19F nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol binding protein II produced in Escherichia coli. An analysis of four tryptophan substitution mutants and their interactions with all-trans-retinol

Rat cellular retinol binding protein (CRBP II) is a 134-amino acid intracellular protein synthesized in the polarized absorptive cells of the intestine. We have previously used 19F nuclear magnetic resonance (NMR) spectroscopy to survey the structural effects of ligand binding on the apoprotein. For these studies, all 4 Trp residues of rat CRBP II were efficiently labeled with 6-fluorotryptophan (6-F-Trp) by inducing its expression in a tryptophan auxotroph of Escherichia coli. Resonances corresponding to 2 of its Trp residues underwent large downfield shifts upon binding of all-trans-retinol and retinal, while resonances corresponding to the other 2 Trp residues underwent only minor perturbations in chemical shifts. To identify which Trp residues undergo changes in their environment upon ligand binding, we have constructed four CRBP II mutants where Trp9, Trp89, Trp107, or Trp110 have been replaced by another hydrophobic amino acid. By comparing the 19F NMR spectrum of each 6-F-Trp-labeled mutant with that of wild type 6-F-Trp CRBP II, we demonstrate that the 19F resonance corresponding to Trp107 undergoes the largest change in chemical shift upon ligand binding (2.0 ppm downfield). This is consistent with the position of this residue predicted from molecular modeling studies. The 19F resonance corresponding to Trp9 also undergoes a downfield change in chemical shift of 0.5 ppm associated with retinol binding even though it is predicted to be removed from the ligand binding site. By contrast, the resonances assigned to Trp89 and Trp110 undergo only minor perturbations in chemical shifts. These results have allowed us to identify residue-specific probes for evaluating the interactions of all-trans-retinol (and other retinoids) with this intracellular binding protein.     

The Nature and Origin of Chemical Shift for Intracellular Water Nuclei in Artemia Cysts

We investigated the possible existence of chemical shift of water nuclei in Artemia cysts using high resolution nuclear magnetic resonance (NMR) methods. The results conducted at 60, 200, and 500 MHz revealed an unusually large chemical shift for intracellular water protons. After correcting for bulk susceptibility effects, a residual downfield chemical shift of 0.11 ppm was observed in fully hydrated cysts. Similar results have been observed for the deuterium and ¹⁷O nuclei.

We have ruled out unusual intracellular pH, diamagnetic susceptibility of intracellular water, or interaction of water molecules with lipids, glycerol, and/or trehalose as possible origins of the residual chemical shift. We conclude that the residual chemical shift observed for water nuclei (¹H, ²H, and ¹⁷O) is due to significant water-macromolecular interactions.

     

Motion at the active site of [(4-fluorophenyl)sulfonyl]chymotrypsin

Fluorine and deuterium NMR relaxation studies have been used to examine the motion of the 4-fluorophenyl ring attached to the active site of [(4-fluorophenyl)sulfonyl]-alpha-chymotrypsin at pH 4. Analysis of the results indicates that rotation about the 2-fold axis of this ring is reasonably rapid, though not as fast as in tosylchymotrypsin. Two-dimensional (2D) nuclear Overhauser effects (NOEs) were used to suggest the shifts of those protons of the enzyme close enough to the fluorine nucleus to lead to relaxation; important proton-fluorine dipolar relaxation contributions arise from protons with shifts of 7.4 +/- 0.3 ppm and between 4.0 and 5.4 ppm. Specific deuteration permits the assignment of the first of these to the protons ortho to the fluorine while serine-189, cysteines-191 and -220, and methionine-192 are suggested as possible bearers of the other protons. The fluorine chemical shift effect observed for the native conformation of this protein is 9 ppm downfield of the shift observed with the denatured protein; this large shift may be the result of van der Waals interactions between the fluorine and one or more of the protons whose signals appear in the 2D NOE experiments.     

