinity of homeodomains to their cognate DNA. The data obtained here with the Antennapedia homeodomain-DNA complex clearly show that nucleic acid isotope-labeling can support detailed conformational characterization of DNA in complexes with proteins, which will be indispensable for structure determinations of complexes containing globally distorted DNA conformations.

NMR solution structure of the pathogenesis-related protein P14a.

The nuclear magnetic resonance (NMR) structure of the 15 kDa pathogenesis-related protein P14a, which displays antifungicidal activity and is induced in tomato leaves as a response to pathogen infection, was determined using 15N/13C doubly labeled and unlabeled protein samples. In all, 2030 conformational constraints were collected as input for the distance geometry program DIANA. After energy-minimization with the program OPAL the 20 best conformers had an average root-mean-square deviation value relative to the mean coordinates of 0.88 A for the backbone atoms N, C(alpha) and C', and 1.30 A for all heavy atoms. P14a contains four alpha-helices (I to IV) comprising residues 4 to 17, 27 to 40, 64 to 72 and 93 to 98, a short 3(10)-helix of residues 73 to 75 directly following helix III, and a mixed, four-stranded beta-sheet with topology +3x, -2x, +1, containing the residues 24-25, 53 to 58, 104 to 111 and 117 to 124. These regular secondary structure elements form a novel, complex alpha + beta topology in which the alpha-helices I, III and IV and the 3(10)-helix are located above the plane defined by the beta-sheet, and the alpha-helix II lies below this plane. The alpha-helices and beta-strands are thus arranged in three stacked layers, which are stabilized by two distinct hydrophobic cores associated with the two layer interfaces, giving rise to an "alpha-beta-alpha sandwich". The three-dimensional structure of P14a provides initial leads for identification of the so far unknown active sites and the mode of action of the protein, which is of direct interest for the generation of transgenic plants with improved host defense properties.

The NMR solution structure of the pheromone Er-2 from the ciliated protozoan Euplotes raikovi.

The NMR structure of the pheromone Er-2 from the ciliated protozoan Euplotes raikovi has been determined in aqueous solution. The structure of this 40-residue protein was calculated with the distance geometry program DIANA from 621 distance constraints and 89 dihedral angle constraints; the program OPAL was employed for the energy minimization. For a group of 20 conformers used to characterize the solution structure, the average pairwise RMS deviation from the mean structure calculated for the backbone heavy atoms N, C alpha, and C' of residues 3-37 was 0.31 A. The molecular architecture is dominated by an up-down-up bundle of 3 short helices of residues 5-11, 14-20, and 23-33, which is similar to the structures of the homologous pheromones Er-1 and Er-10. Novel structural features include a well-defined N-cap on the first helix, a 1-residue deletion in the second helix resulting in the formation of a 3(10)-helix rather than an alpha-helix as found in Er-1 and Er-10, and the simultaneous presence of 2 different conformations for the C-terminal tetrapeptide segment, i.e., a major conformation with the Leu 39-Pro 40 peptide bond in the trans form and a minor conformation with this peptide bond in the cis form.

Pulmonary surfactant-associated polypeptide C in a mixed organic solvent transforms from a monomeric alpha-helical state into insoluble beta-sheet aggregates.

In the 35-residue pulmonary surfactant-associated lipopolypeptide C (SP-C), the stability of the valyl-rich alpha-helix comprising residues 9-34 has been monitored by circular dichroism, nuclear magnetic resonance, and Fourier transform infrared spectroscopy in both a mixed organic solvent and in phospholipid micelles. The alpha-helical form of SP-C observed in freshly prepared solutions in a mixed solvent of CHCl3/CH3OH/0.1 M HCl 32:64:5 (v/v/v) at 10 degrees C undergoes within a few days an irreversible transformation to an insoluble aggregate that contains beta-sheet secondary structure. Hydrogen exchange experiments revealed that this conformational transition proceeds through a transition state with an Eyring free activation enthalpy of about 100 kJ mol(-1), in which the polypeptide segment 9-27 largely retains a helical conformation. In dodecylphosphocholine micelles, the helical form of SP-C was maintained after seven weeks at 50 degrees C. The alpha-helical form of SP-C thus seems to be the thermodynamically most stable state in this micellar environment, whereas its presence in freshly prepared samples in the aforementioned mixed solvent is due to a high kinetic barrier for unfolding. These observations support a previously proposed pathway for in vivo synthesis of SP-C through proteolytic processing from a 21-kDa precursor protein.

