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Biomedical subjects

I M Brereton

Publications and source records attributed to I M Brereton.

At least 19 recordsLinked to original sources
PubMed2001

NMR structure and backbone dynamics of a concatemer of epidermal growth factor homology modules of the human low-density lipoprotein receptor.

The ligand-binding region of the low-density lipoprotein (LDL) receptor is formed by seven N-terminal, imperfect, cysteine-rich (LB) modules. This segment is followed by an epidermal growth factor precursor homology domain with two N-terminal, tandem, EGF-like modules that are thought to participate in LDL binding and recycling of the endocytosed receptor to the cell surface. EGF-A and the concatemer, EGF-AB, of these modules were expressed in Escherichia coli. Correct protein folding of EGF-A and the concatemer EGF-AB was achieved in the presence or absence of calcium ions, in contrast to the LB modules, which require them for correct folding. Homonuclear and heteronuclear 1H-15N NMR spectroscopy at 17.6 T was used to determine the three-dimensional structure of the concatemer. Both modules are formed by two pairs of short, anti-parallel beta-strands. In the concatemer, these modules have a fixed relative orientation, stabilized by calcium ion-binding and hydrophobic interactions at the interface. 15N longitudinal and transverse relaxation rates, and [1H]-15N heteronuclear NOEs were used to derive a model-free description of the backbone dynamics of the molecule. The concatemer appears relatively rigid, particularly near the calcium ion-binding site at the module interface, with an average generalized order parameter of 0.85+/-0.11. Some mutations causing familial hypercholesterolemia may now be rationalized. Mutations of D41, D43 and E44 in the EGF-B calcium ion-binding region may affect the stability of the linker and thus the orientation of the tandem modules. The diminutive core also provides little structural stabilization, necessitating the presence of disulfide bonds. The structure and dynamics of EGF-AB contrast with the N-terminal LB modules, which require calcium ions both for folding to form the correct disulfide connectivities and for maintenance of the folded structure, and are connected by highly mobile linking peptides.

Amino Acid Sequence↗
PubMed2000

Three-dimensional NMR structure of the sixth ligand-binding module of the human LDL receptor: comparison of two adjacent modules with different ligand binding specificities.

The sixth ligand-binding module of the low-density lipoprotein receptor contributes to the binding of apolipoprotein B100-containing lipoproteins. 1H NMR spectroscopy, DYANA and X-PLOR structure calculations were used to determine that this module has a well defined structure with a backbone conformation similar to other modules. Structures from calculations that simulated the presence of a calcium ion showed increased resolution without large increases in energy, increased deviations from idealised geometry or violations of experimental constraints. Investigation of the surface properties of this module indicates there are significant differences from the fifth module, which binds apolipoprotein E-containing lipoproteins in addition to apolipoprotein B100-containing lipoproteins.

Algorithms↗
PubMed2000

NMR structure of a concatemer of the first and second ligand-binding modules of the human low-density lipoprotein receptor.

The ligand-binding domain of the human low-density lipoprotein receptor consists of seven modules, each of 40-45 residues. In the presence of calcium, these modules adopt a common polypeptide fold with three conserved disulfide bonds. A concatemer of the first and second modules (LB(1-2)) folds efficiently in the presence of calcium ions, forming the same disulfide connectivities as in the isolated modules. The three-dimensional structure of LB(1-2) has now been solved using two-dimensional 1H NMR spectroscopy and restrained molecular dynamics calculations. No intermodule nuclear Overhauser effects were observed, indicating the absence of persistent interaction between them. The near random-coil NH and H alpha chemical shifts and the low phi and psi angle order parameters of the four-residue linker suggest that it has considerable flexibility. The family of LB(1-2) structures superimposed well over LB1 or LB2, but not over both modules simultaneously. LB1 and LB2 have a similar pattern of calcium ligands, but the orientations of the indole rings of the tryptophan residues W23 and W66 differ, with the latter limiting solvent access to the calcium ion. From these studies, it appears that although most of the modules in the ligand-binding region of the receptor are joined by short segments, these linkers may impart considerable flexibility on this region.

