Regulatory role of sphingomyelin metabolites in hypoxia-induced vascular smooth muscle cell proliferation

The rotating frame nuclear magnetic resonance relaxation rate R(1rho) in the blood and cell lysate was studied at 4.7T to provide reference values for in vivo modeling and to address the mechanisms contributing to net relaxation. A strong dependence on oxygenation, hematocrit, and spin lock field strength B(1) (0.2-1.6G) was observed in whole blood, whereas in lysate the effects were severely attenuated. The results were further compared to transverse relaxation rate R(2). A good agreement in low-field asymptotes of these two relaxation rates was found. R(1rho) field dispersion was fitted to Lorenzian line shape and resulted in correlation times around 40 micros. The dispersion behavior was related to motional properties of intracellular hemoglobin and effects of susceptibility shift interface across the cell membrane induced by compartmentalization of Hb into cells in blood.     

Active site dynamics in the lead-dependent ribozyme

Conformational dynamics are an important property of ribozymes and other RNA molecules but there is currently only limited information on the relationship between dynamics and RNA function. A recent structural study of the lead-dependent ribozyme, known as the leadzyme, showed significant dynamics at the active site and indicated that a structural rearrangement is required for the reaction to proceed from the ground to the transition state. In this work, microsecond-to-millisecond dynamics of the leadzyme are probed by analysis of the power dependence of (13)C NMR relaxation times in the rotating frame (T(1)(rho)). These results revealed a wide range of conformational dynamics for various residues in the leadzyme. For residue A25 in the active site, the power dependence of T(1)(rho) yielded an exchange lifetime similar to that previously measured by line-shape analysis, and provides an important calibration of this T(1)(rho) methodology for probing the dynamics of macromolecules. Strong evidence was also found for a previously suggested dynamic network of hydrogen bonds stabilizing the GAAA tetraloop motif. Within the active site of the leadzyme, internal motions are observed on a wide variety of time scales, suggesting a complex landscape of accessible states, and potential correlations between observed motions and catalytic function are discussed. These results demonstrate that the power dependence of (13)C T(1)(rho) relaxation times provides a valuable method for probing dynamics in nucleic acids.     

Separating the contributions to 15N transverse relaxation in a fibronectin type III domain

In proteins, dynamic mobility is an important feature of structure, stability, and biomolecular recognition. Uniquely sensitive to motion throughout the milli- to picosecond range, rates of transverse relaxation, R2, are commonly obtained for the characterization of chemical exchange, and the construction of motional models that attempt to separate overall and internal mobility. We have performed an in-depth study of transverse relaxation rates of backbone 15N nuclei in TNfn31–90, the third fibronectin type III domain from human tenascin. By combining the results of spin-echo (CPMG) and off-resonance T1 experiments, we present R2 rates at effective field strengths of 2 to 40 krad/s, obtaining a full spectrum of 16 independent R2 data points for most residues. Collecting such a large number of replicate measurements provides insight into intrinsic uncertainties. The median standard deviation in R2 for non-exchanging residues is 0.31, indicating that isolated measurements may not be sufficiently accurate for a precise interpretation of motional models. Chemical exchange events on a timescale of 570 s were observed in a cluster of residues at the C terminus. Rates of exchange for five other residues were faster than the sampled range of frequencies and could not be determined. Averaged 'exchange free' transverse relaxation rates, R20, were used to calculate the diffusion tensor for rotational motion. Despite a highly asymmetric moment of inertia, the narrow angular dispersion of N-H vectors within the sandwich proves insufficient to define deviations from isotropic rotation. Loop residues provide exclusive evidence for axially symmetric diffusion (Dpar/Dper=1.55).     

Proton exchange as a relaxation mechanism for T1 in the rotating frame in native and immobilized protein solutions.

