Crystallization and X-ray crystallographic studies of wild-type and mutant tryptophan synthase alpha-subunits from Escherichia coli

The alpha-subunit of Escherichia coli tryptophan synthase (aTS), a component of the tryptophan synthase alpha2beta2 complex, is a monomeric 268-residues protein (Mr = 28,600). alphaTS by itself catalyzes the cleavage of indole-3-glycerol phosphate to glyceraldehyde-3-phosphate and indole, which is converted to tryptophan in tryptophan biosynthesis. Wild-type and P28L/Y173F double mutant alpha-subunits were overexpressed in E. coli and crystallized at 298 K by the hanging-drop vapor-diffusion method. X-ray diffraction data were collected to 2.5 angstroms resolution from the wild-type crystals and to 1.8 angstroms from the crystals of the double mutant, since the latter produced better quality diffraction data. The wild-type crystals belonged to the monoclinic space group C2 (a = 155.64 angstroms, b = 44.54 angstroms, c = 71.53 angstroms and beta = 96.39 degrees) and the P28L/Y173F crystals to the monoclinic space group P21 (a = 71.09 angstroms, b = 52.70, c = 71.52 angstroms, and beta = 91.49 degrees). The asymmetric unit of both structures contained two molecules of aTS. Crystal volume per protein mass (V(m)) and solvent content were 2.15 angstroms3 Da(-1) and 42.95% for the wild-type and 2.34 angstroms3 Da(-1) and 47.52% for the double mutant.     

Correlation of surface properties with conformational stabilities of wild-type and six mutant tryptophan synthase alpha-subunits substituted at the same position

The surface properties of wild-type and six mutant alpha-subunits of tryptophan synthase substituted at the same position, 49, which is buried in the interior, were measured by surface tension, foaming and emulsifying properties to correlate the surface properties with the stabilities. The conformational stabilities of the seven alpha-subunits differed dramatically depending on the characteristics of the substituting residues [Yutani et al. (1987) Proc. Natl. Acad. Sci., 84, 4441-4444]. The mutant proteins substituted by isoleucine and phenylalanine in place of glutamic acid at position 49 were more stable than the other proteins and showed higher surface tension and lower foaming and emulsifying properties than the wild-type and other mutant proteins. Good correlations were observed between these surface properties and values of the Gibbs free energy of unfolding in water, of the proteins. This indicates that the surface properties of the alpha-subunits of tryptophan synthase depend closely on the conformational stabilities.     

Proton nuclear magnetic resonance studies of mast cell histamine

The state of histamine in mast cells was studied by 1H NMR spectroscopy. Spectra were measured for histamine in situ in intact mast cells, for histamine in suspensions of mast cell granule matrices that had been stripped of their membranes, and for histamine in solutions of heparin. The 1H NMR spectrum of intact mast cells is relatively simple, consisting predominantly of resonances for intracellular histamine superimposed on a weaker background of resonances from heparin and proteins of the cells. All of the intracellular histamine contributes to the NMR signals, indicating it must be relatively mobile and not rigidly associated with the negatively charged granule matrix. Spectra for intracellular histamine and for histamine in granule matrices are similar, indicating the latter to be a reasonable model for the in situ situation. The dynamics of binding of histamine by granule matrices and by heparin are considerably different; exchange of histamine between the bulk water and the granule matrices is slow on the 1H NMR time scale, whereas exchange between the free and bound forms in heparin solution is fast. The chemical shifts of resonances for histamine in mast cells are pH dependent, decreasing as the intragranule pH increases without splitting or broadening. The results are interpreted to indicate that histamine in mast cells is relatively labile, with rapid exchange between bound histamine and pools of free histamine in water compartments confined in the granule matrix.     