Amino Acid Sequence↗
PubMed1999

NMR studies of the low-density lipoprotein receptor-binding peptide of apolipoprotein E bound to dodecylphosphocholine micelles.

Circular dichroism and NMR spectroscopy have been used to determine the structure of the low-density lipoprotein (LDL) receptor-binding peptide, comprising residues 130-152, of the human apolipoprotein E. This peptide has little persistent three-dimensional structure in solution, but when bound to micelles of dodecylphosphocholine (DPC) it adopts a predominantly alpha-helical structure. The three-dimensional structure of the DPC-bound peptide has been determined by using 1H-NMR spectroscopy: the structure derived from NOE-based distance constraints and restrained molecular dynamics is largely helical. The derived phi and psi angle order parameters show that the helical structure is well defined but with some flexibility that causes the structures not to be superimposable over the full peptide length. Deuterium exchange experiments suggest that many peptide amide groups are readily accessible to the solvent, but those associated with hydrophobic residues exchange more slowly, and this helix is thus likely to be positioned on the surface of the DPC micelles. In this conformation the peptide has one hydrophobic face and two that are rich in basic amino acid side chains. The solvent-exposed face of the peptide contains residues previously shown to be involved in binding to the LDL receptor.

Amino Acid Sequence↗
PubMed1999

Cortical and medullary betaine-GPC modulated by osmolality independently of oxygen in the intact kidney.

Renal osmolyte concentrations are reduced during reflow following ischemia. Osmolyte decreases may follow oxygen depletion or loss of extracellular osmolality in the medulla. Image-guided volume-localized magnetic resonance (MR) microspectroscopy was used to monitor regional osmolytes during hyposmotic shock and hypoxia in the intact rat kidney. Alternate spectra were acquired from 24-microl voxels in cortex and medulla of the isolated perfused kidney. There was a progressive decrease in the combined betaine-glycerophosphorylcholine (GPC) peak intensity of 21% in cortex and 35% in medulla of normoxic kidneys between 60 and 160 min after commencing perfusion. Hypoxia had no significant effect on the betaine-GPC peak intensity in cortex or medulla, despite a dramatic reduction in tubular sodium, potassium, and water reabsorption. The results suggest that cortical and medullary intracellular osmolyte concentrations depend on osmotically regulated channels that are insensitive to oxygen and dissociated from the oxygen-dependent parameters of renal function, the fractional excretion of sodium, the fractional excretion of potassium, and urine-to-plasma inulin concentration ratio.

Animals↗
PubMed1998

Calcium is essential for the structural integrity of the cysteine-rich, ligand-binding repeat of the low-density lipoprotein receptor.

Seven cysteine-rich repeats form the ligand-binding region of the low-density lipoprotein (LDL) receptor. Each of these repeats is assumed to bind a calcium ion, which is needed for association of the receptor with its ligands, LDL and beta-VLDL. The effects of metal ions on the folding of the reduced N-terminal cysteine-rich repeat have been examined by using reverse-phase high-performance liquid chromatography to follow the formation of fully oxidized isomers with different disulfide connectivities. In the absence of calcium many of the 15 possible isomers formed on oxidation, whereas in its presence the predominant product at equilibrium had the native disulfide bond connectivities. Other metals were far less effective at directing disulfide bond formation: Mn2+ partly mimicked the action of Ca2+, but Ba2+, Sr2+, and Mg2+ had little effect. This metal-ion specificity was also observed in two-dimensional 1H NMR spectral studies; only Ca2+ induced the native three-dimensional fold. The two paramagnetic ions, Gd3+ and Mn2+, and Cd2+ did not promote adoption of a well-defined structure, and the two paramagnetic ions did not displace calcium ions. The location of calcium ion binding sites in the repeat was also explored by NMR spectroscopy. The absence of chemical shift changes for the side chain proton resonances of Asp26, Asp36, and Glu37 from pH 3.9 to 6.8 in the presence of calcium ions and their proximal location in the NMR structures implicated these side chains as calcium ligands. Deuterium exchange NMR experiments also revealed a network of hydrogen bonds that stabilizes the putative calcium-binding loop.

Amino Acid Sequence↗
PubMed1997

Regional proton nuclear magnetic resonance spectroscopy differentiates cortex and medulla in the isolated perfused rat kidney.