T1 relaxation in the rotating frame (T1rho) is a sensitive magnetic resonance imaging (MRI) contrast for acute brain insults. Biophysical mechanisms affecting T1rho relaxation rate (R1rho) and R1rho dispersion (dependency of R1rho on the spin-lock field) were studied in protein solutions by varying their chemical environment and pH in native, heat-denatured, and glutaraldehyde (GA) cross-linked samples. Low pH strongly reduced R1rho in heat-denatured phantoms displaying proton resonances from a number of side-chain chemical groups in high-resolution 1H NMR spectra. At pH of 5.5, R1rho dispersion was completely absent. In contrast, in the GA-treated phantoms with very few NMR visible side chain groups, acidic pH showed virtually no effect on R1rho. The present data point to a crucial role of proton exchange on R1rho and R1rho dispersion in immobilized protein solution mimicking tissue relaxation properties.     

Lateral diffusion of the phospholipid molecule in dipalmitoylphosphatidylcholine bilayers. An investigation using nuclear spin--lattice relaxation in the rotating frame

Measurements of the proton NMR spin--lattice relaxation time in the rotating frame (T 1rho) have permitted the explicit determination of the lateral diffusion coefficient of phospholipid molecules in the lamellar mesophase of dipalmitoylphosphatidylcholine at temperatures above the phase-transition temperature. The experimentally observed temperature and frequency dependence of T 1rho for the dipalmitoylphosphatidylcholine protons suggest that intermolecular dipole--dipole relaxation contributions are important. Proton T 1rho experiments involving dilution with deuterated dipalmitoylphosphatidylcholine support the premise that intermolecular dipolar interactions are significant and, concomitantly, that lateral diffusion is the motion modulating that interaction. The lateral diffusion coefficient is determined directly from the dependence of the rotating frame spin--lattice relaxation rate (1/T 1rho) on the strength of the applied radiofrequency field in the spin-locking experiment. A series of experiments with varying concentrations of dipalmitoylphosphatidylcholine in the lamellar mesophase indicates that the lateral diffusion coefficient varies as a function of phospholipid concentration.     

Heteronuclear relaxation in time-dependent spin systems: 15N-T1ρ dispersion during adiabatic fast passage

A novel NMR experiment comprising adiabatic fast passage techniques for the measurement of heteronuclear self-relaxation rates in fully 15N-enriched proteins is described. Heteronuclear self-relaxation is monitored by performing adiabatic fast passage (AFP) experiments at variable adiabaticity (e.g., variation of RF spin-lock field intensity). The experiment encompasses gradient- selection and sensitivity-enhancement. It is shown that transverse relaxation rates derived with this method are in good agreement with the ones measured by the classical Carr–Purcell–Meiboom–Gill (CPMG) sequences. An application of this method to the study of the carboxyl-terminal LIM domain of quail cysteine and glycine-rich protein qCRP2(LIM2) is presented.     

High-resolution solid-state 13C NMR of the LH1 from Rhodospirillum rubrum

High-resolution solid-state 13C NMR spectra of the light-harvesting antenna complex (LH1) from Rhodospirillum rubrum were observed for the first time by cross-polarization (CP), magic angle spinning (MAS) methods with a total elimination of spinning side band technique (TOSS). Chemical shift analysis of the CP/MAS/TOSS 13C NMR spectra confirmed that the LH1 consists mainly of -helices in the solid state. Time constants of cross polarization (TCH) and relaxation time T1 in a rotating frame (T1H) were determined from the experiments at various contact times. Smaller values of TCH were obtained for the carbons attached directly with protons in a rigid state. Relaxation times T1H revealed the dynamic structure of the complex and showed that bacteriochlorophyll a in the LH1 has high internal mobility even in the solid state. The proton spin-lattice relaxation time in a laboratory frame (T1H) determined by the 13C NMR signal amplitude changes suggested that protons in the LH1 proteins have such strong interaction among them that the spins of all protons in the protein can diffuse through spin-lattice-relaxation.     

Study of protein dynamics in solution by measurement of 13Cα-13CO NOE and 13CO longitudinal relaxation

Summary ¹³C-¹³CO homonuclear NOE and ¹³CO T₁ relaxation were measured for a 20 kDa protein using tripleresonance pulse sequences. The experiments were sufficiently sensitive to obtain statistically significant differences in relaxation parameters over the molecule. The ¹³C-¹³CO cross-relaxation rate, obtained from these data, is directly proportional to an order parameter describing local motion and it is largely independent of the local correlation time. It is therefore a relatively straightforward observable for the identification of local dynamics.     