Pulmonary surfactant-associated polypeptide SP-C in lipid micelles: CD studies of intact SP-C and NMR secondary structure determination of depalmitoyl-SP-C(1-17).

The surfactant-associated polypeptide C (SP-C) is a 35-residue lipopolypeptide which is essential for the function of surfactants used for therapy of infant respiratory distress. Modeling based on the recently determined nuclear magnetic resonance (NMR) structure of native SP-C in an organic solvent showed that SP-C could readily insert into fluid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers. The present paper describes further physical-chemical studies of intact SP-C and its N-terminal 17-residue polypeptide fragment, depalmitoyl-SP-C(1-17), in the presence of dodecylphosphocholine micelles. The results obtained provide a link between the NMR solution structure and the behaviour of SP-C in an ordered lipid environment, and thus present new insights for rational design of SP-C analogs for therapeutic purposes.

NMR determination of the secondary structure and the three-dimensional polypeptide backbone fold of the human sterol carrier protein 2.

Nuclear magnetic resonance (NMR) spectroscopy was used to determine the secondary structure and the three-dimensional polypeptide backbone fold of the human sterol carrier protein 2 (hSCP2), which is a basic protein with 123 residues believed to participate in the intracellular transport of cholesterol and various other lipids. Sequence-specific assignments were obtained for nearly all backbone 1H and 15N resonances, as well as for about two-thirds of the side-chain 1H resonances, using uniform 15N-labeling of the protein combined with homonuclear two-dimensional 1H NMR and three-dimensional 15N-correlated 1H NMR. Three alpha-helices comprising the polypeptide segments of residues 9-22, 25-30 and 78-84 were identified by sequential and medium-range nuclear Overhauser effects (NOE). The analysis of long-range backbone-backbone NOEs showed that hSCP2 further contains a five-stranded beta-sheet including the residues 33-41, 47-54, 60-62, 71-76 and 100-102, which is a central feature of the molecular architecture. The first three strands are arranged in an antiparallel fashion, the polypeptide chain then crosses over this three-stranded sheet in a right-handed sense so that the fourth strand is added parallel to the first one. The fifth strand runs antiparallel to the fourth one, so that the overall topology is +1, +1, -3x, -1. The three-dimensional arrangement of the beta-sheet and the first two helices was determined using an input of 625 NOE upper distance constraints and 95 scalar coupling constants for a preliminary structure calculation with the distance geometry program DIANA.

Metabolic flux response to phosphoglucose isomerase knock-out in Escherichia coli and impact of overexpression of the soluble transhydrogenase UdhA.

Blocking glycolytic breakdown of glucose by inactivation of phosphoglucose isomerase (Pgi) in Escherichia coli led to a greatly reduced maximum specific growth rate. Examination of the operational catabolic pathways and their flux ratios using [U-(13)C(6)]glucose-labeling experiments and metabolic flux ratio analysis provide evidence for the pentose phosphate (PP) pathway as the primary route of glucose catabolism in the knock-out mutant. The resulting extensive flux through the PP pathway disturbs apparently the reducing power balance, since overexpression of the recently identified soluble transhydrogenase UdhA improves significantly the growth rate of the Pgi mutant. The presented results provide first evidence that UdhA restores the cellular redox balance by catalyzing electron transfer from NADPH to NADH.

The 2D [31P] spin-echo-difference constant-time [13C, 1H]-HMQC experiment for simultaneous determination of 3J(H3'P) and 3J(C4'P) in 13C-labeled nucleic acids and their protein complexes.

A two-dimensional [31P] spin-echo-difference constant-time [13C, 1H]-HMQC experiment (2D [31P]-sedct-[13C, 1H]-HMQC) is introduced for measurements of 3J(C4'P) and 3J(H3'P) scalar couplings in large 13C-labeled nucleic acids and in DNA-protein complexes. This experiment makes use of the fact that 1H-13C multiple-quantum coherences in macromolecules relax more slowly than the corresponding 13C single-quantum coherences. 3J(C4'P) and 3J(H3'P) are related via Karplus-type functions with the phosphodiester torsion angles beta and epsilon, respectively, and their experimental assessment therefore contributes to further improved quality of NMR solution structures. Data are presented for a uniformly 13C, 15N-labeled 14-base-pair DNA duplex, both free in solution and in a 17-kDa protein-DNA complex.