Volume-localized proton nuclear magnetic resonance spectroscopy was used as an assay of regional biochemistry in the isolated perfused rat kidney. This model eliminated artifacts caused by respiratory and cardiac motion experienced in vivo. Immersion of the kidney under its venous effluent reduced the susceptibility artifacts evoked by tissue-air interfaces. The rapid acquisition with relaxation enhancement imaging sequence was used for scout imaging. This gave excellent spatial resolution of the cortex, outer medulla, and inner medulla. Spectra were then acquired in 10 minutes using the volume-selective multipulse spectroscopy sequence from voxels with a volume of approximately 24 microL located within the cortical or medullary regions. Spectral peaks were assigned by the addition of known compounds to the perfusion medium and by comparison with spectra of protein-free extracts of cortex and medulla. The medullary region spectra were characterized by signals from the osmolytes betaine, glycerophosphorylcholine, and inositol. The spectra from the cortex were more complex and contained lesser contributions from osmolytes.

Animals↗
PubMed1996

Localized 1H NMR spectroscopy of rat spinal cord in vivo.

A movable, actively decoupled surface coil has been employed to obtain a localized 1H NMR spectrum from the lumbosacral spinal cord of a live Lewis rat. A volume selective 'VOSY' normally spelled out as 'volume selective spectroscopy' spectroscopy pulse sequence that incorporates 'phase ramped' selective RF pulses, has been used to minimize random phase jitter in the NMR signal as a result of the large frequency shifts required to locate the voxel in the center of the cord while using intense gradient pulses. Spectra from 13-microliters voxels in healthy rats and in rats inoculated with guinea pig spinal cord and complete Freund's adjuvant, resulting in experimental autoimmune encephalomyelitis, are shown.

Animals↗
PubMed1996

Ionization states of the catalytic residues in HIV-1 protease.

Chemical synthesis was used to prepare the HIV-1 protease specifically 13C-labelled in the catalytically essential Asp 25 in each monomer. The NMR chemical shift of the 13C-enriched homodimeric enzyme was measured in the presence of the inhibitor pepstatin, a mimic of the tetrahedral intermediate formed in enzyme catalysis. In this complex, the catalytic carboxyls do not titrate in the pH range where the enzyme is active; throughout the range pH 2.5-6.5, one Asp 25 side chain is protonated and the other deprotonated. By contrast, in the absence of inhibitor the two Asp side chains are chemically equivalent and both deprotonated at pH6, the optimum for enzymatic activity. These direct observations of the chemical properties of the catalytic apparatus of the enzyme provide concrete information on which to base the design of improved HIV-1 protease inhibitors.

Aspartic Acid↗
PubMed1994

Localized two-dimensional shift correlated spectroscopy in humans at 2 Tesla.

A method for the acquisition of localized 2D shift-correlated spectra, based on the combination of the stimulated-echo volume-selection and gradient-enhanced COSY experiments, is described. The sequence can be modified to perform a number of localized experiments including HOHAHA and DQF-COSY. The method is demonstrated in vivo by presentation of localized COSY and HOHAHA spectra of human tibia marrow, and a localized COSY spectrum of human brain acquired at a field strength of 2 Tesla. Cross peaks corresponding to correlations between coupled groups along the acyl chains of triglycerides are observed in the spectra of marrow. The major cerebral metabolites are represented in the in vivo COSY brain spectrum, including N-acetylaspartate, glutamate/glutamine, total creatine, aspartate, and myo-inositol. Difficulties in the implementation of localized shift-correlation spectroscopy, including water suppression and T2 relaxation, are discussed.

Bone Marrow↗
PubMed1993

Ciguatoxin-2 is a diastereomer of ciguatoxin-3.

