Solution conformation of the synthetic bovine proenkephalin-A209-237 by 1H NMR spectroscopy

Proenkephalin-A has been described to generate enkephalins, opoid peptides, and several derived peptides, which display various biological effects, including antinociception and immunological enhancement. Recently, we have isolated from bovine chromaffin granules a new antibacterial peptide, named enkelytin, which corresponds to the bisphosphorylated form of PEAP209-237 (Goumon, Y., Strub, J. M., Moniatte, M., Nullans, G., Poteur, L., Hubert, P., Van Dorsselaer, A., Aunis, D., and Metz-Boutigue, M. H. (1996) Eur. J. Biochem. 235, 516-525). In this paper, the three-dimensional solution structure of synthetic PEAP209-237 was investigated by NMR. These studies indicate that this peptide, which is unstructured in water, folds into an alpha-helical structure in trifluoroethanol/water (1/1). NMR data revealed two possible three-dimensional models of PEAP209-237. In both models, the proline residue Pro-227 induces a 90 degrees hinge between two alpha-helical segments (Ser-215 to Ser-221 and Glu-228 to Arg-232) leading to an overall L-shaped structure for the molecule. The negative charge of PEAP209-237 and the low amphipathy of the two alpha-helical segments imply new mechanisms to explain the antibacterial activity of enkelytin.     

A protein folding intermediate of ribonuclease T1 characterized at high resolution by 1D and 2D real-time NMR spectroscopy

The rate-limiting step during the refolding of S54G/P55N ribonuclease T1 is determined by the slow trans-->cis prolyl isomerisation of Pro39. We investigated the refolding of this variant by one-dimensional (1D) and two-dimensional (2D) real-time NMR spectroscopy, initiated by a tenfold dilution from 6 M guanidine hydrochloride at 10 degreesC. Two intermediates could be resolved with the 1D approach. The minor intermediate, which is only present early during refolding, is largely unfolded. The major intermediate, with an incorrect trans Pro39 peptide bond, is highly structured with 33 amide protons showing native chemical shifts and native NOE patterns. They could be assigned in a real-time 2D-NOESY (nuclear Overhauser enhancement spectroscopy) by using a new assignment strategy to generate positive and negative signal intensities for native and non-native NOE cross-peaks, respectively. Surprisingly, amide protons with non-native environments are located not only close to Tyr38-Pro39, but are spread throughout the entire protein, including the C-terminal part of the alpha-helix, beta-strands 3 and 4 and several loop regions. Native secondary and tertiary structure was found for the major intermediate in the N-terminal beta-strands 1 and 2 and the C terminus (connected by the disulfide bonds), the N-terminal part of the alpha-helix, and the loops between beta-strands 4/5 and 5/6. Implications of these native and non-native structure elements of the intermediate for the refolding of S54G/P55N ribonuclease T1 and for cis/trans isomerizations are discussed.     

1H NMR spectroscopy studies of Huntington's disease: correlations with CAG repeat numbers

Huntington's disease (HD) is the result of an expanded (CAG) repeat in a gene on chromosome 4. A consequence of the gene defect may be progressive impairment of energy metabolism. We previously showed increased occipital cortex lactate in HD using localized 1H spectroscopy. We have now extended these studies to show an almost threefold elevation in occipital cortex lactate in 31 HD patients as compared with 17 normal control subjects (p < 10(-11)). The spectra in three presymptomatic gene-positive patients were identical to normal control subjects in cortical regions, but three in eight showed elevated lactate in the striatum. Similar to recently reported increases in task-related activation of the striatum in the dominant hemisphere, we found that striatal lactate levels in HD patients were markedly asymmetric (higher on the left side). Markers of neuronal degeneration, decreased N-acetylaspartate (NAA)/creatine and increased choline/creatine levels, were symmetric. Both decreased NAA and increased lactate in the striatum significantly correlated with duration of symptoms. When divided by his or her age, an individual's striatal NAA loss and lactate increase were found to directly correlate with the subject's CAG repeat number, with correlation coefficients of 0.8 and 0.7, respectively. Similar correlations were noted between postmortem cell loss and age versus CAG repeat length. Together, these data provide further evidence for an interaction between neuronal activation and a defect in energy metabolism in HD that may extend to presymptomatic subjects.     

