Saturation Transfer Experiments Research Articles

A water molecule participates in the secondary structure of hyaluronan.

The structure of hyaluronan was investigated in water/dimethyl sulphoxide mixtures by using high-field n.m.r. and space-filling molecular models. The secondary structure previously established in detail in 'dry' dimethyl sulphoxide [Heatley, Scott & Hull (1984) Biochem. J. 220, 197-205] undergoes changes on addition of water, compatible with the incorporation of a water bridge between the uronate carboxylate and acetamido NH groups. Molecular models show that such a configuration is highly probable, and saturation-transfer experiments yield rates of NH proton exchange that support this proposed structure. The existence of two distinct stable configurations for hyaluronan, in water-rich and water-poor conditions respectively, may have biological implications, e.g. during its biosynthesis in cell membranes. There are extensive hydrophobic regions in both forms, which may be important for interactions with e.g., membranes, proteins and itself.

NMR studies of carbonic anhydrase-4-fluorobenzenesulfonamide complexes.

Binding of 4-fluorobenzenesulfonamide to human carbonic anhydrases I and II has been studied by proton, fluorine, and nitrogen-15 nuclear magnetic resonance spectroscopy. All three types of experiments provide evidence that the stoichiometry of the interaction of this inhibitor with both enzymes is 2 mol of inhibitor bound per mole of enzyme. Observations which suggest that the bound forms are involved in an exchange process that is rapid at room temperature but slower at 2 degrees C are described. Nitrogen-15 shift data show that the bound inhibitors are present at the active site as anions. The proton experiments indicate appreciable reorganization of the tertiary structure of the protein upon binding. Saturation-transfer experiments to determine the rate of dissociation of the inhibitor-enzyme complex lead to the conclusion that the dissociation process is more complicated than a simple free-bound equilibrium.

Anisatin: a Crystallographic, N.M.R. and Theoretical Conformation Study

The convulsant compound anisatin has been studied by 1H n.m.r ., and X- ray crystallography, to establish its molecular geometry. The n.m.r . measurements included an analysis of proton-proton vicinal coupling constants and saturation transfer experiments which monitored exchange of the hydroxy groups of anisatin. The former analysis was used to obtain a solution conformation via the Karplus equation while the latter experiments yield information on intramolecular hydrogen bonding. The experimental geometry is compared to that obtained by several theoretical methods, including MINDO/3, MNDO, AM1 and MM2. The AM1 optimized geometry was closest to that of the crystal structure.

Proton magnetic resonance study of lysine-binding to the kringle 4 domain of human plasminogen: The structure of the binding site

The binding of l-Lys, d-Lys and ε-aminocaproic acid (εACA) to the kringle 4 domain of human plasminogen has been investigated via one and two-dimensional 1H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Ligand-kringle association constants ( K a) were determined assuming single site binding. At 295 K, pH ∗ 7.2, d-Lys binds to kringle 4 much more weakly (K a = 1.2m m −1) than does l-Lys (K a = 24.4 m m −1). l-Lys binding to kringle 4 causes the appearance of ring current-shifted high-field resonances within the −1 ≲ δ ≲ 0 parts per million range. The ligand origin of these signals has been confirmed by examining the spectra of kringle 4 titrated with deuterated l-Lys. A systematic analysis of ligand-induced shifts on the aromatic resonances of kringle 4 has been carried out on the basis of 300 MHz two-dimensional chemical shift correlated (COSY) and double quantum correlated spectroscopies. Significant differences in the effect of l-Lys and d-Lys binding to kringle 4 have been observed in the aromatic COSY spectrum. In particular, the His31 H4 and Trp72 H2 singlets and the Phe64 multiplets appear to be the most sensitive to the particular enantiomers, indicating that these residues are in proximity to the ligand C α center. In contrast, the rest of the indole spectrum of Trp72 and the aromatic resonances of Trp62 and Tyr74, which are affected by ligand presence, are insensitive to the optical nature of the ligand isomer. These results, together with two-dimensional proton Overhauser studies and ligand-kringle saturation transfer experiments reported previously, enabled us to generate a model of the kringle 4 ligand-binding site from the crystallographic co-ordinates of the prothrombin kringle 1. The latter, although lacking recognizable lysine-binding capability, is otherwise structurally homologous to the plasminogen kringles.