1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solution: the amide proton environment

The exchangeable amide protons of hyaluronic acid (HA) oligosaccharides and a higher-molecular-weight segment dissolved in H2O at pH 2.5 or 5.5 were examined by H NMR spectroscopy at 250 MHz. The HA segment preparation showed a single amide resonance, near the chemical shift for the amide proton of the monosaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose (beta-GlcNAc). Smaller HA oligosaccharides showed two or three separate amide proton resonances, corresponding in relative peak area to interior or end GlcNAc residues. The interior GlcNAc amide resonance occurred at the same chemical shift as the single resonance of the HA segment. For the end GlcNAc residues, linkage to D-glucuronopyranose (GlcUA) through C1 resulted in an upfield shift relative to the beta-anomer of GlcNAc, whereas linkage through C3 resulted in a downfield shift relative to the corresponding anomer of GlcNAc. These chemical-shift perturbations appeared to be approximately offsetting in the case of linkage at both positions. The amide proton vicinal coupling constant (ca. 9 Hz) was found to be essentially independent of chain length, residue position, or solution pH. These data favor a nearly perpendicular orientation for the acetamido group with respect to the sugar ring, little affected by linkage of GlcNAc to GlcUA. No evidence for the existence of a stable hydrogen bond linking the amide proton with the carboxyl(ate) oxygen of the adjacent uronic acid residue was found. The amide proton resonances for chondroitin, chondroitin 4-sulfate, and dermatan sulfate were compared to that of HA. The chemical shifts of these resonances deviated no more than 0.1 ppm from that of HA. A small dependence on the identity of the adjacent uronic acid residue was noted, based on the observation of two resonances for dermatan sulfate.     

Observation of deuterium-labeled diacetyldeuteroporphyrin incorporated in cyanoferrimyoglobin by deuterium nuclear magnetic resonance.

Sperm whale apomyoglobin was reconstituted with selectively deuterated D₆-2,4-diacetyldeuterohemin in which the ²H label was confined to the methyl groups of the acetyl moieties. A single resonance was observed in ²H NMR of the cyanoferrimyoglobin derivative, with a chemical shift 0.80 ppm downfield of external D₁₂-TMS at pH 6.7. The corresponding chemical shift of D₆-2,4-diacetyldeuterohemin-OMe as the cyanide complex in pyridine-water was 0.96 ppm downfield of external D₁₂-TMS. The prominent HOD peak was well separated at 4.4 ppm downfield. The line width of the porphyrin ²H resonances in both the protein and free solvent environments yields evidence of considerable rotational freedom of the -CD3 groups about their axes.     

Carbon-13 nuclear magnetic resonance spectroscopy of high-spin iron(III) prophyrin compounds

¹³C nuclear magnetic resonance spectra have been obtained for variety of high-spin iron(III) porphyrin compounds and corresponding μ-oxo-bridged dimeric species. Large hyperfine shifts and significant line broadening are observed. The monomeric exhibit hyperfine shifts which are downfield with te exception of an upfield shift for the meso-carbon atom. Possible unpaired spin delocalization mechanisms and prospects for observing ¹³C NMR porphyrin resonances in high-spin ferrihemoproteins are discussed. Spectra reported here provide strategy for incorporation of ¹³C labels in hemoproteins either by biosynthetic or chemical means. The vinyl-CH₂ resonances of iron(III) protoporphyrin IX located 260 parts per million downfield from tetramethylsilane are especially attractive from the standpoint of chemical labeling.     

Interactions between hemoglobin and organic phosphates investigated with 31P nuclear magnetic resonance spectroscopy and ultrafiltration.

1. The chemical shifts (delta) of the phosphates of 2,3-diphosphoglycerate and adenosine triphosphate (ATP) were determined by phosphorus nuclear magnetic resonance (31P NMR) spectroscopy and were found to be displaced downfield following the addition of hemoglobin (3 mM) to a solution of either diphosphoglycerate (5 mM) or ATP (1 mM). 2. The binding of these compounds to hemoglobin was also determined by membrane ultrafiltration. A direct relationship was observed between the change in chemical shift ((delta delta) of the 2-P and 3-P of diphosphoglycerate and the percent diphosphoglycerate bound, when the latter was varied by altering pH, oxygenation state, or total diphosphoglycerate concentration. 3. In comparable studies with ATP binding, a linear relationship between the delta delta values of the gamma-, beta-, and alpha-P of ATP and the percent of ATP bound was not observed when the data from all of the experiments were plotted. NMR signals were not detectible in deoxyhemoglobin solutions containing 1 mM ATP but were seen in solutions containing 3.8 mM ATP. 4. The results indicate that 31P NMR spectroscopy is a promising tool for investigating organic phosphate interactions with hemoglobin.     