C-terminal domain of insulin-like growth factor (IGF) binding protein 6: conformational exchange and its correlation with IGF-II binding

Insulin-like growth factor binding proteins (IGFBPs) function as carriers and regulators of the insulin-like growth factors (IGF-I and -II). Within the family of six binding proteins, IGFBP-6 is unique in having a 20-100-fold higher affinity for IGF-II over IGF-I and appears to act primarily as an inhibitor of IGF-II actions. We have recently determined the solution structure of the C-terminal domain of IGFBP-6 (C-BP-6), which shows the presence of substantial flexible regions, including three loop regions. In this paper, we report results from (15)N relaxation measurements carried out in both the laboratory and rotating frames. Analysis of conventional (15)N relaxation data (R(1), R(2), and steady-state (15)N-[(1)H] nuclear Overhauser effect) indicated that there was a considerable number of residues involved in conformational/chemical exchange. Measurements of off-resonance (15)N R(1)(rho) in the rotating frame and (15)N relaxation dispersion using an in- and antiphase coherence-averaged Carr-Purcell-Meiboom-Gill sequence were thus carried out to gain further insight into the solution dynamics of C-BP-6. Although the off-resonance (15)N relaxation data showed no clear evidence for residues undergoing microsecond motion, the (15)N relaxation dispersion data allowed us to identify 15 residues that clearly exhibit submilli- to millisecond motion. A good correlation was observed between residues exhibiting motion at submilli- to millisecond time scales and those affected by IGF-II binding, as identified through the perturbation of nuclear magnetic resonance (NMR) spectra of C-BP-6 following IGF-II addition. A complete NMR relaxation study of C-BP-6 dynamics in complex with IGF-II was hampered by peak broadening and disappearance of C-BP-6 in the presence of IGF-II. Nonetheless, current results strongly suggest possible conformation switching or population shifting between pre-existing conformations in C-BP-6 upon binding to IGF-II.     

Blood-gas,acid-base and catecholamine responses to lactic acid infusion in larval and adult Ambystoma tigrinum

Larval and adult Ambystoma tigrinum were subjected to acidosis by infusing lactic acid (2 M·g^-1) into the peritoneal cavity. Blood was sampled at intervals to establish the time-course of the ensuing acidosis. Both larvae and adults became significantly acidotic after 1 h. The larval acidosis was more pronounced (-4 pH units versus-2 pH units) than adults due to greater extracellular buffering capacity (higher [HCO₃ ^-]) in adults. Both forms recovered in about 8 h. Larvae showed a typical increase in circulating norepinephrine during the initial stages of the acidosis; adults did not, having significantly lower norepinephrine titer than larvae during the acidosis. Both larvae and adults showed transient increases in PO₂ during the acidosis. The ₁ and ₂ antagonists, timolol and butoxamine respectively, (0.2 g·g^-1) were administered to separate groups of larvae. Butoxamine (₂) delayed the recovery from the acidosis by prolonging the increase in arterial PCO₂ and reversing the recovery of [HCO₃ ^-]. Timolol (₁) did not delay recovery. We conclude that ₂ receptors are involved in the catecholamine responses to acidosis in larvae. Catecholamines appear not to play the same role in adult acid-base disturbances as they seem to in larvae.Abbreviations RBC red blood cell     

Acetylcholine-mediated relaxation in rat thoracic aorta is enhanced following acute exposure to physiological concentrations of 17β-estradiol

We investigated the effects of short-term exposure to low concentrations of 17-estradiol on vasorelaxation using an in vitro rat thoracic aortic ring preparation. Supraphysiological levels of 17-estradiol directly relaxed phenylephrine-contracted rings. Although acute incubation (20 min) with 1-100 nM of the female sex hormone did not have any significant effect on phenylephrine-contracted rings, relaxation evoked by acetylcholine was significantly potentiated. In contrast, calcium ionophore A23187-elicited endothelium-dependent relaxation as well as cromakalim- and sodium nitroprusside-mediated endothelium-independent relaxation was unchanged following the same regime with 17-estradiol. These results demonstrate that short-term treatment with physiologically relevant levels of 17-estradiol, which on their own have no effect, enhances endothelium-dependent relaxation by acetylcholine. The vascular effects observed herein may partly account for some of the improved acute vasodilatory responses reported with 17-estradiol on blood flow in humans.     