Proton magnetic resonance spectroscopic studies of proteins containing deuterated tryptophan residues

Summary The deuteration of the tryptophan residues of hen egg white lysozyme, bovine-lactalbumin and bovine-lactoglobulin in d-TFA has been studied by PMR spectroscopy. It is found that short times of exposure to d-TFA allow selective deuteration at the C-2 position with only a small amount of deuteration at the C-5 position, as expected from studies on model peptides described in the previous paper. The proteins studied essentially regained their native structures after the treatment, except for broadening and shifting of the histidine resonances in the case of-lactalbumin. Selective deuteration at the tryptophan C-2 position was readily observed by difference spectroscopy of the denatured protein, but PMR difference spectra of the same proteins in benign solvents did not contain resonances from all of the exchanged protons. Some resonances could not be observed because of line broadening, which causes the resonances to fall below the limit of sensitivity of detection at 100 MHz. Deuteration by brief exposure to d-TFA should be useful for the identification of tryptophan resonances in the PMR spectra of native proteins.The deuteration of all the aromatic protons of tryptophan residues in proteins by immersion in d-TFA for 4 hours at room temperature was studied. This technique is unlikely to be of general use for the simplification of the aromatic region of the PMR spectra of native proteins because of the degradation of tryptophan residues which results from the acid treatment.An invited article.     

Proton magnetic resonance studies of ultraviolet-irradiated apurinic acid

In apurinic acid, a single-stranded polydeoxyribonucleotide easily obtained upon depurination of DNA, the proton resonances arising from thymine and cytosine are readily observable in aqueous solution of 25°C. Two methyl thymine resonances, centered at 1.88 ppm and separated by 0.045 ppm, are observed. We attribute the downfield methyl resonance to thymines with no pyrimidine nearest neighbors and the upfield methyl resonance to thymines having pyrimidine neighbors in the 3′ and/or 5′ positions. Upon ultraviolet irradiation, the upfield methyl and thymine H-6 resonances decrease in amplitude and two methyl resoances appear at 1.63 and 1.52 ppm, corresponding, respectively, to cytosine-thymine and thymine-thymine cyclobutane dimers. Photoreversal eliminates these two minor methyl resonances from the pmr spectrum. We conclude that apurinic acid provides a suitable model system for pmr studies of chemically modified pyrimidine bases in DNA.     

Proton nuclear magnetic resonance studies on the iodide binding by horseradish peroxidase

Binding of an iodide ion to horseradish peroxidase was studied by following the hyperfine-shifted proton nuclear magnetic resonance signals of the enzyme. For the enzyme in an iodide-free solution, the spectra of hyperfine-shifted methyl region were only slightly affected by varying pH. In the presence of iodide (200 mM), however, both chemical shifts and line widths of the heme peripheral 1- and 8-methyl proton signals were markedly affected by the pH change from 7 to 4 and broadened at pH 4. From the change in peak heights of these signals at various concentrations of iodide, the dissociation constant of the iodide to the enzyme was calculated to be about 100 mM at pH 4.0. The peak derived from the proximal histidyl imidazole N epsilon-H proton was not perturbed by the addition of 200 mM iodide at pH 4.0 and 7.1. The rate of oxidation of iodide with hydrogen peroxide catalyzed by the enzyme was increased with decreasing pH, indicating the participation of an ionizable group with the pKa value of 4.0. Optical difference spectrum studies showed that iodide exerts no effect both at pH 4.0 and 7.4 on the binding affinity of resorcinol which is associated with the enzyme in the vicinity of the heme peripheral 8-CH3 group. These results suggest that an iodide ion binds to the enzyme at almost equal distance from the heme peripheral 1- and 8-methyl groups at the distal side of the heme and that the interaction becomes stronger in acidic medium with protonation of the ionizable group with the pKa value of 4.0.     

Proton nuclear magnetic resonance studies of Rhodospirillum rubrum cytochrome.

