1H, 13C, and 15N NMR backbone assignments and chemical-shift-derived secondary structure of glutamine-binding protein of Escherichia coli.

1H, 13C, and 15N NMR assignments of the backbone atoms and beta-carbons have been made for liganded glutamine-binding protein (GlnBP) of Escherichia coli, a monomeric protein with 226 amino acid residues and a molecular weight of 24935 Da. GlnBP is a periplasmic binding protein which plays an essential role in the active transport of L-glutamine through the cytoplasmic membrane. The assignments have been obtained from three-dimensional triple-resonance NMR experiments on a 13C, 15N uniformly labeled sample as well as specifically labeled samples. Results from the 3D triple-resonance experiments, HNCO, HN(CO)CA, HN(COCA)HA, HNCA, HN(CA)HA, HN(CA)CO, and CBCA(CO)NH, are the main sources used to make the resonance assignments. Other 3D experiments, such as HNCACB, COCAH, HCACO, HCACON, and HOHAHA-HMQC, have been used to confirm the resonance assignments and to extend connections where resonance peaks are missing in some of the experiments mentioned above. We have assigned more than 95% of the polypeptide backbone resonances of GlnBP. The result of the standard manual assignment is in agreement with that predicted by an automated probailistic method developed in our laboratory. A solution secondary structure of the GlnBP-Gln complex has been proposed based on chemical shift deviations from random coil values. Eight alpha-helices and 10 beta-strands are derived using the Chemical Shift Index method.

Production of human normal adult and fetal hemoglobins in Escherichia coli.

A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha-globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma-globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.

Restoring allosterism with compensatory mutations in hemoglobin.

Abnormal human hemoglobins (HBs) with amino acid substitutions in the alpha 1 beta 2 interface have very high oxygen affinity and greatly reduced cooperativity in O2 binding compared to normal human Hb. In such abnormal Hbs with mutations at position beta 99, the intersubunit hydrogen bonds between Asp-beta 99 and Tyr-alpha 42 and between Asp-beta 99 and Asn-alpha 97 are broken, thus destabilizing the deoxyquaternary structure of these Hbs. A molecular dynamics method has been used to design compensatory amino acid substitutions in these Hbs that can restore their allosteric properties. We have designed a compensatory mutation in a naturally occurring mutant Hb, Hb Kempsey (Asp-beta 99-->Asn), and have produced it using our Escherichia coli expression plasmid pHE2. We have determined the O2 binding properties of this recombinant double mutant Hb, Hb(Asp-beta 99-->Asn and Tyr-alpha 42-->Asp) and have used 1H NMR spectroscopy to investigate the tertiary structures around the heme groups and the quaternary structure in the alpha 1 beta 2 subunit interface. Our results clearly show that the Tyr-alpha 42-->Asp replacement can substantially compensate for the functional defect of Hb Kempsey caused by the Asp-beta 99-->Asn substitution. The structural and functional information derived from this recombinant Hb provides insights into the structural basis of allosterism and the design of compensatory amino acid substitutions to restore the functional properties of other abnormal HBs associated with hemoglobinopathies.

Crystals of glutamine-binding protein in various conformational states.

Crystals of glutamine-binding protein (GlnBP) in various conformational states have been obtained. Crystals of the ligand-free "open" state (denoted form B) have unit cell dimensions a = 86.3 A, b = 86.3 A, c = 81.5 A, alpha = beta = gamma = 90 degrees and diffract to about 2.3 A resolution. An analysis of the intensity data using an Requiv plot indicates that the crystal system is orthorhombic, space group P2(1)2(1)2(1). Crystals of the ligand-bound "open" state (form B*) are obtained by soaking form B crystals with glutamine (Gln) and diffract to about 1.9 A. Crystals of the GlnBP-Gln complex in a ligand-bound "closed" state (form C) belong to space group P2(1)2(1)2(1) with a = 62.0 A, b = 65.7 A and c = 121.8 A and diffract to about 2.3 A. Crystals of a selenomethionyl GlnBP (form B') are isomorphous to form B crystals and diffract to about 2.1 A resolution.

An investigation of the ligand-binding site of the glutamine-binding protein of Escherichia coli using rotational-echo double-resonance NMR.