Proton magnetic resonance studies of the states of ionization of histidines in native and modified subtilisins

A technique was developed to exchange the backbone -N-H protons in D2O in the native subtilisins Carlsberg and BPN (Novo) that resulted in clearly resolved proton resonances in the aromatic region of the nuclear magnetic resonance spectrum. pH titration curves for four of the five histidine C2-H resonances in subtilisin Carlsberg and five of the six in subtilisin BPN between 7.5 and 8.8 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonic acid sodium salt provided microscopic pKa's between 6.3 and 7.2 for both sources of the enzyme at ambient (approximately 22 degrees C) probe temperature. A resonance that titrated with a pKapp of 7.35 +/- 0.05 was observed in the 1H spectra only of the diisopropylphosphoryl derivatives of the subtilisins from both sources. The 31P NMR pH titration of the same preparations under identical conditions of solvent (D2O) and temperature gave a pKapp = 7.40 +/- 0.05 of the single titratable resonance. Both observations must pertain to His-64 at the active center. A resonance smaller than the others and titrating with a pKapp of 7.2 could also be observed in the native enzymes. This resonance was assigned to the catalytic center histidine since its pK corresponded to that derived from kinetic studies. No major perturbations in the chemical shifts or the pK's derived from the pH dependence of the observed resonances were apparent in the presence of saturating concentrations of the two putative transition-state analogues phenylboronic acid and bis [3,5-(trifluoromethyl)phenyl]boronic acid and in monoisopropylphosphorylsubtilisin.(ABSTRACT TRUNCATED AT 250 WORDS)     

15N-labeled tRNA. Identification of 4-thiouridine in Escherichia coli tRNASer1 and tRNATyr2 by 1H-15N two-dimensional NMR spectroscopy

Uridine is uniquely conserved at position 8 in elongator tRNAs and binds to A14 to form a reversed Hoogsteen base pair which folds the dihydrouridine loop back into the core of the L-shaped molecule. On the basis of 1H NMR studies, Hurd and co-workers (Hurd, R. E., Robillard, G. T., and Reid, B. R. (1977) Biochemistry 16, 2095-2100) concluded that the interaction between positions 8 and 14 is absent in Escherichia coli tRNAs with only 3 base pairs in the dihydrouridine stem. We have taken advantage of the unique 15N chemical shift of N3 in thiouridine to identify 1H and 15N resonances for the imino units of S4U8 and s4U9 in E. coli tRNASer1 and tRNATyr2. Model studies with chloroform-soluble derivatives of uridine and 4-thiouridine show that the chemical shifts of the protons in the imino moieties move downfield from 7.9 to 14.4 ppm and from 9.1 to 15.7 ppm, respectively; whereas, the corresponding 15N chemical shifts move downfield from 157.5 to 162.5 ppm and from 175.5 to 180.1 ppm upon hydrogen bonding to 5'-O-acetyl-2',3'-isopropylidene adenosine. The large difference in 15N chemical shifts for U and s4U allows one to unambiguously identify s4U imino resonances by 15N NMR spectroscopy. E. coli tRNASer1 and tRNATyr2 were selectively enriched with 15N at N3 of all uridines and modified uridines. Two-dimensional 1H-15N chemical shift correlation NMR spectroscopy revealed that both tRNAs have resonances with 1H and 15N chemical shifts characteristic of s4UA pairs. The 1H shift is approximately 1 ppm upfield from the typical s4U8 resonance at 14.8 ppm, presumably as a result of local diamagnetic anisotropies. An additional s4U resonance with 1H and 15N shifts typical of interaction of a bound water or a sugar hydroxyl group with s4U9 was discovered in the spectrum of tRNATyr2. Our NMR results for tRNAs with 3-base pair dihydrouridine stems suggest that these molecules have an U8A14 tertiary interaction similar to that found in tRNAs with 4-base pair dihydrouridine stems.     