Remodeling of HDL by phospholipid transfer protein: demonstration of particle fusion by 1H NMR spectroscopy

There is evidence that phospholipid transfer protein (PLTP) can increase reverse cholesterol transport by inducing favorable subclass distribution in the high density lipoprotein (HDL) fraction. This includes generation of initial cholesterol acceptor particles, pre beta-HDL, and of enlarged particles that are rapidly cleared from the circulation. However, partly because of methodological difficulties, the mechanisms behind the PLTP-mediated interconversion of HDL particles are not fully understood. In this communication, we describe the use of a novel methodology, based on 1H NMR spectroscopy, to study the PLTP-induced size changes in the HDL particles. In accordance with native gradient gel electrophoresis, the 1H NMR data revealed a gradual production of enlarged HDL particles in the HDL3+ PLTP mixtures. In addition, according to a physical model for lipoprotein particles, relating the frequency shifts observable with NMR to the size of the lipoprotein particles, the NMR data demonstrated that PLTP-mediated HDL remodeling involves fusion of the HDL particles.     

Neuronal laterality in Parkinson''s disease with unilateral symptom by in vivo 1H magnetic resonance spectroscopy

PURPOSE: The aim of the present study was to investigate carcinoembryonic antigen (CEA), CA19.9, and CA72.4 in the serum and gastric juice of patients with gastric cancer. METHODS: Serum and gastric juice tumor markers CEA, CA19.9, and CA72.4 were measured in 59 patients who had gastric adenocarcinomas and were undergoing curative gastrectomy. The same markers were measured in 47 patients with benign gastric disorders and in 40 healthy subjects. The correlation between the serum and gastric juice levels of tumor markers and several clinicopathological factors were evaluated by univariate analysis. The significance of the tumor markers as prognostic factors was assessed both by univariate and multivariate analysis. RESULTS: The positivity rates of serum CEA, CA19.9, and CA72.4 were 57.6%, 38.9%, and 18.6% respectively. The positivity rates of gastric juice CEA, CA19.9, and CA72.4 were 62.7%, 30.5%, and 23.7% respectively. The combination of serum and gastric juice markers gave a positivity of 81.3%. There was no correlation between serum and gastric juice level of each tumor marker. Positivity of gastric juice markers did not correlate with prognosis. A significant difference in prognosis was observed between patients positive and negative for serum CEA and CA19.9. Multivariate analysis also revealed that serum CEA and CA19.9 levels were independent prognostic factors. CONCLUSIONS: Levels of both serum and gastric juice tumor markers continue to have only limited diagnostic usefulness in gastric cancer patients. CEA and CA19.9 in the preoperative sera are good prognostic factors, whereas the presence of tumor markers in the gastric juice does not play any prognostic role.     

1H MAS and 1H[23Na] double resonance NMR studies on the modification of surface hydroxyl groups of gamma-alumina by sodium

The modification of surface hydroxyl groups with sodium in a series of Na2CO3-gamma-Al2O3 catalysts was investigated as a function of both the Na2CO3 loading and the calcination temperature by means of 1H magic angle spinning (MAS) and 1H(23Na) spin-echo double resonance NMR techniques. The 1H NMR experiments revealed that sodium ions are homogeneously distributed over the alumina surface and closely coordinated with the surface hydroxyl groups. In the catalysts calcined at 250 degrees C, the acidic hydroxyl groups (with a chemical shift of 2.0 ppm) are preferentially associated with sodium ions at low Na2CO3 coverages (5 and 10%), while both the acidic and the basic (0 ppm) hydroxyl groups are accessible for sodium ions at high coverages (15 and 20%). The coordination causes a low-field shift of about 2 ppm in the 1H MAS spectra, and a broad signal at 4.5 ppm appears. It is interesting that the 4.5 ppm signal is completely suppressed in the 1H(23Na) MAS experiments, providing direct evidence that a strong interaction exists between adsorbed sodium ions and the surface hydroxyl groups. Increasing the calcination temperature to 450 degrees C results in preferential removal of the acidic hydroxyl groups, and only the most basic hydroxyl groups remain when the calcination temperature is raised to 600 degrees C. This is attributed to the formation of the coordinated species. [formula: see text] which enhances the acidity of the surface hydroxyl groups and prompts their dehydroxylation, especially at high calcination temperature. Correlation of the 1H MAS NMR results and catalytic activity measurements indicates that the basic hydroxyl groups are essential for the carbonyl sulfide hydrolysis reaction.     