31P-NMR studies of phosphate transfer rates in T47D human breast cancer cells

The concentration of phosphates and the kinetics of phosphate transfer reactions were measured in the human breast cancer cell line, T47D, using 31P-NMR spectroscopy. The cells were embedded in agarose filaments and perifused with oxygenated medium during the NMR measurements. The following phosphates were identified in spectra of perifused cells and of cell extracts: phosphorylcholine (PC), phosphorylethanolamine (PE), the glycerol derivatives of PC and PE, inorganic phosphate (P i), phosphocreatine (PCr), nucleoside triphosphate (primarily ATP) and uridine diphosphate glucose. The rates of the transfers: PC → γATP (0.2 mM/s), P i → γATP (0.2 mM/s) and the conversion βATP → βADP (1.3 mM/s) were determined from analysis of data obtained in steady-state saturation transfer and inversion recovery experiments. Data from spectrophotometric assays of the specific activity of creatine kinase (approx. 0.1 μmol/min per mg protein) and adenylate kinase (approx. 0.4 μmol/min per mg protein) suggest that the βATP → βADP rate is dominated by the latter reaction. The ratio between the rate of ATP synthesis from P i and the rate of consumption of oxygen atoms (4 · 10 −3 mM/s) was approx. 50. This high value and preliminary measurements of the rate of lactate production from glucose, indicated that aerobic glycolysis is the main pathway of ATP synthesis.

The use of transmembrane differences in saturation transfer for measuring fast membrane transport; application to H 13C0 3− exchange across the human erythrocyte

We consider the NMR signal intensity of a solute B, that is both in exchange across the membrane of cells in suspension and also in chemical exchange with another solute C, when the nuclei of C are saturated with selective radiofrequency radiation. This extension of the classical saturation transfer experiment potentially enables the measurement of fast unidirectional membrane transport rates of solutes that are in chemical equilibrium, when the chemical shifts of the intra- and extracellular solute are identical. The application of the theory and experimental methods to a biological system is illustrated by using 13C NMR with the H 13 CO 3 − 13 CO 2 exchange system in human erythrocytes. In the buffer conditions employed the estimated value of the exchange rate of H 13C0 3 − was comparable to values estimated with NMR and other non-NMR methods.

Substrate-induced alterations of high energy phosphate metabolism and contractile function in the perfused heart.

The bioenergetic basis by which the Krebs cycle substrate pyruvate increased cardiac contractile function over that observed with the Embden-Meyerhof substrate glucose was investigated in the isovolumic guinea pig heart. Alterations in the content of the high energy phosphate metabolites and the rate of high energy phosphate turnover were measured by 31P NMR. These were correlated to the changes in contractile function and rates of myocardial oxygen consumption. Maximum left ventricular developed pressure (LVDP) and high energy phosphates were observed with 16 mM glucose or 10 mM pyruvate. In hearts perfused with 16 mM glucose, the intracellular phosphocreatine (PCr) concentration was 15.2 +/- 0.6 mM with a PCr/Pi ratio of 10.3 +/- 0.9. The O2 consumption was 5.35 mumol/g wet weight/min, and these hearts exhibited a LVDP of 97 +/- 3.7 mm Hg at a constant paced rate of 200 beats/min. In contrast, when hearts were switched to 10 mM pyruvate, the PCr concentration was 18.3 +/- 0.4 mM, the PCr/Pi ratio was 30.4 +/- 2.2, the O2 consumption was 6.67 mumol/g wet weight/min, and the LDVP increased to 125 +/- 3.3 mm Hg. From NMR saturation transfer experiments, the steady-state flux of ATP synthesis from PCr was 4.9 mumol/s/g of cell water during glucose perfusion and 6.67 mumol/s/g of cell water during pyruvate perfusion. The flux of ATP synthesis from ADP was measured to be 0.99 mumol/s/g of cell water with glucose and calculated to be 1.33 mumol/s/g of cell water with pyruvate. These results suggest that pyruvate quite favorably alters myocardial metabolism in concert with the increased contractile performance. Thus, as a mechanism to augment myocardial performance, pyruvate appears to be unique.