Characterization of the broad resonance in 31P NMR spectra of excised rat brain

31P NMR spectra of excised rat brain showed a broad resonance between-12 and -13 ppm. Subcellular fractions of brain, rich in membranes, exhibited the broad resonance and it was also present in isolated myelin, the major membrane component of brain. However, it was absent in brain cytosol (161,100 X g supernatant). Raising the temperature of the brain above 50 degrees C caused a gradual downfield chemical shift of the broad resonance, to about -1 ppm at 90 degrees C. An even larger downfield shift was produced by halothane or deoxycholate with concomitant narrowing of the line width of this resonance. Vesicles prepared from the phospholipids of excised brain or isolated myelin showed the broad resonance, and halothane produced the same downfield shift and peak sharpening in brain phospholipid vesicles as that in the intact brain. The chemical shift anisotropy was estimated to be 45 ppm for both myelin and the brain, as characteristic for biological membranes. The T1 and T2 relaxation times of the perpendicular 31P chemical shift tensor component of the broad resonance were 0.66 sec and 1.6 msec, respectively, in the same range as those for other biological membranes. Halothane-treatment of the brain increased both the T1 and T2 times considerably, as expected from the disruption of the phospholipid bilayer in a membrane. These data indicate that the broad resonance in the 31P NMR spectrum of excised rat brain originates exclusively from the phosphate head group of membrane bound phospholipids. Similar broad resonances were found in autopsied human brain and porcine spinal cord and to a lesser extent in excised rat liver and kidney.     

Phosphorus-31 Fourier transform nuclear magnetic resonance study of mononucleotides and dinucleotides. 1. Chemical shifts.

A phosphorus-31 nuclear magnetic resonance (NMR) study of adenine, uracil, and thymine mononucleotides, their cyclic analogues, and the corresponding dinucleotides is reported. From the pH dependence of phosphate chemical shifts, pKa values of 6.25-6.30 are found for all 5'-mononucleotides secondary phosphate ionization, independently from the nature of the base and the presence of a hydroxyl group at the 2' position. Conversely, substitution of a hydrogen atom for a 2'-OH lowers the pKa of 3'-monoribonucleotides from 6.25 down to 5.71-5.85. This indication of a strong influence of the 2'-hydroxyl group on the 3'-phosphate is confirmed by the existence of a 0.4 to 0.5 ppm downfield shift induced by the 2'-OH on the phosphate resonance of 3'-monoribonucleotides, and 3',5'-cyclic nucleotides and dinucleotides with respect to the deoxyribosyl analogues. Phosphate chemical shifts and titration curves are affected by the ionization and the type of the base. Typically, deviations from the theoretical Henderson-Hasselbalch plots are observed upon base titration. In addition, purine displays a more deshielding influence than pyrimidine on the phosphate groups of most of the mononucleotides (0.10 to 0.25 ppm downfield shift) with a reverse situation for dinucleotides. These effects together with the importance of stereochemical arrangement (furanose ring pucker, furanose-phosphate backbone conformation, O-P-O bond angle) on the phosphate chemical shifts are discussed.     

Diastereomeric phosphonate ester adducts of chymotrypsin: 31P-NMR measurements

Generation of diastereomeric phosphonate ester adducts of chymotrypsin was evidenced for the first time by ³¹P NMR and spectrophotometric kinetic measurements. ³¹P NMR signals were recorded for 4-nitrophenyl 2-propyl methylphosphonate (IMN) at 32.2 ppm and for its hydrolysis product at 26.3 ppm downfield from phosphoric acid. The inhibition of α-chymotrypsin at pH > 8.0 by the faster reacting enantiomer of IMN or 2-propyl methylphosphonochloridate (IMCl), or other phosphonate ester analogs of these compounds, all caused a ～6.0 ppm downfield shift of the ³¹P signal to the 39–40 ppm region. IMN, when applied below the stoichiometric amount of chymotrypsin, under the same conditions, generated two signals, at 39.0 and at 37.4 ppm. Scans accumulated in hourly intervals showed the decomposition of both diastereomers, with approximate half-lives of 12 h at pH 8.0 and 22°C, into a species with a resonance at 35.5 ppm. The most likely reaction to account for the appearance of this new peak is the enzymic dealkylation of the isopropyl group from the covalently bound phosphonate ester. We base this conclusion mostly on the similarity of the upfield shift to the hydrolysis of phosphonate esters. Contrary to experience with phosphate ester adducts of serine proteases, no signal was detected higher than 25.0 ppm downfield from phosphoric acid for several phosphonate ester adducts of chymotrypsin and in no case did the resonance for the adduct shift further downfield in the course of the experiments. © 1993 Wiley-Liss, Inc.     