Solid state NMR and X-ray diffraction studies of alpha-D-galacturonic acid monohydrate

Crystalline alpha-D-galacturonic acid monohydrate has been studied by 13C CPMAS NMR and X-ray crystallography. The molecular dynamics were investigated by evaluating 13C spin-lattice relaxation in the rotating frame (T1rho) and chemical-shift-anisotropy properties of each carbon. Only limited molecular motions can be detected in the low frequency (< 10(4) Hz) range by 13C relaxation time measurements (T1rho) and changes of chemical shift anisotropy properties as a function of temperature. X-ray analysis (at both ambient temperature and 150 K) shows that the acid has the usual chair-shaped, pyranose ring conformation, and that the acid and water molecules are linked, through all their O-H groups, in an extensively hydrogen-bonded lattice.     

Imazalil-cyclomaltoheptaose (beta-cyclodextrin) inclusion complex: preparation by supercritical carbon dioxide and 13C CPMAS and 1H NMR characterization

An inclusion complex between imazalil (IMZ), a selected fungicide, and cyclomaltoheptaose (beta-cyclodextrin, betaCD) was obtained using supercritical fluid carbon dioxide. The best preparation conditions were determined, and the inclusion complex was investigated by means of 1H NMR spectroscopy in aqueous solution and 13C CPMAS NMR spectroscopy in the solid state. Information on the geometry of the betaCD/IMZ complex was obtained from ROESY spectroscopy, while the dynamics of the inclusion complex in the kilohertz range was obtained from the proton spin-lattice relaxation times in the rotating frame, T(1rho) (1H).     

Two-dimensional measurement of proton T1rho relaxation in unlabeled proteins: mobility changes in alpha-bungarotoxin upon binding of an acetylcholine receptor peptide

A method for the measurement of proton T(1)(rho) relaxation times in unlabeled proteins is described using a variable spin-lock pulse after the initial nonselective 90 degrees excitation in a HOHAHA pulse sequence. The experiment is applied to alpha-bungarotoxin (alpha-BTX) and its complex with a 25-residue peptide derived from the acetylcholine receptor (AChR) alpha-subunit. A good correlation between high T(1)(rho) values and increased local motion is revealed. In the free form, toxin residues associated with receptor binding according to the NMR structure of the alpha-BTX complex with an AChR peptide and the model for alpha-BTX with the AChR [Samson, A. O., et al. (2002) Neuron 35, 319-332] display high mobility. When the AChR peptide binds, a decrease in the relaxation times and the level of motion of residues involved in binding of the receptor alpha-subunit is exhibited, while residues implicated in binding gamma- and delta-subunits retain their mobility. In addition, the quantitative T(1)(rho) measurements enable us to corroborate the mapping of boundaries of the AChR determinant strongly interacting with the toxin [Samson, A. O., et al. (2001) Biochemistry 40, 5464-5473] and can similarly be applied to other protein complexes in which peptides represent one of the two interacting proteins. The presented method is advantageous because of its simplicity, generality, and time efficiency and paves the way for future investigation of proton relaxation rates in small unlabeled proteins.     

Determination of 3J(H infi supN,C infi sup′ ) coupling constants in proteins with the C′-FIDS method

Summary We introduce the C-FIDS-¹H,¹⁵N-HSQC experiment, a new method for the determination of ³J(H _infi ^supN ,C _infi ^sup ) coupling constants in proteins, yielding information about the torsional angle . It relies on the ¹H,¹⁵N-HSQC or HNCO experiment, two of the the most sensitive heteronuclear correlation experiments for isotopically labeled proteins. A set of three ¹H,¹⁵N-HSQC or HNCO spectra are recorded: a reference experiment in which the carbonyl spins are decoupled during t₁ and t₂, a second experiment in which they are decoupled exclusively during t₁ and a third one in which they are coupled in t₁ as well as t₂. The last experiment yields an E.COSY-type pattern from which the ²J(H _infi ^supN ,C _infi-1 ^sup ) and ¹J(N_i,C _infi-1 ^sup ) coupling constants can be extracted. By comparison of the coupled multiplet (obtained from the second experiment) with the decoupled multiplet (obtained from the first experiment) convoluted with the ²J(H _infi ^supN ,C _infi-1 ^sup ) coupling, the ³J(H _infi ^supN ,C _infi ^sup ) coupling can be found in a one-parameter fitting procedure. The method is demonstrated for the protein rhodniin, containing 103 amino acids. Systematic errors due to differential relaxation are small for ⁿJ(H^N,C) couplings in biomacromolecules of the size currently under NMR spectroscopic investigation.     