Rhodospirillum rubrum cytochrome c2 was studied by proton nuclear magnetic resonance at 220 MHz. Assignments were made to the resonances of heme c by double-resonance techniques and by temperature-dependence studies. The aromatic resonances of Trp-62 and Tyr-70 of ferrocytochrome c2 were identified by spin-decoupling experiments. The resonances of the Met-91 methyl group of the ferri- and ferrocytochromes were assigned by saturation-transfer experiments. The assignments are compared to those made for cytochromes c. A pH titration showed that the methionine methyl resonance of ferricytochrome c2 shifted with a pK of 6.25 and disappeared above pH 9. No histidine CH resonances that titrated normally over the neutral pH range were observed in the spectrum of either oxidation state of the protein. The possible origins of the ionizations at pH 6.25 and 9 are discussed.     

Proton and carbon-13 nuclear magnetic resonance studies of rhodopsin-phospholipid interactions

Proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra of rhodopsin-phospholipid membrane vesicles and sonicated disk membranes are presented and discussed. The presence of rhodopsin in egg phosphatidylcholine vesicles results in homogeneous broadening of the methylene and methyl resonances. This effect is enhanced with increasing rhodopsin content and decreased by increasing temperature. The proton NMR data indicate the phospholipid molecules exchange rapidly (less than 10(-3) s) between the bulk membrane lipid and the lipid in the immediate proximity of the rhodopsin. These interactions result in a reduction in either or both the frequency and amplitude of the tilting motion of the acyl chains. The 13C NMR spectra identify the acyl chains and the glycerol backbone as the major sites of protein lipid interaction. In the disk membranes the saturated sn-1 acyl chain is significantly more strongly immobilized than the polyunsaturated sn-2 acyl chain. This suggest a membrane model in which the lipid molecules preferentially solvate the protein with the sn-1 chain, which we term an edge-on orientation. The NMR data on rhodopsin-asolectin membrane vesicles demonstrate that the lipid composition is not altered during reconstitution of the membranes from purified rhodopsin and lipids in detergent.     

Proton nuclear magnetic resonance studies of intact native bovine parathyroid hormone

Native intact bovine PTH was studied by proton nuclear magnetic resonance (NMR) techniques, at pH 3.5 and pH 6.3. The 1H-NMR spectra had good resolution and many multiplet structures were observed. Assignment of the NMR resonances corresponding to specific amino acids was approached using 1H chemical shifts, coupling constants, and pH dependence in the one-dimensional spectra and the 1H-1H connectivities revealed in two-dimensional homonuclear correlated spectroscopy (COSY) experiments. All the aromatic proton resonances were assigned. Two histidine residues had lower pK than the other two. The methyl groups of two residues were moved significantly downfield: using COSY and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) correlations, these were assigned to an alanine residue close to both Trp-23 and Tyr-43, and a valine residue in close spatial proximity to Trp-23. The NOESY spectrum also showed cross-peaks between the residues of the upfield valine-leucine-isoleucine methyl envelope. Many of the H alpha protons moved upfield as the pH was increased. These results indicate that intact native PTH exists in a preferred conformation in solution at pH 6.5. Our studies have provided new information on the three-dimensional spatial proximity of several amino acids along the polypeptide chain. The observed interactions are consistent with the currently accepted model suggesting that the hormone has two separate structural domains associated with the amino- and carboxy-terminal regions of the molecule respectively. The potential implications of this model for the expression of biological activity are discussed.     

Effect of single amino acid substitutions on the protease susceptibility of tryptophan synthase alpha subunit

In order to explore the correlation between protease susceptibility and conformational stability of a protein, the proteolytic degradation by trypsin, subtilisin and pronase P of the wild-type alpha subunit of tryptophan synthase from Escherichia coli and of its two mutant proteins was studied by measuring circular dichroism at 222 nm at various pH values at 37 degrees C. The mutant proteins are substituted by Gln or Met in place of Glu at position 49. The single amino acid substitutions at position 49 significantly affected susceptibility of this protein to the three proteases. Dependence of protease susceptibility of the wild-type and the two mutant proteins on pH was characteristic of each protein and similar for the three proteases. Comparison of the present results with the conformational stabilities of the three proteins previously measured shows that the order of resistance to the proteases among the three proteins coincides with the order of the values of unfolding Gibbs energy change, suggesting that protein degradation depends upon the conformational stability of a protein.     