Kinetics of tautomerism in 2-substituted 5,10,15,20-tetraphenylporphyrins: directionality of proton transfer between the inner nitrogens

This paper describes a study of the kinetics of proton transfer in 2-substituted 5,10,15,20-tetraphenylporphyrins. Variable-temperature NMR studies on 16 2-substituted porphyrins showed that the position of the tautomeric equilibrium is altered by the substituent and that the tautomerism can be readily monitored by variable-temperature NMR spectroscopy. Activation parameters for the tautomerism were determined by saturation transfer experiments and line shape analysis and compared to the values reported for the unsubstituted 5,10,15,20-tetraphenylporphyrin.

Mechanism of oligonucleotide loop formation in solution.

We have studied the tridecadeoxynucleotide CGCGAATTACGCG (I), which contains an additional A at position 9 compared to the dodecanucleotide of which the crystal structure has been determined. Sequence I exhibits no distinct melting curve and also has a concentration-dependent pattern of peaks on reverse-phase chromatography. This behavior is explained by a slow equilibration between loop and duplex forms in solution. We have characterized this equilibrium by proton NMR spectroscopy and shown that it is fully reversible by monitoring the two thymine methyl resonances, each of which occurs in two environments. Lower temperature and higher concentration favor the duplex; the midpoint of the transition is such that the loop predominates at room temperature. We have measured the van't Hoff enthalpy of formation of the duplex and the activation energy by temperature-jump and saturation-transfer experiments. The results are compared with those for the 17-mer sequence CGCGCGAATTACGCGCG (II), which contains two additional base pairs in the stem of the loop. The thermodynamic parameters and the effect of increasing salt concentration on the rate of conversion of the loop and duplex forms lead us to presume that the mechanism of interconversion involves complete strand separation and re-formation rather than cruciform formation and branch migration.

1H, 13C and 31P NMR studies of the stereochemistry of chiral (4R, 6R)‐2‐substituted‐1,3,2‐dioxaphosphorinanes

Abstract13C, 1H and 31P NMR investigations have been carried out to determine the ring stereochemistry and phosphorus configuration for four chiral (4R, 6R)‐2‐substituted‐1,3,2‐dioxaphosphorinane derivatives 1–4 (2‐substituent = Cl, Ph, OMe and O‐t‐Bu, respectively). 1H NMR spectra, double resonance, 2D‐J experiments and spin simulation proved the chair character of the ring. Depending on the position of R, two diastereomers may exist, but in most cases only one predominating form was detected. Dynamic processes were observed for 1 and 2. Saturation transfer experiments proved the existence of ring and/or phosphorus inversions.

Nuclear spin coherence transfer in photochemical reactions

The idea of labeling nuclear spin magnetization in the study of chemical reactions dates back to the famous saturation transfer experiment of Forsen and Hoffman. This experiment, designed for chemically exchanging systems, has seen many applications, recently, for instance, in /sup 31/P NMR studies of cell metabolism. The two-dimensional analogue of it has been proposed by Jeener et al. (2D exchange spectroscopy). Sagdeev and co-workers have applied radiofrequency labeling techniques to the study of one-sided chemical reactions. All these experiments involve z-magnetization labeling either through saturation or inversion of nuclear spins. The authors propose here a two-dimensional experiment, whereby use is made of xy-magnetization labeling of nuclear spins. It can be used to study one-sided chemical reactions where the chemical change is brought about in a pulsed fashion, such as in flash photolysis. Comparison with a similar 2D experiment based on z-magnetization transfer shows that the coherence transfer experiment is inherently more sensitive. Furthermore a comparison of both may be particularly informative in situations where dephasing occurs (e.g., in the presence of paramagnetic intermediates), since the decay of xy-magnetization may be quite different from that of z-magnetization. In the latter case the two methods provide complementary information.

Selective excitation in solid-state NMR in the presence of multiple-pulse line narrowing

A procedure is described and analyzed which allows selective excitation in solids in the presence of multiple-pulse dipolar decoupling and magic-angle sample spinning. Two experimental schemes are discussed: (a) Selective inversion-recovery technique for the measurement of spin diffusion and exchange rates of abundant nuclei, and (b) saturation-transfer experiments for investigating the phase structure of heterogeneous materials.