Glutamine-binding protein (GlnBP) is an essential component of the glutamine transport system in Escherichia coli. Rotational-echo double-resonance (REDOR) solid-state nuclear magnetic resonance (NMR) has been used to determine internuclear distances in the complex of GlnBP and its ligand, L-glutamine. REDOR, combined with strategically placed isotopic labels, is effective in obtaining model-independent internuclear distances and thus detailed structural information on the ligand-binding site of GlnBP. The existence of a single histidine residue (His156) in the binding site has provided an excellent probe for distance measurements between protein and ligand. REDOR distances up to 6.3 A have been observed between 13C labels in L-glutamine and 15N labels in His156. These results have unambiguously determined the ligand orientation with respect to the imidazole ring of His156, which is an important first step in refining the ligand-binding-site model of GlnBP in general. The measured distances were also used as constraints in restrained molecular dynamics calculations of the complex using the unliganded crystal structure of GlnBP as the starting point. The simulations clearly show consistency between calculated distances and those measured by REDOR.

Multidimensional 1H and 15N NMR investigation of glutamine-binding protein of Escherichia coli.

Specific and uniform 15N labelings along with site-directed mutagenesis of glutamine-binding protein have been utilized to obtain assignments of the His156, Trp32 and Trp220 residues. These assignments have been made not only to further study the importance of these 3 amino acid residues in protein-ligand and protein-protein interactions associated with the active transport of L-glutamine across the cytoplasmic membrane of Escherichia coli, but also to serve as the starting points in the sequence-specific backbone assignment. The assignment of H epsilon 2 of His156 refines the earlier model where this particular proton forms an intermolecular hydrogen bond to the delta-carbonyl of L-glutamine, while assignments of both Trp32 and Trp220 show the variation in local structures which ensure the specificity in ligand binding and protein-protein interaction. Using 3D NOESY-HMQC NMR, amide connectivities can be traced along 8-9 amino acid residues at a time. This paper illustrates the usefulness of combining 15N isotopic labeling and multinuclear, multidimensional NMR techniques for a structural investigation of a protein with a molecular weight of 25,000.

Resolution of fluorescence intensity decays of the two tryptophan residues in glutamine-binding protein from Escherichia coli using single tryptophan mutants.

Time correlated single photon counting measurements of tryptophan (Trp) fluorescence intensity decay and other spectroscopic studies were performed on glutamine-binding protein (GlnBP) from Escherichia coli. Using site-specifically mutated forms of the protein in which tyrosine (Tyr) and phenylalanine (Phe) substitute for the Trp residues at positions 32 and 220, we have examined whether wild-type (Wtyp) intensity decay components may be assigned to specific Trp residues. Results indicate that: (a) two exponential intensity decay components are recovered from the Wtyp protein (6.16 ns, 0.46 ns); (b) the long decay component arises from Trp-220 and comprises greater than 90% of the total fluorescence emission; (c) the short component arises from Trp-32 and is highly quenched; (d) all four single-Trp mutants exhibit multiexponential intensity decays, yet equimolar mixtures of two single-Trp mutants yield only two decay components which are virtually indistinguishable from the Wtyp protein; (e) the recovery of additional components in protein mixtures is obscured by statistical noise inherent in the technique of photon counting; (f) various spectroscopic measurements suggest that Trp-Trp interactions occur in the Wtyp protein, but the Wtyp intensity decay may be closely approximated by a linear combination of intensity decays from single-Trp mutants; and (g) inferences derived independently from fluorescence and NMR spectroscopy which pertain to the presence of Trp-Trp interactions and the relative solvent exposure of the two Trp residues are in agreement.

Proton nuclear magnetic resonance studies on glutamine-binding protein from Escherichia coli. Formation of intermolecular and intramolecular hydrogen bonds upon ligand binding.