A hairpin-loop conformation in tandem repeat sequence of the ice nucleation protein revealed by NMR spectroscopy

We report two cases of acromegaly due to pituitary adenoma without any other endocrinopathy in a family. The patients had high plasma GH and were improved by transsphenoidal adenomectomy. Acromegaly is usually a clinical syndrome of sporadic nonfamilial occurrence. The familial occurrence of acromegaly not associated with multiple endocrine neoplasia is very rare. Our patients are unlikely to be associated with the multiple endocrine neoplasia type 1 syndrome. Here we describe two patients with acromegaly, a father and his daughter, and review familial cases reported.     

Clinical correlation of neuropsychological tests with 1H magnetic resonance spectroscopy in hepatic encephalopathy

The mortality from coronary and cerebrovascular diseases is higher in Finnmark County than in other Norwegian counties. In a population-based cohort study, we compared the incidence of myocardial infarction, stroke, and diabetes mellitus in different ethnic groups in Finnmark. A total of 10,622 subjects of Norse, Sami, and Finnish origin were followed for 14 years. During approximately 150,000 person-years, we identified 509 and 84 cases of myocardial infarction, 107 and 75 cases of stroke, and 96 and 73 cases of clinical diabetes mellitus among men and women, respectively. A total of 533 men and 199 women died. Norse subjects born outside of Finnmark had the most favorable risk factor levels and, in general, the lowest incidence of disease. Men of Finnish origin had a higher incidence rate of all endpoints than other men, and Finnish women had a higher incidence rate of myocardial infarction than other women. Sami women were more obese but did not have a higher diabetes mellitus incidence than other women. After adjustment for major cardiovascular risk factors and height, most ethnic differences were attenuated.     

Dexamethasone and dexamethasone phosphate detected by 1H and 19F NMR spectroscopy in the aqueous humour

To apply nuclear magnetic resonance (NMR) spectroscopy to study the penetration of dexamethasone phosphate into the aqueous humour from rabbit following topical administration. After topical administration of 0.1%, 1.0% and 10% dexamethasone phosphate solutions, respectively, samples of aqueous humour were aspirated, freeze-dried, redissolved in deuterium oxide and analyzed by high resolution 1H and 19F NMR spectroscopy. In order to study the lipophilic and hydrophilic metabolites of the drug, samples obtained after application of 1% dexamethasone phosphate were extracted with methanol/chloroform, and then extracted with perchloric acid. In all samples obtained from eyes denuded of the corneal epithelium prior to administration of dexamethasone, signals corresponding to the chemical shifts of the drug were identified in 19F NMR spectra. In the experiments performed with 1% dexamethasone phosphate, both dexamethasone and dexamethasone phosphate were detected in the aqueous humour. Using 10% dexamethasone phosphate solutions, signals from the drug were detected in 1H NMR spectra simultaneously with signals from about twenty other substances present in the aqueous humour. NMR spectroscopy appears to be a valuable method for studying dexamethasone metabolism and penetration into ocular tissues. It provides simultaneous detection of both the drug metabolites and other substances in the sample and might offer a complementary approach to other analytical methods.     