Proton nuclear magnetic resonance studies of the effects of ligand binding on tryptophan residues of selectively deuterated dihydrofolate reductase from Lactobacillus casei

We have prepared a selectively deuterated dihydrofolate reductase in which all the aromatic protons except the C(2) protons of tryptophan have been replaced by deuterium and have examined the 1H NMR spectra of its complexes with folate, trimethoprim, methotrexate, NADP+, and NADPH. One of the four Trp C(2)-proton resonance signals (signal P at 3.66 ppm from dioxane) has been assigned to Trp-21 by examining the NMR spectrum of a selectively deuterated N-bromosuccinimide-modified dihydrofolate reductase. This signal is not perturbed by NADPH, indicating that the coenzyme is not binding close to the 2 position of Trp-21. This contrasts markedly with the 19F shift (2.7 ppm) observed for the 19F signal of Trp-21 in the NADPH complex with the 6-fluorotryptophan-labeled enzyme. In fact the crystal structure of the enzyme . methotrexate . NADPH shows that the carboxamide group of the reduced nicotinamide ring is near to the 6 position of Trp-21 but remote from its 2 position. The nonadditivity of the 1H chemical-shift contributions for signals tentatively assigned to Trp-5 and -133 indicates that these residues are influenced by ligand-induced conformational changes.     

Proton and iodine-127 nuclear magnetic resonance studies on the binding of iodide by lactoperoxidase

Interaction of an iodide ion with lactoperoxidase was studied by the use of 1H NMR, 127I NMR, and optical difference spectrum techniques. 1H NMR spectra demonstrated that a major broad hyperfine-shifted signal at about 60 ppm, which is ascribed to the heme peripheral methyl protons, was shifted toward high field by adding KI, indicating the binding of iodide to the active site of the enzyme; the dissociation constant was estimated to be 38 mM at pH 6.1. The binding was further detected by 127I NMR, showing no competition with cyanide. Both 1H NMR and 127I NMR revealed that the binding of iodide to the enzyme is facilitated by the protonation of an ionizable group with a pKa value of 6.0-6.8, which is presumably the distal histidyl residue. Optical difference spectra showed that the binding of an aromatic donor molecule to the enzyme is slightly but distinctly affected by adding KI. On the basis of these results, it was suggested that an iodide ion binds to lactoperoxidase outside the heme crevice but at the position close enough to interact with the distal histidyl residue which possibly mediates electron transport in the iodide oxidation reaction.     

Structures of wild-type and P28L/Y173F tryptophan synthase alpha-subunits from Escherichia coli

The alpha-subunit of tryptophan synthase (alphaTS) catalyzes the cleavage of indole-3-glycerol phosphate to glyceraldehyde-3-phosphate and indole, which is used to yield the amino acid tryptophan in tryptophan biosynthesis. Here, we report the first crystal structures of wild-type and double-mutant P28L/Y173F alpha-subunit of tryptophan synthase from Escherichia coli at 2.8 and 1.8A resolution, respectively. The structure of wild-type alphaTS from E. coli was similar to that of the alpha(2)beta(2) complex structure from Salmonella typhimurium. As compared with both structures, the conformational changes are mostly in the interface of alpha- and beta-subunits, and the substrate binding region. Two sulfate ions and two glycerol molecules per asymmetric unit bind with the residues in the active sites of the wild-type structure. Contrarily, double-mutant P28L/Y173F structure is highly closed at the window for the substrate binding by the conformational changes. The P28L substitution induces the exposure of hydrophobic amino acids and decreases the secondary structure that causes the aggregation. The Y173F suppresses to transfer a signal from the alpha-subunit core to the alpha-subunit surface involved in interactions with the beta-subunit and increases structural stability.     