(13)C, (31)P and (15)N NMR studies of the ligand exchange reactions of auranofin and chloro(triethylphosphine)gold(I) with thiourea.

The interaction of thiourea (Tu) with auranofin (Et(3)PAuSATg) and its analogue, Et(3)PAuCl has been studied using (13)C, (31)P and (15)N NMR spectroscopy. It is observed that Tu is able to replace both the ligands, Et(3)P and SATg(-) simultaneously from gold(I) in auranofin, forming [Et(3)P-Au-Tu](+) and Tu-Au-SATg complexes. However, no separate resonances for these species were observed either due to their rapid exchange with auranofin and thus giving only the average resonances or because the chemical shifts of either two species are same so that they cannot be resolved. The displaced SATg(-) is oxidized to its disulfide, (SATg)(2). However, some of the displaced Et(3)P is oxidized to Et(3)PO while the remaining reacts with Tu to form Et(3)P-Tu species, characterized by delta 31P of 1.0 ppm, assigned after an independent reaction between Et(3)P and Tu. In an experiment using a 0.05 M solution of auranofin, the Et(3)PO resonance appeared in auranofin spectrum after 4 days of addition of 1.0 equivalent of Tu, showing that the reaction is slow. A resonance for free Et(3)P is also detected in 31P NMR on the addition of CN(-). It is also observed that Tu reacts with Et(3)PAuCl to form [Et(3)P-Au-Tu](+) via displacement of Cl(-), consistent with an upfield shift of 6.2 ppm in >C [double bond] S resonance of Tu in (13)C NMR. In (15)N NMR, a smaller downfield, instead of an upfield shift, in NH(2) resonance of Tu on its addition to auranofin and Et(3)PAuCl indicates that it is not binding to gold(I) through nitrogen.     

Proton nuclear magnetic resonance assignments of thyroid hormone and its analogues

1H NMR data of a series of thyroid hormone analogues, e.g., thyroxine (T4), 3,5,3'-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3), 3,3'-diiodothyronine (3,3'-T2), 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), 3-monoidothyronine (3-T1), 3'-monoiodothyronine (3'-T1), and thyronine (TO) in dimethylsulfoxide (DMSO) have been obtained on a 300 MHz spectrometer. The chemical shift and coupling constant are determined and tabulated for each aromatic proton. The inner tyrosyl ring protons in T4, T3, and 3,5-T2 have downfield chemical shifts with respect to those of the outer phenolic ring protons. Four-bond cross-ring coupling has been observed in all the monoiodinated rings. However, this long-range coupling does not exist in T4, diiodinated on both rings, and T0, containing no iodines on the rings. There is no evidence that at 30 degrees C these iodothyronines have any motional constraint in DMSO solution. In addition to identification of the hormones, the potential use of some characteristic peaks as probes in binding studies is discussed.     

Cross-peptide bond 13C--15N coupling constants by 13C and J cross-polarization 15N NMR

Comparative 13C--15N coupling constants are reported for the linear dipeptide tBoc-L-[U-13C]Ala-[15N]GlyOMe and the corresponding cyclic diketopiperazine, both in dimethylsulfoxide (DMSO) and, upon removal of the tBoc group, in water solutions. Spectra were obtained by 13C NMR and by the first application of J cross-polarization (JCP) 15N NMR, which greatly reduces the time required to accumulate 15N NMR spectra. In DMSO there was evidence for the formation of complexed species which were not present in water. The values obtained for the cross-peptide bond coupling constant 2J13C alpha--15N were consistently less (by 2.2 Hz in DMSO, 4.3 Hz in water) for the cyclic than for the linear peptide, which may be related to the cross-peptide bond conformation. The 15N resonance for the cyclic peptide was shifted only 2 ppm downfield from the linear peptide chemical shift value in both solvents.     

