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.

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.

Aromatic ring-flipping in supercooled water: implications for NMR-based structural biology of proteins.

We have characterized, for the first time, motional modes of a protein dissolved in supercooled water: the flipping kinetics of phenylalanyl and tyrosinyl rings of the 6 kDa protein BPTI have been investigated by NMR at temperatures between -3 and -16.5 degrees C. At T = -15 degrees C, the ring-flipping rate constants of Tyr 23, Tyr 35, and Phe 45 are smaller than 2 s(-1), i.e., flip-broadening of aromatic NMR lines is reduced beyond detection and averaging of NOEs through ring-flipping is abolished. This allows neat detection of distinct NOE sets for the individual aromatic (1)H spins. In contrast, the rings of Phe 4, Tyr 10, Tyr 21, Phe 22, and Phe 33 are flipping rapidly on the chemical shift time scale with rate constants being in the range from approximately 10(2) to 10(5) s(-1) even at T = -15 degrees C. Line width measurements in 2D [(1)H,(1)H]-NOESY showed that flipping of the Phe 4 and Phe 33 rings is, however, slowed to an extent that the onset of associated line broadening in the fast exchange limit is registered. The reduced ring-flipping rate constant of Phe 45 in supercooled water allowed very precise determination of Eyring activation enthalpy and entropy from cross relaxation suppressed 2D [(1)H,(1)H]-exchange spectroscopy. This yielded DeltaH = 14 +/- 0.5 kcal.mol(-1) and DeltaS = -4 +/- 1 cal.mol(-1).K(-1), i.e., values close to those previously derived by Wagner and Wüthrich for the temperature range from 4 to 72 degrees C (DeltaH = 16 +/- 1 kcal.mol(-1) and DeltaS = 6 +/- 2 cal.mol(-1).K(-1)). The preservation of the so far uniquely low value for DeltaS indicates that the distribution of internal motional modes associated with the ring flip of Phe 45 is hardly affected by lowering T well below 0 degrees C. Hence, if a globular protein does not cold denature, aromatic flipping rates, and thus likely also the rates of other conformational and/or chemical exchange processes occurring in supercooled water, can be expected to be well estimated from activation parameters obtained at ambient T. This is of keen interest to predict the impact of supercooling for future studies of biological macromolecules, and shows that our approach enables one to conduct NMR-based structural biology at below 0 degrees C in an unperturbed aqueous environment. A search of the BioMagResBank indicated that the overwhelming majority of the Phe and Tyr rings (>95%) are flipping rapidly on the chemical shift time scale at ambient T, while our data for BPTI and activation parameters available for ring-flipping in Iso-2-cytochrome c reveal that in these smaller proteins a total of six out of seventeen rings ( approximately 35%) are "frozen in" at T = -15 degrees C. This suggests that a large fraction of Tyr and Phe rings in globular proteins that are flipping rapidly on the chemical shift time scale at ambient T can be effectively slowed in supercooled water. The present investigation demonstrates that supercooling of protein solutions appears to be an effective means to (i) harvest potential benefits of stalled ring-flipping for refining NMR solution structures, (ii) recruit additional aromatic rings for investigating protein dynamics, and (iii) use multiple slowly flipping rings to probe cold denaturation. The implications for NMR-based structural biology in supercooled water are addressed.

Central carbon metabolism of Saccharomyces cerevisiae explored by biosynthetic fractional (13)C labeling of common amino acids.

Aerobic and anaerobic central metabolism of Saccharomyces cerevisiae cells was explored in batch cultures on a minimal medium containing glucose as the sole carbon source, using biosynthetic fractional (13)C labeling of proteinogenic amino acids. This allowed, firstly, unravelling of the network of active central pathways in cytosol and mitochondria, secondly, determination of flux ratios characterizing glycolysis, pentose phosphate cycle, tricarboxylic acid cycle and C1-metabolism, and thirdly, assessment of intercompartmental transport fluxes of pyruvate, acetyl-CoA, oxaloacetate and glycine. The data also revealed that alanine aminotransferase is located in the mitochondria, and that amino acids are synthesized according to documented pathways. In both the aerobic and the anaerobic regime: (a) the mitochondrial glycine cleavage pathway is active, and efflux of glycine into the cytosol is observed; (b) the pentose phosphate pathways serve for biosynthesis only, i.e. phosphoenolpyruvate is entirely generated via glycolysis; (c) the majority of the cytosolic oxaloacetate is synthesized via anaplerotic carboxylation of pyruvate; (d) the malic enzyme plays a key role for mitochondrial pyruvate metabolism; (e) the transfer of oxaloacetate from the cytosol to the mitochondria is largely unidirectional, and the activity of the malate-aspartate shuttle and the succinate-fumarate carrier is low; (e) a large fraction of the mitochondrial pyruvate is imported from the cytosol; and (f) the glyoxylate cycle is inactive. In the aerobic regime, 75% of mitochondrial oxaloacetate arises from anaplerotic carboxylation of pyruvate, while in the anaerobic regime, the tricarboxylic acid cycle is operating in a branched fashion to fulfill biosynthetic demands only. The present study shows that fractional (13)C labeling of amino acids represents a powerful approach to study compartmented eukaryotic systems.

