Patricia F. Cottam

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.

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.

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.

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.

Multidimensional1H and15N NMR investigation of glutamine-binding protein ofEscherichia coli

SummarySpecific and uniform15N labelings along with site-directed mutagenesis of glutamine-binding protein have been utilized to obtain assignments of the His156, Trp32 and Trp.220 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 ofl-glutamine across the cytoplasmic membrane ofEscherichia coli, but also to serve as the starting points in the sequence-specific backbone assignment. The assignment of H2 of His156 refines the earlier, model where this particular proton formas an intermolecular hydrogen bond to the δ-carbonyl ofl-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 combining15N isotopic labeling and multinuclear, multidimensional NMR techniques for a structural investigation of a protein with a molecular weight of 25 000.

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

Abstract1H, 13C, and 15N NMR assignments of the backbone atoms and β-carbons have been madefor liganded glutamine-binding protein (GlnBP) of Escherichia coli, a monomeric protein with226 amino acid residues and a molecular weight of 24,935 Da. GlnBP is a periplasmicbinding protein which plays an essential role in the active transport of L-glutamine throughthe cytoplasmic membrane. The assignments have been obtained from three-dimensionaltriple-resonance NMR experiments on a 13C,15N uniformly labeled sample as well asspecifically 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 themain sources used to make the resonance assignments. Other 3D experiments, such asHNCACB, COCAH, HCACO, HCACON, and HOHAHA-HMQC, have been used to confirmthe resonance assignments and to extend connections where resonance peaks are missing insome of the experiments mentioned above. We have assigned more than 95% of thepolypeptide backbone resonances of GlnBP. The result of the standard manual assignment isin agreement with that predicted by an automated probabilistic method developed in ourlaboratory. A solution secondary structure of the GlnBP–Gln complex has beenproposed based on chemical shift deviations from random coil values. Eight α-helices and10 β-strands are derived using the Chemical Shift Index method.

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.