Ciguatoxin-2, a major ciguatoxin present in the flesh and viscera of ciguateric fishes, has been shown by 1H nuclear magnetic resonance studies (2-dimensional homonuclear Hartman Hahn, nuclear Overhauser effect and decoupling difference experiments) to be a diastereomer of ciguatoxin-3, differing only in stereochemistry at carbon 52 (a quaternary carbon). This difference accounts for the significant changes in the chemical shift of resonances for protons in this region of ciguatoxin-2. Differences between ciguatoxin-1, -2 and -3 involve modifications at only one end of the ciguatoxins (ring M) and modest differences in potency, indicating that this ring contributes to, but is not critical for, high affinity binding of the ciguatoxins to voltage-dependent sodium channels. It is proposed that ciguatoxin-2 originates from a different precursor to the precursor (presumably gambiertoxin-4b) for ciguatoxin-1 and -3, and that both precursors are produced by a common biosynthetic pathway in Gambierdiscus toxicus.

Base Sequence↗
PubMed1993

Respiratory triggered imaging with an optical displacement sensor.

Motion of abdominal organs with respiration is a major problem in NMR spectroscopy and imaging thereof. Triggering each phase-encoding step with respiration or gating a number of phase-encoding steps is one approach to the problem. The design of a sensor for small animal experiments has not been as simple. An optical device, implemented with polymer optical fibres is described, along with associated hardware and electronics which can act as a trigger for small animal NMR experiments. A brief description of a similar device for human application is also given. 2DFT spin-echo and B0 susceptibility images, both triggered and untriggered, are presented to validate the technique.

Abdomen↗
PubMed1992

Measurement of the T2 relaxation time of ethanol and cerebral metabolites, in vivo.

The SPACE volume selection technique was combined with a spin-echo sequence to measure the transverse relaxation time of the resonances of ethanol and cerebral metabolites in the dog brain, in vivo. The method was extended to measure brain metabolite T2 values in the rat using 1H NMR microspectroscopy. The T2 decays for the resonances of the metabolites N-acetylaspartate, creatine/phosphocreatine, and choline/phosphorylcholine were found to be biexponential with long T2 components of 490, 260, and 350 ms for the dog and 490, 220, and 355 ms for the rat brain, respectively. The existence of a second T2 component may originate from J-coupled nonresolved metabolite resonances. The relaxation decay for the ethanol triplet could be fitted to a single exponential giving a T2 relaxation time of 335 ms. However, given the large errors in the measurement of ethanol peak intensities at short echo times because of overlapping lipid signal and the effects of J-modulation, a biexponential decay with a long T2 component of 335 ms cannot be ruled out. Ambiguities regarding the reported partial detection of the 1H NMR signal of ethanol in the brain are discussed.

Animals↗
PubMed1992

Application of self-refocusing band selective RF pulses for spectroscopic localization.

A new self-refocusing slice selection 90 degrees pulse is presented and its incorporation in the SPACE localization sequence described. Experimental comparisons are made with the self-refocusing pulse reported by Geen (H. Geen, S. Wimperis and R. Freeman, J. Magn. Reson. 85, 620 (1990)). The main source of localization error in the SPACE sequence is traced to the hard pi/2 pulse and the development of a shaped-pulse version of the sequence is described. This required the calculation of a slice-selective pulse capable of rotating coherent transverse magnetization to the z-axis. The RF power requirements for these experiments are also discussed.

Magnetic Resonance Spectroscopy↗
PubMed1991

The visibility of the 1H NMR signal of ethanol in the dog brain.

In vivo, high-resolution, volume-selected 1H NMR spectroscopy was used to monitor the concentration of ethanol in the dog brain following intravenous injection of ethanol. Equilibration of ethanol in the body water should result in approximately equivalent concentrations of ethanol in the blood and brain. However, the mean equilibrium brain ethanol concentration determined using N-acetylaspartate as an internal standard was only 23 +/- 5% of the blood ethanol concentration. The disparity between blood and brain ethanol concentrations was attributed to underestimation of the ethanol concentration due to overlapping resonances with NAA and to T2 attenuation or possible nondetection of the 1H signal from ethanol bound at the surface of cell membranes and partitioned into the hydrophobic core of membrane lipids.

Animals↗
PubMed1990

High-field localized in vivo proton spectroscopy on micro volumes.

A water-suppressed volume-selected in vivo 1H spectrum of 0.2 ml of a rat brain has been obtained at 200 MHz using the SPACE localization method. Good signal-to-noise and spectral resolution were obtained by averaging 256 acquisitions. The spectrum shows little T2 weighting effect.

Animals↗