Proton nuclear magnetic resonance studies have revealed several structural and dynamic properties of the glutamine-binding protein of Escherichia coli. When this protein binds L-glutamine, six low-field, exchangeable proton resonances appear in the region from +5.5 to +10 parts per million downfield from water (or +10.2 to +14.7 parts per million downfield from the methyl proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate). This suggests that the binding of L-glutamine induces specific conformational changes in the protein molecule, involving the formation of intermolecular and intramolecular hydrogen bonds between the glutamine-binding protein and L-glutamine, and within the protein molecule. The oxygen atom of the gamma-carbonyl group of L-glutamine is likely to be involved in the formation of an intermolecular hydrogen bond between the ligand and the binding protein. We have shown that at least one phenylalanine and one methyl-containing residue are spatially close to this intermolecular hydrogen-bonded proton. The intermolecular and intramolecular hydrogen-bonded protons of the ligand-protein complex undergo solvent exchange. The local conformations around these intermolecular and intramolecular hydrogen bonds are quite stable when subjected to pH and temperature variations. From these results, the utility of proton nuclear magnetic resonance spectroscopy for investigating such binding proteins has been shown, and a picture of the ligand-binding process can be drawn.

Molecular genetic, biochemical and nuclear magnetic resonance studies on the role of the tryptophan residues of glutamine-binding protein from Escherichia coli.

The results of molecular genetic, biochemical and nuclear magnetic resonance studies on glutamine-binding protein of Escherichia coli suggest that the only two tryptophan residues, at positions 32 and 220, in the protein molecule are likely to be involved in (or sensitive to) interactions with the membrane-bound protein components of the glutamine transport system. It has been found that both tryptophan residues have limited motional freedom, are located away from the surface of the protein molecule and are not close to the ligand-binding site. Their presence, however, is required for the optimal transport of L-glutamine across the cytoplasmic membrane, though not essential for the ligand-binding process. The relevance of these results to the structure and function of the glutamine-binding protein in the glutamine transport system is discussed.

Formation of intermolecular and intramolecular hydrogen bonds in histidine-binding protein J of Salmonella typhimurium upon binding L-histidine. A proton nuclear magnetic resonance study.

Histidine-binding protein J of Salmonella typhimurium has been chosen as a model system for a proton nuclear magnetic resonance spectroscopic investigation of binding protein-ligand interaction. This interaction is involved in the recognition step of the osmotic shock-sensitive active transport systems. When J protein binds L-histidine, four new, low-field, exchangeable proton resonances appear in the region +7 to +12 parts per million downfield from the water proton resonance (or +11.7 to +16.7 parts per million downfield from the methyl proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate). Due to their chemical shift range and other properties, they indicate the formation of both intra- and intermolecular hydrogen bonds. Experiments with 15N-labeled compounds confirm this conclusion. The specificity of the hydrogen-bond formation is demonstrated by observing the effects of substrate analogs, temperature, pH, and mutations on the exchangeable proton resonances. Proton-proton nuclear Overhauser effect measurements suggest that two of these exchangeable proton resonances (at +7.2 and +10.6 parts per million from H2O) are most likely from intramolecular hydrogen-bonded protons, while the other two (at +7.1 and +9.5 parts per million from H2O) are intermolecular hydrogen bonds. Our finding of L-histidine-induced hydrogen-bond formation in histidine-binding protein J in the solution state is an excellent demonstration of the production of specific conformational changes in a periplasmic binding protein upon binding of ligand.

Preliminary crystallographic analysis of glutamine-binding protein from Escherichia coli.

Glutamine-binding protein from Escherichia coli, an essential component in the active transport of L-glutamine across the cytoplasmic membrane, has been crystallized by vapor diffusion in the presence of ammonium sulfate. The crystals exhibit pseudo-tetragonal symmetry with cell constants a = 77.5 A, b = 78.5 A and c = 90.2 A. Analysis of the diffraction data indicates that the space group is P2(1)2(1)2(1). There are two molecules per asymmetric unit and the solvent content is estimated to be 53%.

Biochemical and morphological properties of membranes of unsaturated fatty acid auxotrophs of Salmonella typhimurium: effects of fluorinated myristic acids.