Nucleotide and AP5A complexes of porcine adenylate kinase: A 1H and 19F NMR study

Proton and fluorine nuclear magnetic resonance spectroscopies (NMR) were used as methods to investigate binary complexes between porcine adenylate kinase (AK1) and its substrates. We also studied the interaction of fluorinated substrate analogues and the supposed bisubstrate analogue P1,P5-bis(5'-adenosyl) pentaphosphate (AP5A) with AK1 in the presence of Mg2+. The chemical shifts of the C8-H, C2-H, and ribose C1'-H resonances of both adenosine units in stoichiometric complexes of AK1 with AP5A in the presence of Mg2+ could be determined. The C2-H resonance of one of the adenine bases experiences a downfield shift of about 0.8 ppm on binding to the enzyme. The chemical shift of the His36 imidazole C2-H was changed in the downfield direction on ATP-Mg2+ and, to a lesser extent, AMP binding. 19F NMR chemical shifts of 9-(3-fluoro-3-deoxy-beta-D-xylofuranosyl)adenine triphosphate (3'-F-X-ATP)-Mg2+ and 9-(3-fluoro-3-deoxy-beta-D-xylofuranosyl)adenine monophosphate (3'-F-X-AMP) bound to porcine adenylate kinase could be determined. The different chemical shifts of the bound nucleotides suggest that their mode of binding is different. Free and bound 3'-F-X-AMP are in fast exchange with respect to their 19F chemical shifts, whereas free and bound 3'-F-X-ATP are in slow exchange on the NMR time scale in the absence as well as in the presence of Mg2+. This information could be used to determine the apparent dissociation constants of the nucleotides and the 3'-F-X analogues in the binary complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

19F NMR studies on 8-fluoroflavins and 8-fluoro flavoproteins

The 19F NMR spectra of the oxidized and reduced forms of 8-fluororiboflavin, 8-fluoro-FAD, and the 8-fluoroflavin-reconstituted flavoproteins flavodoxin, riboflavin binding protein, D-amino acid oxidase, p-hydroxybenzoate hydroxylase, Old Yellow Enzyme, anthranilate hydroxylase, general acyl-CoA dehydrogenase, glucose oxidase, and L-lactate oxidase were measured. For the proteins studied the oxidized resonances appeared over a 10.1-ppm range, while the reduced resonances were spread over 10.3 ppm. Reduction caused an upfield shift of about 27 ppm for the free 8-fluoroflavins and most of the 8-fluoro flavoproteins. The notable exception was 8-fluoro-FMN flavodoxin, which was shifted 37.6 ppm, indicating an unusually high electron density in the benzene ring. Ligand binding to the oxidized 8-fluoro flavoproteins caused either upfield or downfield shifts of 1.5-5 ppm, depending on the protein/ligand combination. The 8-fluoro-FAD anthranilate hydroxylase resonance was shifted downfield and split into two peaks in the presence of anthranilate. The 8-fluoro-FMN Old Yellow Enzyme resonance was shifted upfield upon complexation with charge-transfer-forming, para-substituted phenolates. The upfield shift increased from less than 1 to 5 ppm as the electron-donating capacity of the phenolate increased. Complexation of native Old Yellow Enzyme with 2,4-difluorophenol caused the fluorine resonances of the ligand to shift and split into two pairs of signals. Each pair of signals was associated with a different isozyme of Old Yellow Enzyme.     

Carbon-13 NMR studies on [4-13C] uracil labelled E. coli transfer RNA1(Val1).

