Publications by authors named "Wand A"

Selective incorporation of 13C into the methyl groups of protein side chains is described as a means for simplifying the measurement and interpretation of 13C relaxation parameters. High incorporation (> 90%) is accomplished by using pyruvate (3-13C, 99%) as the sole carbon source in the growth media for protein overexpression in E. coli.

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A maleimide nitroxide spin-label (MAL-6) linked to a cysteine in the hydrophobic core and a coproporphyrin I (CP) appended on the N-terminus of a synthetic helix-loop-helix peptide ([alpha2]) have been used to examine the designed self-association of a four-helix bundle ([alpha2]2), focusing on the bundle topology and stability and the rotational dynamics of the spin-label. Gel-permeation chromatography demonstrated that the [alpha2] peptide and the peptide modified with a spin-label ([MAL-6-alpha2]), a coproporphyrin ([CP-alpha2]) and a coproporphyrin plus a spin-label ([CP-MAL-6-alpha2]) self-associate into four helix bundles in solution as designed. Circular dichroism (CD) spectra prove that all these peptides are highly alpha-helical, confirmed for [alpha2]2 by Fourier transform infrared (FTIR) spectroscopic analysis.

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Proper positioning and assessment of abnormalities and complications of the above-mentioned devices have a significant impact on the management of critically ill patients in the intensive care unit (ICU). The timely assessment of new or rapidly evolving findings is critical. Optimal radiographic technique, availability of images to the clinicians, and rapid reporting by the radiologist all serve to maximize the efficacy of bedside chest radiography in the ICU.

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A simple model is used to illustrate the relationship between the dynamics measured by NMR relaxation methods and the local residual entropy of proteins. The expected local dynamic behavior of well-packed extended amino acid side chains are described by employing a one-dimensional vibrator that encapsulates both the spatial and temporal character of the motion. This model is then related to entropy and to the generalized order parameter of the popular "model-free" treatment often used in the analysis of NMR relaxation data.

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A model for the solution structure of horse heart ferricytochrome c has been determined by nuclear magnetic resonance spectroscopy combined with hybrid distance geometry-simulated annealing calculations. Forty-four highly refined structures were obtained using a total of 1671 distance constraints based on the observed magnitude of nuclear Overhauser effects and 58 torsion angle restrains based on the magnitude of determined J-coupling constants. The model incorporates six long-lived water molecules detected by pseudo-two-dimensional NOESY-TOCSY spectra.

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Recent advances in NMR methods now allow protein complexes to be studied in great detail in a wide range of solution conditions. Isotope-enrichment strategies, resonance-assignment approaches and structural-determination methods have evolved to the point where almost any type of complex involving proteins of reasonable size may be studied in a straightforward way. A variety of isotope editing and filtering strategies underlie these powerful methodologies.

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The use of random, fractional 13C-enrichment combined with low pass filtration has allowed the determination of NMR relaxation parameters at an unprecedented number of sites within recombinant human ubiquitin. Essentially complete 1H, 13C, and 15N resonance assignments for the protein are reported. Carbon spin lattice and heteronuclear NOE relaxation data have been analyzed in the context of the Lipari-Szabo "model free" formalism.

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The interaction of apocalmodulin (apoCaM) with a peptide (Neurop) based on the primary sequence of the calmodulin-binding domain of neuromodulin has been studied by fluorescence spectroscopy. The 1:1 complex (12 microM) formed between apoCaM and the Neurop peptide is extremely sensitive to salt and is half dissociated in less than 0.1 M KCl, suggesting that electrostatic interactions contribute strongly to complex formation.

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Purpose: To investigate the anatomic basis for the juxtaphrenic peak (JP) in upper and/or middle lobe volume loss through radiographic and computed tomographic (CT) correlation.

Materials And Methods: Chest radiographs and CT scans were reviewed in 32 patients with upper or middle lobe volume loss. The study included 33 cases of volume loss: 12 affected the left upper lobe; 12, the right upper lobe; five, the right upper and middle lobes; and four, the middle lobe.

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In summary, the chest radiograph has only moderate accuracy in visualizing opacification caused by cardiopulmonary abnormalities and may be quite nonspecific as to etiology, whereas it has high diagnostic accuracy for detecting malpositioning of tubes and lines. While focal parenchymal abnormalities are usually visualized on chest radiographs, identification of concomitant abnormalities when ARDS or PE already exist is more difficult. Atelectasis, aspiration, pneumonia, pulmonary hemorrhage, pulmonary thromboembolism, atypical cardiogenic edema, asymmetric ARDS, and neoplasms may be indistinguishable.