In order to investigate the utility of the fluorine-19 nucleus as a spectroscopic probe, a fluorinated analog of myristic acid has been incorporated into the membrane lipids of an unsaturated fatty acid auxotroph of Salmonella typhimurium. It is capable of supporting limited growth at temperatures above 37 degrees C. Freeze-fracture electron microscopic examinations of the membrane ultrastructure show a temperature and fatty acid supplement-dependent segregation of intramembranous protein particles into distinct patches in the auxotrophic membrane leaving intramembranous protein-denuded areas. The occurrence of these patches seems to be related to the phase separation of membrane lipids. Corresponding changes in the transport and accumulation of methyl thio-beta-D-galactopyranoside and tetracycline are observed. However, transport of histidine does not appear to be dependent on the physical state of the membrane lipids. The auxotroph shows differences in growth and morphological characteristics from those of the wild type. Functions of both inner and outer membranes are shown to be affected as a response to the fatty acid chain composition of the lipids.

Fluorine-19 nuclear magnetic resonance study of 5-fluorotryptophan-labeled histidine-binding protein J of Salmonella typhimurium.

Fluorine-19 nuclear magnetic resonance has been used to investigate the histidine-binding protein J from Salmonella typhimurium. The protein has been labeled with fluorine-19 by growing the bacterial cells of a tryptophan auxotroph in the presence of 5-fluorotryptophan. Incorporation of up to 70% was achieved. The binding of L-histidine to the 19F-labeled protein is not affected by the isotopic labeling. The protein contains one tryptophan residue, giving rise to a single 19F resonance. Upon binding L-histidine to 19F-labeled histidine-binding protein J, the observed 19F resonance is shifted downfield by about 0.6 parts per million, indicating a conformational change of the protein molecule and a more hydrophobic environment for the 19F nucleus. Additional fluorescence experiments confirm that the tryptophan residue is located inside the hydrophobic core of the protein. 19F spin-lattice relaxation times of the 19F-labeled protein as a function of temperature show no difference between the free protein and the protein-histidine complex. However, the linewidth for the free protein is much larger than that of the protein-substrate complex. This can be explained by slow fluctuations between different conformations of the free protein molecule having slightly different 19F chemical shifts. Both with and without the substrate, the tryptophan residue is immobile inside the protein molecule as shown by the total disappearance of the 19F signal upon broadband irradiation at the 1H frequency. Also, the 19F spin-lattice relaxation times indicate that the protein is a rather rigid structure, in which rapid motions of the tryptophan residue on the time scale of 10(-8) second are not prominent.

A high-resolution 1H-NMR investigation of the histidine-binding protein J of Salmonella typhimurium. Substrate-induced conformational changes.

High-resolution 1H-NMR spectroscopy at 600 MHz has been used to investigate the conformational transitions of the histidine-binding protein J of Salmonella typhimurium in solution as a function of pH and of L-histidine concentration. The dissociation constant for the binding of L-histidine to histidine-binding protein J increases from 6.0 X 10(-8) to 5.1 X 10(-7) M in going from pH 5.57 to 8.00. The conformation of this protein as observed by 1H-NMR also changes over this range of pH. However, when L-histidine is bound, the changes in conformation with pH are much smaller. Also, the pK for the single histidyl residue in histidine-binding protein J changes from 6.75 in the absence of L-histidine to 6.52 when L-histidine is bound. Earlier work in this laboratory resulted in the identification of several proton resonances believed to be at or near the L-histidine-binding site. Two of these resonances have been assigned to a tyrosine and the single histidyl residue in the histidine-binding protein J molecule.

A biochemical study of the reconstitution of D-lactate dehydrogenase-deficient membrane vesicles using fluorine-labeled components.

Fluorine-19 labeled compounds have been incorporated into lipids and proteins of Escherichia coli. 19F-Labeled membrane vesicles, prepared by growing a fatty acid auxotroph of a D-lactate dehydrogenase-deficient strain on 8,8-difluoromyristic acid, can be reconstituted for oxidase and transport activities by binding exogenous D-lactate dehydrogenase. 19F-Labeled D-lactate dehydrogenases prepared by addition of fluorotryptophans to a tryptophan-requiring strain are able to reconstitute D-lactate dehydrogenase-deficient membrane vesicles. Thus, lipid and protein can be labeled independently and used to investigate protein-lipid interactions in membranes.

A biophysical study of protein-lipid interactions in membranes of Escherichia coli. Fluoromyristic acid as a probe.