In this paper we describe carbon-13 nuclear magnetic resonance results on 13C-enriched purified transfer RNAI(VAL) from from E. coli SO-187, a uracil requiring auxotroph. The organism was grown on uracil 90% 13C-enriched at the carbonyl C4 position. Transfer RNAI(Val) was purified from bulk tRNA by sequential chromatography on columns of BD cellulose, DEAE-Sephadex A-50 and reverse gradient sepharose 4B. Dihydrouridine, 4-thiouridine, and uridine 5-oxyacetic acid located at discrete positions in the polymer backbone were tentatively assigned in the highly resolved 25 MHz 13C-spectra. Chemical shift versus temperature plots reveal differential thermal perturbation of the ordered solution structure, evident in the large dispersion (ca 3-4 ppm) of the uridine C4 resonances. Over the range 26-68 degrees C, V in the anticodon displays the largest downfield shift. Whereas several uridine residues rapidly shift downfield between 50-68 degrees, one moves upfield beginning at 37 degrees. The results are qualitatively compared with proton NMR analysis of the three dimensional structure.     

Analysis of phosphate metabolites, the intracellular pH, and the state of adenosine triphosphate in intact muscle by phosphorus nuclear magnetic resonance.

31P nuclear magnetic resonance spectra recorded from intact muophosphate, and the sugar phosphates. Quantitation of these metabolites by 31P nuclear magnetic resonance was in good agreement with values obtained by chemical analyses. The spectra obtained from various muscles showed considerable variation in their phosphorus profile. Thus, differences could be detected between (a) normal and diseased muscle; (b) vertebrates and invertebrates; (c) different species of the same animal. The time course of change in phosphate metabolites in frog muscle showed that ATP level remains unchanged until phosphocreatine is nearly depleted. Comparative studies revealed that under anaerobic conditions the Northern frog maintains its ATP content for 7 hours, while other types of amphibian, bird, and mammalian muscles begin to show an appreciable decay in ATP after 2 hours. Several lines of evidence indicated that ATP forms a complex with magnesium in the muscle water: (a) the phosphate resonances of ATP in the muscle were shifted downfield as compared to those in the alkaline earth metal-free perchloric acid extract of the muscle; (b) the coupling constants of ATP measured in various live muscles closely corresponded to those for MgATP in a solution resembling the composition of the muscle water; (c) in the muscle the gamma-phosphate group of ATP exhibited no shift change over a period of 10 hours under conditions where resonances of other phosphate compounds could be titrated. This behavior is similar to that of MgATP in model solutions in the physiological pH range, and it is different from that of CaATP. The chemical shifts of the phosphate metabolites were determined in several relevant solutions as a function of pH. Under all conditions only inorganic orthophosphate showed an invariant titration curve. From the chemical shift of inorganic phosphate observed during aging of intact muscle the intracellular pH of frog muscle was estimated to be 7.2.     

Structure and protein environment of the retinal chromophore in light- and dark-adapted bacteriorhodopsin studied by solid-state NMR

Our previous solid-state 13C NMR studies on bR have been directed at characterizing the structure and protein environment of the retinal chromophore in bR568 and bR548, the two components of the dark-adapted protein. In this paper, we extend these studies by presenting solid-state NMR spectra of light-adapted bR (bR568) and examining in more detail the chemical shift anisotropy of the retinal resonances near the ionone ring and Schiff base. Magic angle spinning (MAS) 13C NMR spectra were obtained of bR568, regenerated with retinal specifically 13C labeled at positions 12-15, which allowed assignment of the resonances observed in the dark-adapted bR spectrum. Of particular interest are the assignments of the 13C-13 and 13C-15 resonances. The 13C-15 chemical resonance for bR568 (160.0 ppm) is upfield of the 13C-15 resonance for bR548 (163.3 ppm). This difference is attributed to a weaker interaction between the Schiff base and its associated counterion in bR568. The 13C-13 chemical shift for bR568 (164.8 ppm) is close to that of the all-trans-retinal protonated Schiff base (PSB) model compound (approximately 162 ppm), while the 13C-13 resonance for bR548 (168.7 ppm) is approximately 7 ppm downfield of that of the 13-cis PSB model compound. The difference in the 13C-13 chemical shift between bR568 and bR548 is opposite that expected from the corresponding 15N chemical shifts of the Schiff base nitrogen and may be due to conformational distortion of the chromophore in the C13 = C14-C15 bonds.(ABSTRACT TRUNCATED AT 250 WORDS)