Dissection of central carbon metabolism of hemoglobin-expressing Escherichia coli by 13C nuclear magnetic resonance flux distribution analysis in microaerobic bioprocesses.

Escherichia coli MG1655 cells expressing Vitreoscilla hemoglobin (VHb), Alcaligenes eutrophus flavohemoprotein (FHP), the N-terminal hemoglobin domain of FHP (FHPg), and a fusion protein which comprises VHb and the A. eutrophus C-terminal reductase domain (VHb-Red) were grown in a microaerobic bioreactor to study the effects of low oxygen concentrations on the central carbon metabolism, using fractional (13)C-labeling of the proteinogenic amino acids and two-dimensional [(13)C, (1)H]-correlation nuclear magnetic resonance (NMR) spectroscopy. The NMR data revealed differences in the intracellular carbon fluxes between E. coli cells expressing either VHb or VHb-Red and cells expressing A. eutrophus FHP or the truncated heme domain (FHPg). E. coli MG1655 cells expressing either VHb or VHb-Red were found to function with a branched tricarboxylic acid (TCA) cycle. Furthermore, cellular demands for ATP and reduction equivalents in VHb- and VHb-Red-expressing cells were met by an increased flux through glycolysis. In contrast, in E. coli cells expressing A. eutrophus hemeproteins, the TCA cycle is running cyclically, indicating a shift towards a more aerobic regulation. Consistently, E. coli cells displaying FHP and FHPg activity showed lower production of the typical anaerobic by-products formate, acetate, and D-lactate. The implications of these observations for biotechnological applications are discussed.

Protein NMR spectroscopy in structural genomics.

Protein NMR spectroscopy provides an important complement to X-ray crystallography for structural genomics, both for determining three-dimensional protein structures and in characterizing their biochemical and biophysical functions.

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.

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.

Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism.

The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional (13)C-(1)H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. coli cells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.

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.

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.

NMR structure of the chimeric hybrid duplex r(gcaguggc).r(gcca)d(CTGC) comprising the tRNA-DNA junction formed during initiation of HIV-1 reverse transcription.

A high-quality NMR solution structure of the chimeric hybrid duplex r(gcaguggc).r(gcca)d(CTGC) was determined using the program DYANA with its recently implemented new module FOUND, which performs exhaustive conformational grid searches for dinucleotides. To ensure conservative data interpretation, the use of 1H-1H lower distance limit constraints was avoided. The duplex comprises the tRNA-DNA junction formed during the initiation of HIV-1 reverse transcription. It forms an A-type double helix that exhibits distinct structural deviations from a standard A-conformation. In particular, the minor groove is remarkably narrow, and its width decreases from about 7.5 A in the RNA/RNA stem to about 4.5 A in the RNA/DNA segment. This is unexpected, since minor groove widths for A-RNA and RNA/DNA hybrid duplexes of approximately 11 A and approximately 8.5 A, respectively, were previously reported. The present, new structure supports that reverse transcriptase-associated RNaseH specificity is related primarily to conformational adaptability of the nucleic acid in 'induced-fit'-type interactions, rather than the minor groove width of a predominantly static nucleic acid duplex.

Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica.

Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13C labeling with a mixture of 90% unlabeled and 10% uniformly 13C-labeled glycerol. The resulting 13C-labeling patterns in the amino acids were analyzed by two-dimensional 13C,1H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya.

Bioreaction network topology and metabolic flux ratio analysis by biosynthetic fractional 13C labeling and two-dimensional NMR spectroscopy.

Biosynthetically directed fractional 13C labeling of the proteinogenic amino acids is achieved by feeding a mixture of uniformly 13C-labeled and unlabeled carbon source compounds into a bioreaction network. Analysis of the resulting labeling pattern enables both a comprehensive characterization of the network topology and the determination of metabolic flux ratios. Attractive features with regard to routine applications are (i) an inherently small demand for 13C-labeled source compounds and (ii) the high sensitivity of two-dimensional [13C,1H]-correlation nuclear magnetic resonance spectroscopy for analysis of 13C-labeling patterns. A user-friendly program, FCAL, is available to allow rapid data analysis. This novel approach, which recently also has been employed in conjunction with metabolic flux balancing to obtain reliable estimates of in vivo fluxes, enables efficient support of metabolic engineering and biotechnology process design.

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.