Investigation of dynamic equilibria using NMR-spectroscopy III. Saturation transfer experiments on the hydration of pyridine-4-carbaldehyde

Spin saturation experiments can be performed by pulse techniques. Different experiments with monoexponential as well as with biexponential decay functions are shown. Such experiments are very useful for the estimation of rate constants of chemical reactions in the time range of 0.1 to 1 seconds, where classical methods as well as relaxation kinetics are unfavourable. The application of spin saturation experiments on the hydration of pyridine-4-carbaldehyde is shown.

Structure and ligand exchange reactions of the complexes CpUCl 3L 2 in solution (L = hexamethylphosphoramide, tetrahydrofuran, triphenylphosphine oxide)

The dynamic behaviour in solution of the compounds CpUCl 3L 2 (L = HMPA, I: THF, II; OPPh 3, III) was studied by 1H and 31P NMR (variable temperature and spin saturation transfer experiments). Two isomers of I in equilibrium (IA and IB) were detected in CDCl 3 ( E a = 92 kJ mol −1). The two HMPA ligands of the major (70%) isomer IA are relatively cis in a mer pseudo-octahedral configuration whereas in IB (30%), the two ligands are in trans equatorial positions. For all the complexes CpUCl 3L 2, the equatorial ligand of isomer A was found to exchange with free added L molecules. In the case of I ( k = 7.0 ± 0.2 mol −1 1 s −1 at 260 K), this reaction proceeded by an associative mechanism; at 240 K, the intermediate CpUCl 3L 3 was observed. Addition of chloride ions to a solution of I led to the formation of CpUCl 4(HMPA) −.

In vivo 31P nuclear magnetic resonance saturation transfer measurements of phosphate exchange reactions in the yeast Saccharomyces cerevisiae

31P saturation transfer techniques have been used to measure phosphate kinetics in the yeast Saccharomyces cerevisiae. The phosphate consumption rate observed in acetate grown mid-log cells was combined with measurements of O 2 consumption to yield P/O ratios of 2.2 and 2.9, for cells respiring on glucose and ethanol, respectively. However, no phosphate consumption activity was observed in saturation transfer experiments on anaerobic glucose fed cells. The phosphate consumption rates measured by saturation transfer in cells respiring on glucose and ethanol was attributed to the unidirectional rates of mitochondria! ATP synthesis.

Nuclear magnetic resonance studies on oligo-deoxyribonucleotides containing thedam methylation site GATC. Adenine methylation of d(GGATCC) does not change the helix geometry

We have studied the conformation of two hexanucleotides d(GGATCC) and d(GGm6ATCC) using proton nuclear magnetic resonance. Nuclear Overhauser effect measurements show that d(GGATCC) assumes a normal right handed B helix. The single and double strand resonances are in fast exchange on a proton nuclear magnetic resonance time scale. For d(GGm6ATCC), up to the Tm separate resonances are observed for each state, indicating slow exchange, though above the Tm it becomes more rapid. The orientation of the adenosine methyl-amino group, preferentiallycis to N1, hinders base pair formation.The connectivities of the resonances of the two states were established by saturation transfer experiments. At 0°C irradiation of the m6 A-T imino proton gives an nuclear Overhauser effect to AH2 showing that base pairing is Watson-Crick. Intra and interresidue nuclear Overhauser effects starting from the 3′ terminus show that the helix is right handed and in the B-form.The results on the two oligomers demonstrate that adenosine methylation induces little or no change in the conformation of the helix, but reduces the Tm from 45° to 32°C and slows the opening and closing of the m6A.T base pair by a factor of about 100.

Mechanism of action of thrombin on fibrinogen: NMR evidence for a beta-bend at or near fibrinogen A alpha Gly(P5)-Gly(P4).