Delayed fluorescence from the photosynthetic reaction center measured by electronic gating of the photomultiplier

The decay of the delayed fluorescence (920 nm) of reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides R26 in the P(+)Q(A)(-) charge-separated state (P and Q(A) are the primary donor and quinone, respectively) has been monitored in a wide (100 ns to 100 ms) time range. The photomultiplier (Hamamatsu R3310-03) was protected from the intense prompt fluorescence by application of gating potential pulses (-280 V) to the first, third, and fifth dynodes during the laser pulse. The gain of the photomultiplier dropped transiently by a factor of 1 x 10(6). The delayed fluorescence showed a smooth but nonexponential decay from 100 ns to 1 ms that was explained by the relaxation of the average free energy between P* and P(+)Q(A)(-) changing from -580 to -910 meV. This relaxation is due to the slow protein response to charge separation and can be described by a Kohlrausch relaxation function with time constant of 65 micros and a stretching exponent of alpha = 0.45.     

NMR (13)C-relaxation study of base and sugar dynamics in GCAA RNA hairpin tetraloop

Intramolecular dynamics of a 14-mer RNA hairpin including GCAA tetraloop was investigated by (13)C NMR relaxation. R(1) and R(1rho) relaxation rates were measured for all protonated base carbons as well as for C1' carbons of ribose sugars at several magnetic field strengths. The data has been interpreted in the framework of modelfree analysis [G. Lipari and A. Szabo. J Am Chem Soc 104, 4546-4559 (1982); G. Lipari and A. Szabo. J Am Chem Soc 104, 4559-4570 (1982)] characterizing the internal dynamics of the molecule by order parameters and correlation times for fast motions on picosecond to nanosecond time scale and by contributions of the chemical exchange. The fast dynamics reveals a rather rigid stem and a significantly more flexible loop. The cytosine and the last adenine bases in the loop as well as all the loop sugars exhibit a significant contribution of conformational equilibrium on microsecond to millisecond time scale. The high R(1rho) values detected on both base and sugar moieties of the loop indicate coordinated motions in this region. A semiquantitative analysis of the conformational equilibrium suggests the exchange rates on the order of 10(4) s(-1). The results are in general agreement with dynamics studies of GAAA loops by NMR relaxation and fluorescent spectroscopy and support the data on the GCAA loop dynamics obtained by MD simulations.     

C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1

Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.     

Low-frequency motion in membranes. The effect of cholesterol and proteins

Nuclear magnetic resonance (NMR) relaxation techniques have been used to study the effect of lipid-protein interactions on the dynamics of membrane lipids. Proton enhanced (PE) 13C-NMR measurements are reported for the methylene chain resonances in red blood cell membranes and their lipid extracts. For comparison similar measurements have been made of phospholipid dispersions containing cholesterol and the polypeptide gramicidin A+. It is found that the spin-lattice relaxation time in the rotating reference frame (T1 rho) is far more sensitive to protein, gramicidin A+ or cholesterol content than is the laboratory frame relaxation time (T1). Based on this data it is concluded that the addition of the second component to a lipid bilayer produces a low-frequency motion in the region of 10(5) to 10(7) Hz within the membrane lipid. The T1 rho for the superimposed resonance peaks derived from all parts of the phospholipid chain are all influenced in the same manner suggesting that the low frequency motion involves collective movements of large segments of the hydrocarbon chain. Because of the molecular co-operativity implied in this type of motion and the greater sensitivity of T1 rho to the effects of lipid-protein interactions generally, it is proposed that these low-frequency perturbations are felt at a greater distance from the protein than those at higher frequencies which dominate T1.