NMR scalar couplings across Watson-Crick base pair hydrogen bonds in DNA observed by transverse relaxation-optimized spectroscopy.

This paper describes the NMR observation of 15N---15N and 1H---15N scalar couplings across the hydrogen bonds in Watson-Crick base pairs in a DNA duplex, hJNN and hJHN. These couplings represent new parameters of interest for both structural studies of DNA and theoretical investigations into the nature of the hydrogen bonds. Two dimensional [15N,1H]-transverse relaxation-optimized spectroscopy (TROSY) with a 15N-labeled 14-mer DNA duplex was used to measure hJNN, which is in the range 6-7 Hz, and the two-dimensional hJNN-correlation-[15N,1H]-TROSY experiment was used to correlate the chemical shifts of pairs of hydrogen bond-related 15N spins and to observe, for the first time, hJHN scalar couplings, with values in the range 2-3.6 Hz. TROSY-based studies of scalar couplings across hydrogen bonds should be applicable for large molecular sizes, including protein-bound nucleic acids.

NMR with 13C, 15N-doubly-labeled DNA: the Antennapedia homeodomain complex with a 14-mer DNA duplex.

Nearly complete 1H, 13C and 15N NMR assignments have been obtained for a doubly labeled 14-base pair DNA duplex in solution both in the free state and complexed with the uniformly 15N-labeled Antennapedia homeodomain. The DNA was either fully 13C, 15N-labeled or contained uniformly 13C, 15N-labeled nucleotides only at those positions which form the protein-DNA interface in the previously determined NMR solution structure of the Antennapedia homeodomain-DNA complex. The resonance assignments were obtained in three steps: (i) identification of the deoxyribose spin systems via scalar couplings using 2D and 3D HCCH-COSY and soft-relayed HCCH-COSY; (ii) sequential assignment of the nucleotides via 1H-1H NOEs observed in 3D 13C-resolved NOESY; and (iii) assignment of the imino and amino groups via 1H-1H NOEs and 15N-1H correlation spectroscopy. The assignment of the duplex in the 17 kDa protein-DNA complex was greatly facilitated by the fact that 1H signals of the protein were filtered out in 13C-resolved spectroscopy and by the excellent carbon chemical shift dispersion of the DNA duplex. Comparison of corresponding 13C chemical shifts of the free and the protein-bound DNA indicates conformational changes in the DNA upon complex formation.

In pre-sterol carrier protein 2 (SCP2) in solution the leader peptide 1-20 is flexibly disordered, and residues 21-143 adopt the same globular fold as in mature SCP2.

The preform of the rabbit sterol carrier protein 2 (pre-rSCP2) was cloned, the uniformly 15N-labelled protein expressed in Escherichia coli and studied by three-dimensional 15N-resolved nuclear magnetic resonance spectroscopy. In spite of its low solubility in aqueous solution of only approximately 0.3 mM, sequential 15N and 1H backbone resonance assignments were obtained for 105 out of the 143 residues. From comparison of the sequential and medium-range nuclear Overhauser effects (NOEs) in the two proteins, all regular secondary structures previously determined in mature human SCP2 (hSCP2) [Szyperski et al. (1993) FEBS Lett. 335: 18-26] were also identified in pre-rSCP2. Near-identity of the backbone 15N and 1H chemical shifts and 1:1 correspondence of 24 long-range NOEs to backbone amide groups in the two proteins show that the residues 21-143 adopt the same globular fold in pre-rSCP2 and mature hSCP2. The N-terminal 20-residue leader peptide of pre-rSCP2 is flexibly disordered in solution and does not observably affect the conformation of the polypeptide segment 21-143.

Structure comparison of human glioma pathogenesis-related protein GliPR and the plant pathogenesis-related protein P14a indicates a functional link between the human immune system and a plant defense system.

The human glioma pathogenesis-related protein (GliPR) is highly expressed in the brain tumor glioblastoma multiforme and exhibits 35% amino acid sequence identity with the tomato pathogenesis-related (PR) protein P14a, which has an important role for the plant defense system. A molecular model of GliPR was computed with the distance geometry program DIANA on the basis of a P14a-GliPR sequence alignment and a set of 1,200 experimental NMR conformational constraints collected with P14a. The GliPR structure is represented by a group of 20 conformers with small residual DIANA target function values, low AMBER-energies after restrained energy-minimization with the program OPAL, and an average rms deviation relative to the mean of 1.6 A for the backbone heavy atoms. Comparison of the GliPR model with the P14a structure lead to the identification of a common partially solvent-exposed spatial cluster of four amino acid residues, His-69, Glu-88, Glu-110, and His-127 in the GliPR numeration. This cluster is conserved in all known plant PR proteins of class 1, indicating a common putative active site for GliPR and PR-1 proteins and thus a functional link between the human immune system and a plant defense system.