The following synthetic linear A alpha fibrinogen-like peptides were studied by NMR spectroscopy in aqueous solution: Ac-Asp(P10)-Phe(P9)-Leu-Ala-Glu-Gly(P5)-Gly(P4)-Gly(P3)-Val- Arg(P1)-Gly-(P1)-Pro-Arg(P3')-Val-NHCH3 (F-8), Ac-Phe-Leu-Ala-Glu-Gly-Gly(P4)-Gly(P3)-Val-Arg-Gly-Pro-NHCH3 (F-6), Ac-Leu-Ala-Glu-Gly-Gly(P4)-Gly(P3)-Val-Arg-Gly-Pro-NHCH3 (F-7), and Ac-Gly-Gly(P4)-Gly-(P3)-Val-Arg-Gly-Pro-NHCH3 (F-9). The temperature dependence of the amide proton chemical shift is smaller by approximately 22% for the Gly(P3) amide proton in F-9, F-6, and F-8 and is similarly smaller for the Gly(P4) amide proton in F-6 and F-8, but not F-9, relative to the other amide protons in these peptides. The exchange rates with solvent water for the Gly(P3) amide proton in each of these four peptides were determined by solvent spin saturation transfer experiments. The exchange rate constant for the Gly(P3) amide proton of F-8 was half that of the rate constant determined for this proton in F-9, F-7, and F-6. In conjunction with previously reported data for the rate of hydrolysis of the Arg(P1)-Gly(P1') bond by thrombin, these results suggest that there is a beta-bend at Gly(P5)-Gly(P4), possibly stabilized by salt links between Asp(P10) and Arg(P3') and between phosphorylated Ser(P14) and Arg(P7'), which brings Phe(P9) close to the hydrolyzable Arg-Gly bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer simulation of the P 31 NMR equations governing the creatine kinase reaction

A method for solving the Bloch equations that govern magnetization transfer NMR experiments is presented. It requires the numerical evaluation of a matrix exponential and lends itself to computer simulation. It is a simple, versatile method for evaluating P31-NMR magnetization transfer experiments designed to measure biochemical exchange rates. We apply the method to the saturation and inversion transfer experiments and find that the "initial slope" method of determining flux is subject to at least two interpretations. This verifies the accepted concept that biochemical models representing compartmentation and competing reaction hypotheses cannot be reliably distinguished by simply selecting values for NMR and biochemical parameters that give a "best fit" to experimental data. However, our results do indicate that a controlled manipulation of biochemical exchange rate may distinguish between these two models.

Sensitivity of two-dimensional NMR experiments for studying chemical exchange

The conditions required to optimize the sensitivity of two-dimensional experiments for the study of chemical exchange are discussed in detail and compared with results for selective saturation transfer experiments. Details of phase-cycling procedures are also given and a new method of data handling to produce pure absorption lineshapes is presented. The inherent sensitivity of two-dimensional spectra is compared with ordinary NMR spectra with particular regard to the significance of using discrete Fourier transforms. In conclusion, the superior sensitivity of two-dimensional methods over that of conventional techniques is demonstrated for the study of exchange processes in complex molecules.

Saturation transfer electron paramagnetic resonance of spin-labeled muscle fibers: Dependence of myosin head rotational motion on sarcomere length

We have investigated the orientation and rotational mobility of spin-labeled myosin heads in muscle fibers as a function of the sarcomere length in the absence of ATP. An iodoacetamide spin label was used to label selectively two-thirds of the sulfhydryl-1 groups in glycerinated rabbit psoas muscle. Conventional electron paramagnetic resonance experiments were used to determine the orientation distribution of the probes relative to the fiber axis, and saturation transfer experiments were used to detect sub-millisecond rotational motion. When fibers are at sarcomere length 2.3 μm (full overlap), spin-labeled heads have a high degree of orientational order. The probes are in a single, narrow orientation distribution (full width 15 °), and they exhibit no detectable sub-millisecond rotational motion. When fibers are stretched (sarcomere length increased), either before or after labeling, disorder and microsecond mobility increase greatly, in proportion to the fraction of myosin heads that are no longer in the overlap zone between the thick and thin filaments. Saturation transfer difference spectra show that a fraction of myosin heads equal to the fraction outside the overlap zone have much more rotational mobility than those in fibers at full overlap, and almost as much as in synthetic myosin filaments. The most likely interpretation is that some of the probes, corresponding approximately to the fraction of heads in the overlap zone, remain oriented and immobile, while the rest are highly disordered (angular spread >90 °) and mobile (microsecond rotational motion). Thus, it appears that myosin heads are rigidly immobilized by actin, but they rotate through large angles on the microsecond time-scale when detached from actin, even in the absence of ATP.