Proton magnetic resonance studies on peptide fragments of troponin-C containing single calcium-binding sites

Proton magnetic resonance spectroscopy has been employed to study the solution conformation of three cleavage fragments of troponin-C, each containing a single Ca(II)-binding site and corresponding to different regions in the primary sequence; viz. CB8 (residues 46–77), CB9 (residues 85–134) and TH2 (residues 121–159). Although all three peptides lack a well-defined tertiary fold in the absence of metal ions, several spectral features indicate the presence of local conformational constraints in each apopeptide. Ca(II) binding led to spectral changes consistent with increased restriction of backbone motility and the adoption of a more compact conformation. Studies using paramagnetic ions as conformational probes support current views concerning the nature of the ligands at the metal binding sites.The nature and kinetics of the structural influence of metal binding suggest that the conformational constraints existing in the CB8 apo-peptide provide an adequate Ca(II)-binding configuration. In contrast, the CB9 and TH2 peptides exhibit spectral changes consistent with an increased local structure in the region of helix E (residues 94–102) in the case of CB9 and helix H (residues 148–159) in the case of TH2. In CB9, conformation changes also appear to be transmitted to a portion of the sequence (residues 87–93) preceding helix E, a putative site of interaction between troponin-C and troponin-I. These data are discussed with reference to the contribution of long-range (interdomain) interactions within troponin-C and the modulation of troponin subunit protein-protein interactions by Ca(II) binding.     

Proton nuclear magnetic resonance studies of histidines in horse carbonic anhydrase I

The 250 MHz 1H-NMR spectrum of horse carbonic anhydrase I (or B) (carbonate hydro-lyase, EC 4.2.1.1) was measured as a function of pH under various conditions. Eight resonances corresponding to histidine C-2 protons and four resonances corresponding to histidine C-4 protons were identified and assigned to individual histidine residues in the enzyme molecule. Substantial similarities between horse and human carbonic anhydrases I were demonstrated. While the human enzyme has three titratable histidine residues in its active site, the horse enzyme has only two, His-67 in the human enzyme being replaced by Gln in the horse enzyme (Jabusch, J.R., Bray, R.P. and Deutsch, H.F. (1980) J. Biol. Chem. 255, 9196-9204). This substitution has small but significant effects on the behaviour of the other active-site histidines. His-64 and His-200. However, His-64 has an anomalously low pKa value also in horse isoenzyme I, as previously observed in human isoenzyme I (Campbell, I.D., Lindskog, S. and White, A.I. (1974) J. Mol. Biol. 90, 469-489).     

Proton nuclear magnetic resonance studies on bulge-containing DNA oligonucleotides from a mutational hot-spot sequence

A series of bulge-containing and normal double-helical synthetic oligodeoxyribonucleotides, of sequence corresponding to a frame-shift mutational hot spot in the lambda C1 gene, are compared by proton magnetic resonance spectroscopy at 500 MHz. The imino proton resonances of d(GATGGGCAG).d(CTGCCCCATC), d(GATGGGCAG).d(CTGCCCCATC), and d(GATGGGCAG).d(CTGACCCATC) are assigned by one-dimensional nuclear Overhauser effect spectroscopy. Nonselective T1 inversion-recovery experiments are used to determine exchangeable proton lifetimes and to compare helix stability and dynamics of the three duplexes. An extra adenosine flanking the internal G.C base pairs has a strongly localized effect on helix stability, but the destabilizing effect of an extra cytidine in a C tract is delocalized over the entire G.C run. These data lead to the conclusion that the position of the bulge migrates along the run in the fast-exchange limit on the NMR time scale. Rapid migration of the bulge defect in homopolymeric sequences may help rationalize both frame-shift mutagenesis and translational frame shifting. We estimate that the unfavorable free energy of a localized bulge defect is 2.9-3.2 kcal/mol, in good agreement with earlier estimates for RNA helices.