Biosynthetically directed fractional 13C-labeling of proteinogenic amino acids. An efficient analytical tool to investigate intermediary metabolism.

Biosynthetically directed fractional 13C labeling of proteinogenic amino acids is achieved by expression of proteins on a minimal medium which contains a mixture of [13C6]glucose and glucose with natural isotope abundance as the sole carbon source. Subsequent hydrolysis of the proteins yields the free amino acids. The observation of 13C-13C spin-spin scalar coupling fine structures in sensitive two-dimensional heteronuclear [13C,1H]-correlation spectroscopy (2D [13C,1H]-COSY) allows one to identify non-random 13C-labeling patterns arising from the incorporation of intact two-carbon and three-carbon fragments from a single source molecule of glucose into the amino acids. Since 2D [13C,1H]-COSY suffices to resolve all relevant resonances, the mixture of amino acids can be analyzed without further separation of its components. Probabilistic equations relate the observed multiplet intensities of the 13C fine structures to the relative abundance of the intact carbon fragments. They enable a quantitative analysis of the carbon flux in the network of biosynthetic pathways, thus using the proteinogenic amino acids as probes to study intermediary metabolism. This paper shows that biosynthetically directed fractional 13C labeling of amino acids provides an efficient analytical tool to quantitatively investigate glycolysis, pyruvate metabolism, pentose phosphate pathway, tricarboxylic acid cycle and C1 metabolism. Possible applications of the method include both the exploration of unknown biosynthetic pathways and the rapid elucidation of the response of a known biosynthetic reaction network to changes in growth conditions or genetic manipulations. In conjunction with the relatively low costs for isotopes, manpower and NMR instrument time, this makes biosynthetic fractional 13C labeling of proteinogenic amino acids particularly attractive to support process design and metabolic engineering in biotechnology, since screening procedures become feasible which enable a systematic characterization of the cell's metabolic state as a function of parameters that are involved in the optimization of biotechnological processes.

Deuterium isotope effects on the central carbon metabolism of Escherichia coli cells grown on a D2O-containing minimal medium.

Isotope effects on the central carbon metabolism due to the addition of variable amounts of D2O (0 to 70%) were investigated with biosynthetically directed fractional 13C-labeling for Escherichia coli BL21(DE3) cells during exponential growth on a M9 minimal medium containing a mixture of 70% unlabeled and 30% uniformly 13C-labeled glucose as the sole carbon source. The resulting 13C-labeling patterns in the amino acids were analysed by two-dimensional [13C,1H]-correlation spectroscopy. With the aforementioned growth conditions, higher D2O contents resulted in an increase of the anaplerotic supply of the tricarboxylic acid cycle via carboxylation of phosphoenolpyruvate when compared to the influx of acetyl-CoA. Furthermore, the addition of D2O affected the C1 metabolic pathways that involve Ser and Gly. Otherwise the E. coli cells showed identical topologies of the active biosynthetic pathways in H2O and at elevated D2O contents, and the metabolic flux ratios characterizing glycolysis and the pentose phosphate pathway were not measurably affected by the addition of D2O. Cells that had been adapted for growth in D2O exhibited the same response to the presence of D2O in the nutrient medium as non-adapted cells. Implications of these data for the preparation of recombinant deuterated proteins for NMR studies are discussed.

3D 13C-15N-heteronuclear two-spin coherence spectroscopy for polypeptide backbone assignments in 13C-15N-double-labeled proteins.

The pulse sequence of a new constant-time 3D triple-resonance experiment, ct-HA[CAN]HN, is presented. This experiment delineates exclusively scalar connectivities and uses 13C alpha-15N heteronuclear two-spin coherence to overlay the chemical shift evolution periods of the 13C alpha and 15N nuclei, thereby providing the four resonance frequencies of the alpha-proton, the alpha-carbon, the amide nitrogen, and the amide proton of a given amino acid residue in three dimensions. This experiment promises to be a valid alternative to 4D experiments, providing the same information on intraresidue polypeptide backbone connectivities in 13C-15N-double-labeled proteins.