Fluorine-19 nuclear magentic resonance spectroscopy and transport assays have been used to investigate and compare the membrane properties of unsaturated fatty acid auxotrophs of two strains of Escherichia coli, K1060B5 and ML 308-225-UFA-8. A fluorinated analog of myristic acid, 8, 8-difluoromyristic acid, can be incorporated into the membrane phospholipids by substitution for oleate in the growth medium. Growth for one generation on 8, 8-difluoromyristate results in a 20% content of fluorinated fatty acid in the membranes, changes in the protein to lipid ratio, and altered transport of methyl beta-D-thiogalactopyranoside. The differences in membrane composition and transport behavior seen in oleate supplemented E. coli K1060B5 relative to ML 308-225-UFA-8 are enhanced by the incorporation of 8, 8-difluoromyristate. The phase transition behavior becomes distinctly different and some differences in lipid organization persist above the transition temperature. Concomitantly, the rate and extent of concentration of methyl beta-D-thiogalactopyranoside are reduced two-fold more in E. coli K1060B5 compared to ML 308-225-UFA-8. Such behavior suggests that these fluorinated fatty acid supplemented strains of E. coli are useful to study subtle differences in protein-lipid interactions and their effects on the function of membrane-bound enzymes.

A proton nuclear magnetic resonance investigation of histidine-binding protein J of Salmonella typhimurium: a model for transport of L-histidine across cytoplasmic membrane.

Genetic evidence suggests that the high-affinity L-histidine transport in Salmonella typhimurium requires the participation of a periplasmic binding protein (histidine-binding protein J) and two other proteins (P and Q proteins). The histidine-binding protein J binds L-histidine as the first step in the high-affinity active transport of this amino acid across the cytoplasmic membrane. High-resolution proton nuclear magnetic resonance spectroscopy at 600 MHz is used to investigate the conformations of this protein in the absence and presence of substrate. Previous nuclear magnetic resonance results reported by this laboratory have shown that there are extensive spectral changes in this protein upon the addition of L-histidine. When resonances from individual amino acid residues of a protein can be resolved in the proton nuclear magnetic resonance spectrum, a great deal of detailed information about substrate-induced structural changes can be obtained. In order to gain a deeper insight into the nature of these structural changes, deuterated phenylalanine or tyrosine has been incorporated into the bacteria. Proton nuclear magnetic resonance spectra of selectively deuterated histidine-binding protein J were obtained and compared to the normal protein. Several of the proton resonances have been assigned to the various aromatic amino acid residues of this protein. A model for the high-affinity transport of L-histidine across the cytoplasmic membrane of S typhimurium is proposed. This model, which is a version of the pore model, assumes that both P and Q proteins are membrane-bound and that the interface between these two proteins forms the channel for the passage of substrate. The histidine-binding protein J serves as the "key" for the opening of the channel for the passage of L-histidine. In the absence of substrate, this channel or gate is closed owing to a lack of appropriate interactions among these three proteins. The channel can be opened upon receiving a specific signal from the "key"; namely, the substrate-induced conformational changes in the histidine-binding protein J molecule. This model is consistent with available experimental evidence for the high-affinity transport of L-histidine across the cytoplasmic membrane of S typhimurium.

Fluorine-19 nuclear magnetic resonance studies of Escherichia coli membranes.

Several fluorinated fatty acids of the general structure CH3(CH2)13--mCF2(CH2)m--2COOH are incorporated biosynthetically as unsaturated fatty acid analogues into the phospholipids of Escherichia coli. Under optimum conditions an unsaturated fatty acid autotroph, K1060B5, can be grown so that 50% of the total phospholipid fatty acids are 8,8-difluoromyristate. Conditions are found for which more than 20% of the fatty acids are fluorinated before a decrease in growth rate is observed. We have used 19F nuclear magnetic resonance to examine membranes isolated from E. coli grown under the latter conditions. A comparison is made with spectra of aqueous dispersions of extracted E. coli phospholipids and model multilayer phospholipid membranes. An explanation of the 19F resonance line shape in these membrane systems and the relationship to a molecular order parameter is given. It is apparent that 19F nuclear magnetic resonance is more sensitive to the degree of ordering or fluidity of phospholipids than spin labels or fluorescent probes. For instance, a dramatic effect of membrane protein on lipid fluidity can be seen. Finally, this method can be used to measure the proportion of frozen and fluid lipid in biological membranes at temperatures within the span of the gel-to-lipid phase transition.