Binding of nucleotides by the mitochondrial ADP/ATP carrier as studied by 1H nuclear magnetic resonance spectroscopy

Thromboxane A2 (TXA2) is a potent inducer of vasoconstriction and platelet aggregation. Large scale expression of TXA2 synthase (TXAS) is very useful for studies of the reaction mechanism, structural/functional relationships, and drug interactions. We report here a heterologous system for overexpression of human TXAS. The TXAS cDNA was modified by replacing the sequence encoding the first 28 amino acid residues with a CYP17 amino-terminal sequence and by adding a polyhistidine tag sequence prior to the stop codon; the cDNA was inserted into the pCW vector and co-expressed with chaperonins groES and groEL in Escherichia coli. The resulting recombinant protein was purified to electrophoretic homogeneity by affinity, ion exchange, and hydrophobic chromatography. UV-visible absorbance (UV-Vis), magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) spectra indicate that TXAS has a typical low spin cytochrome P450 heme with an oxygen-based distal ligand. The UV-Vis and EPR spectra of recombinant TXAS were essentially identical to those of TXAS isolated from human platelets, except that a more homogenous EPR spectrum was observed for the recombinant TXAS. The recombinant protein had a heme:protein molar ratio of 0.7:1 and a specific activity of 12 micromol of TXA2/min/mg of protein at 23 degreesC. Furthermore, it catalyzed formation of TXA2, 12-hydroxy-5,8,10-heptadecatrienoic acid, and malondialdehyde in a molar ratio of 0.94:1.0:0.93. Spectral binding titrations showed that bulky heme ligands such as clotrimazole bound strongly to TXAS (Kd approximately 0.5 microM), indicating ample space at the distal face of the heme iron. Analysis of MCD and EPR spectra showed that TXAS was a typical low spin hemoprotein with a proximal thiolate ligand and had a very hydrophobic distal ligand binding domain.     

Conformational study of a collagen peptide by 1H NMR spectroscopy: observation of the 14N-1H spin-spin coupling of the Arg guanidinium moiety in the triple-helix structure

A 36-month-old girl had a 3-week history of proptosis of the right eye. Computed tomography showed an ill-defined homogeneous mass filling the intraconal space. Histopathologic examination and immunohistochemistry findings of an incisional biopsy specimen were consistent with malignant undifferentiated tumor with rhabdoid features. Despite chemotherapy (a combination of vincristine sulfate and dactinomycin) and radiotherapy, massive orbital recurrence occurred 6 months later and orbital exenteration was performed. The recurrent tumor was composed entirely of pleomorphic epithelial cells with prominent nucleoli and many filamentous cytoplasmic inclusions. Immunohistochemical staining showed positive immunoreactivity for vimentin, cytokeratin, and epithelial membrane antigen, and negative immunoreactivity for muscle-specific antigen, melanoma, neural, and histiocytic markers. Electron microscopy excluded myogenic differentiation and showed that the filamentous cytoplasmic inclusions were composed of whorls of intermediate filaments. Aggressive chemotherapy with a combination of vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide phosphate was continued after exenteration. At 17 months' follow-up, orbital debulking surgery with externalization of the maxillary sinus was performed because of massive tumor recurrence in the right orbit and growth into the maxillary sinus. The child died 23 months after initial diagnosis from tumor invasion into the central nervous system. Extrarenal rhabdoid tumor is a rare orbital mass that carries a poor prognosis.     

1H magnetic resonance spectroscopy of the lentiform nucleus in primary focal hand dystonia

Several radiologic findings point toward the lentiform nucleus as a possible site of lesion in primary dystonia. Histologic examinations, however, have shown inconsistent results. 1H-magnetic resonance spectroscopy (MRS) has proved helpful to assess neuronal degeneration in a variety of basal ganglia disorders. MRS data of dystonia patients are, however, lacking so far. 1H-MRS centered on the lentiform nuclei was performed in 14 patients with primary focal hand dystonia and in 12 healthy control subjects using a 1.5-T MR imager. No statistically significant differences of N-acetylaspartate/creatine and lactate/creatine ratios were found between patients and control subjects. Based on these data, the authors found no evidence that primary focal dystonia was associated with a conspicuous loss of lentiform nucleus neurons or a marked disturbance of the aerobic metabolism, although minor abnormalities cannot be excluded because of the possibly limited sensitivity of the method.     