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The interaction of apocalmodulin (apoCaM) with a peptide (Neurop) based on the primary sequence of the calmodulin-binding domain of neuromodulin has been studied by nuclear magnetic resonance (NMR) methods. The NMR spectra of both apocalmodulin and its 1:1 complex with the Neurop peptide have been assigned by triple resonance and nuclear Overhauser effect-(NOE-) based strategies. ApoCaM displays many of the same basic structural features as calcium-saturated calmodulin.

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Amide hydrogen exchange has been used to examine the structural dynamics and energetics of the interaction of a peptide corresponding to the calmodulin binding domain of smooth muscle myosin light chain kinase with calcium-saturated calmodulin. Heteronuclear NMR 15N-1H correlation techniques were used to quantitate amide proton exchange rates of both 15N-labeled and unlabeled amide protons of the smMLCK peptide complexed to calmodulin. Hydrogen exchange slowing factors were determined for 18 of the 19 amide hydrogens and found to span 6 orders of magnitude.

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Determination of the high resolution solution structure of a protein using nuclear magnetic resonance (NMR) spectroscopy requires that resonances observed in the NMR spectra be unequivocally assigned to individual nuclei of the protein. With the advent of modern, two-dimensional NMR techniques arose methodologies for assigning the 1H resonances based on 2D, homonuclear 1H NMR experiments. These include the sequential assignment strategy and the main chain directed strategy.

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The existence of structural water in the interior of both oxidized and reduced horse-heart cytochrome c in solution is demonstrated using nuclear magnetic resonance spectroscopy. Six water molecules have been located in ferrocytochrome c and five in ferricytochrome c, with residence times greater than a few hundred picoseconds. Two water molecules are located in the haem crevice, one of which is found to undergo a large change in position with a change of oxidation state.

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A model for the solution structure of horse heart ferrocytochrome c has been determined by nuclear magnetic resonance spectroscopy combined with hybrid distance geometry-simulated annealing calculations. Forty-four highly refined structures were obtained using a total of 1940 distance constraints based on the observed magnitude of nuclear Overhauser effects and 85 torsional angle restraints based on the magnitude of determined J-coupling constants. The all-residue root mean square deviation about the average structure is 0.

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Calmodulin binds to amphiphilic, helical peptides of a variety of amino-acid sequences. These peptides are usually positively charged, although there is spectroscopic evidence that at least one neutral peptide binds. The complex between calmodulin and one of its natural target peptides, the binding site for calmodulin on smooth muscle myosin light-chain kinase (RS20), has been investigated by crystallography and NMR which have characterized the interactions between the ligand and the protein.

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A water-soluble, 62-residue, di-alpha-helical peptide has been synthesized which accommodates two bis-histidyl haem groups. The peptide assembles into a four-helix dimer with 2-fold symmetry and four parallel haems that closely resemble native haems in their spectral and electrochemical properties, including haem-haem redox interaction. This protein is an essential intermediate in the synthesis of molecular 'maquettes', a novel class of simplified versions of the metalloproteins involved in redox catalysis and in energy conversion in respiratory and photosynthetic electron transfer.

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The introduction of rate matrix analysis to protocols for the refinement of solution structures of biopolymers on the basis of NMR-derived structural constraints has greatly enhanced the self-consistency of the general approach. However, current implementations of this strategy appear not to consider several issues arising from various commonly employed experimental conditions and sample characteristics which can prevent the quantitative interpretation of NOESY spectra of proteins. Here, a number of these effects are considered, including the influence of nonequilibrium populations generated by over-pulsing and solvent presaturation, the presence of heteronuclei, and the equilibration of exchangeable sites with deuterium in solvent.

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The apoprotein is an important intermediate on the folding pathways of many haem proteins, yet a detailed structure of such an intermediate has remained elusive. Here we present the structure of apocytochrome b562 obtained by NMR spectroscopy. The apoprotein has a topology similar to the holoprotein.

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The main chain dynamics of a peptide corresponding to the smooth muscle myosin light chain kinase calmodulin-binding domain bound to calcium-saturated calmodulin have been studied by 15N relaxation techniques. Laboratory and rotating-frame spin lattice relaxation times and nuclear Overhauser effects have been determined for nine amide 15N sites in the peptide using two-dimensional NMR spectroscopy. The global motion of the 1:1 complex is shown to be isotropic and is characterized by a correlation time of 10 ns rad-1.

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We examine the statistical and other considerations which determine the validity and reproducibility of stereospecific hydrogen NMR assignments obtained by the floating prochirality method. In this method, the assignment of a prochiral configuration of hydrogens at selected centers is allowed to 'float' during the structure refinement, and the distribution of prochiral orientations in highly refined structures is subjected to statistical analysis. The underlying statistical basis for this approach is examined and potential limitations of current approaches are identified.

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