Unusual helix-containing greek keys in development-specific Ca(2+)-binding protein S. 1H, 15N, and 13C assignments and secondary structure determined with the use of multidimensional double and triple resonance heteronuclear NMR spectroscopy

Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone and side-chain 1H, 15N, and 13C resonance assignments of calcium loaded Myxococcus xanthus protein S (173 residues). Of the range of constant-time triple resonance experiments recorded, HNCACB and CBCA(CO)NH, which correlate C alpha and C beta with backbone amide resonances of the same and the succeeding residue respectively, proved particularly useful in resolving assignment ambiguities created by the 4-fold internal homology of the protein S amino acid sequence. Extensive side-chain 1H and 13C assignments have been obtained by analysis of HCCH-TOCSY and 15N-edited TOCSY-HMQC spectra. A combination of NOE, backbone amide proton exchange, 3JNH alpha coupling constant, and chemical shift data has been used to show that each of the protein S repeat units consists of four beta-strands in a Greek key arrangement. Two of the Greek keys contain a regular alpha-helix between the third and fourth strands, resulting in an unusual and possibly unique variation on this common folding motif. Despite similarity between two nine-residue stretches in the first and third domains of protein S and one of the Ca(2+)-binding sequences in bovine brain calmodulin [Inouye, S., Franceschini, T., & Inouye, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6829-6833], the protein S topology in these regions is incompatible with an EF-hand calmodulin-type Ca(2+)-binding site.     

1H and 15N resonance assignments and secondary structure of cellular retinoic acid-binding protein with and without bound ligand

Sequence-specific assignments for the 1H and 15N backbone resonances of cellular retinoic acid-binding protein (CRABP), with and without the bound ligand, have been obtained. Most of the side-chain resonances of both apo- and holo-CRABP have also been assigned. The assignments have been obtained using two-dimensional homonuclear and heteronuclear NMR data, and three-dimensional 1H-15N TOCSY-HMQC and NOESY-HMQC experiments. The secondary structure, deduced from nuclear Overhauser effects, amide H/D exchange rates and H alpha chemical shifts, is analogous in both forms of the protein and is completely consistent with a model of CRABP that had been constructed by homology with the crystal structure of myelin P2 protein [Zhang et al. (1992) Protein Struct. Funct. Genet., 13, 87-99]. This model comprises two five-stranded beta-sheets that form a sandwich or beta-clam structure, and a short N-terminal helix-turn-helix motif that closes the binding cavity between the two sheets. Comparison of the data obtained for apo- and holo-CRABP indicates that a region around the C-terminus of the second helix is much more flexible in the apo-protein. Our data provide experimental evidence for the hypothesis that the ligand-binding mechanism of CRABP, and of other homologous proteins that bind hydrophobic ligands in the cytoplasm, involves opening of a portal to allow entry of the ligand into the cavity.     

Modification of ribonuclease T1 specificity by random mutagenesis of the substrate binding segment

Attempts to modify the guanine specificity of ribonuclease T1 (RNase T1) by rationally designed amino acid substitutions failed so far. Therefore, we applied a semirational approach by randomizing the guanine binding site. A combinatorial library of approximately 1.6 million RNase T1 variants containing permutations of 6 amino acid positions within the recognition loop was screened on RNase indicator plates. The specificity profiles of 180 individual clones showing RNase activity revealed that variant K41S/N43W/N44H/Y45A/E46D (RNaseT1-8/3) exhibits an altered preference toward purine nucleotides. The ApC/GpC preference in the cleavage reaction of this variant was increased 4000-fold compared to wild-type. Synthesis experiments of dinucleoside monophosphates from cytidine and the corresponding 2'3'-cyclic diesters using the reverse reaction of the transesterification step showed a 7-fold higher ApC synthesis rate of RNase 8/3 than wild-type, whereas the GpC synthesis rates for both enzymes were comparable. This study shows that site-directed random mutagenesis is a powerful additional tool in protein design in order to achieve new enzymatic specificities.     

Poisoning with methanol and ethylene glycol:1H NMR spectroscopy as an effective clinical tool for diagnosis and quantification

1. Renal haemodynamics, lithium and sodium clearance were measured in 14 patients treated with recombinant interleukin-2 for metastatic renal cell carcinoma. 2. Patients were studied before and after 72 h of continuous intravenous infusion of recombinant interleukin-2 (18x10(6) i.u..24 h-1.m-2) and 48 h post therapy. Cardiac output was measured by impedance cardiography. Effective renal plasma flow and glomerular filtration rate were determined by the renal clearances of 131I-hippuran and 99mTc-diethylenetriaminepenta-acetic acid (DTPA) respectively. Renal clearance of lithium (CLi) was used as an index of proximal tubular outflow. 3. Treatment caused a transient decrease in mean arterial blood pressure and systemic vascular resistance, but cardiac output remained unchanged. Renal blood flow decreased and renal vascular resistance increased during and after treatment. Sodium clearance decreased from 1.10 (0.63/1.19) ml/min to 0.17 (0.18/0.32) ml/min (P=0.003). Glomerular filtration rate remained unchanged, whereas the median CLi decreased from 26 (17/32) ml/min to 17 (10/21) ml/min (P=0.008). Calculated absolute proximal reabsorption rate of water increased from 63 (40/69) ml/min to 71 (47/82) ml/min (P=0.04). The urinary excretion rate of thromboxane B2 and the ratio between excretion rates of thromboxane B2 and 6-keto-prostaglandin-F1alpha increased by 98% (P=0.022) and 175% (P=0.022) respectively. 4. The study suggests a specific recombinant interleukin-2-induced renal vasoconstrictor effect. Changes in renal prostaglandin synthesis may contribute to the decrease in renal blood flow. The lithium clearance data suggest that an increased proximal tubular reabsorption rate may contribute to the decreased sodium clearance during recombinant interleukin-2 treatment.     

1H NMR spectroscopy of the binuclear Cu(II) active site of Streptomyces antibioticus tyrosinase

The 600 MHz 1H NMR spectrum of tyrosinase (31 kDa) of Streptomyces antibioticus in the oxidized, chloride-bound form is reported. The downfield part of the spectrum (15-55 ppm) exhibits a large number of paramagnetically shifted signals. The paramagnetism is ascribed to a thermally populated triplet state. The signals derive from six histidines binding to the metals through their Nepsilon atoms. There is no evidence for endogenous bridges. The exchange coupling, -2J, amounts to 298 cm(-1). In the absence of chloride the peaks broaden. This is ascribed to a slowing down of the electronic relaxation. The exchange coupling decreases to -2J=103 cm(-1).     

Characterization of the three-dimensional solution structure of human profilin: 1H, 13C, and 15N NMR assignments and global folding pattern

Human profilin is a 15-kDa protein that plays a major role in the signaling pathway leading to cytoskeletal rearrangement. Essentially complete assignment of the 1H, 13C, and 15N resonances of human profilin have been made by analysis of multidimensional, double- and triple-resonance nuclear magnetic resonance (NMR) experiments. The deviation of the 13C alpha and 13C beta chemical shifts from their respective random coil values were analyzed and correlate well with the secondary structure determined from the NMR data. Twenty structures of human profilin were refined in the program X-PLOR using a total of 1186 experimentally derived conformational restraints. The structures converged to a root mean squared distance deviation of 1.5 A for the backbone atoms. The resultant conformational ensemble indicates that human profilin is an alpha/beta protein comprised of a seven-stranded, antiparallel beta-sheet and three helices. The secondary structure elements for human profilin are quite similar to those found in Acanthamoeba profilin I [Archer, S. J., Vinson, V. K., Pollard, T. D., & Torchia, D. A. (1993), Biochemistry 32, 6680-6687], suggesting that the three-dimensional structure of Acanthamoeba profilin I should be analogous to that determined here for human profilin. The structure determination of human profilin has facilitated the sequence alignment of lower eukaryotic and human profilins and provides a framework upon which the various functionalities of profilin can be explored. At least one element of the actin-binding region of human profilin is an alpha-helix. Two mechanisms by which phosphatidylinositol 4,5-bisphosphate can interfere with actin-binding by human profilin are proposed.