Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible.

The backbone dynamics of Ca(2+)-saturated recombinant Drosophila calmodulin has been studied by 15N longitudinal and transverse relaxation experiments, combined with 15N(1H) NOE measurements. Results indicate a high degree of mobility near the middle of the central helix of calmodulin, from residue K77 through S81, with order parameters (S2) in the 0.5-0.

Dynamics of methyl groups in proteins as studied by proton-detected 13C NMR spectroscopy. Application to the leucine residues of staphylococcal nuclease.

This paper describes the application of recently developed nuclear magnetic resonance (NMR) pulse sequences to obtain information about the internal dynamics of isotopically enriched hydrophobic side chains in proteins. The two-dimensional spectra provided by the pulse sequences enable one to make accurate measurements of nuclear Overhauser effects (NOE) and longitudinal (T1) and transverse (T2) relaxation times of enriched methyl carbons in proteins. Herein, these techniques are used to investigate the internal dynamics of the 11 leucine side chains of staphylococcal nuclease (SNase), a small enzyme having Mr = 16.

The role of the flow rate of the pneumohydraulic system on post-sphincter of Oddi manometry pancreatitis.

Manometry of sphincter of Oddi (SO) carries a risk of acute pancreatitis by a mechanism not yet clearly understood. This study attempted to evaluate the role of the flow rate of the perfusion system in the development of acute pancreatitis. During the past 60 months, we have performed 81 manometry studies of SO in 79 patients, 61 women and 18 men, who were referred for recurrent attacks of abdominal pain suggestive of SO dysfunction.

Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy.

Heteronuclear 2D and 3D NMR experiments were carried out on recombinant Drosophila calmodulin (CaM), a protein of 148 residues and with molecular mass of 16.7 kDa, that is uniformly labeled with 15N and 13C to a level of greater than 95%. Nearly complete 1H and 13C side-chain assignments for all amino acid residues are obtained by using the 3D HCCH-COSY and HCCH-TOCSY experiments that rely on large heteronuclear one-bond scalar couplings to transfer magnetization and establish through-bond connectivities.

Improved three-dimensional 1H-13C-1H correlation spectroscopy of a 13C-labeled protein using constant-time evolution.

An improved version of the three-dimensional HCCH-COSY NMR experiment is described that correlates the resonances of geminal and vicinal proton pairs with the chemical shift of the 13C nucleus attached to one of the protons. The experiment uses constant-time evolution of transverse 13C magnetization which optimizes transfer of magnetization and thus improves the sensitivity of the experiment over the original scheme. The experiment is demonstrated for calmodulin complexed with a 26-residue peptide comprising the binding site of skeletal muscle myosin light chain kinase.

[Recovery after ambulatory vasectomy].

The recovery after outpatient vasectomy, with particular attention to general postoperative complaints, was investigated by means of a questionnaire. A material of 115 patients who were submitted to operation during the period 1.1.

Triple-resonance multidimensional NMR study of calmodulin complexed with the binding domain of skeletal muscle myosin light-chain kinase: indication of a conformational change in the central helix.

Heteronuclear 3D and 4D NMR experiments have been used to obtain 1H, 13C, and 15N backbone chemical shift assignments in Ca(2+)-loaded calmodulin complexed with a 26-residue synthetic peptide (M13) corresponding to the calmodulin-binding domain (residues 577-602) of rabbit skeletal muscle myosin light-chain kinase. Comparison of the chemical shift values with those observed in peptide-free calmodulin [Ikura, M., Kay, L.

Solution structure of a polypeptide dimer comprising the fourth Ca(2+)-binding site of troponin C by nuclear magnetic resonance spectroscopy.

The structure of a 39 amino acid proteolytic fragment of rabbit skeletal troponin C containing the fourth Ca(2+)-binding site has been determined by an approach involving nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations. Hydrodynamic and NMR evidence establishes unambiguously that the fragment forms a stable dimer in solution in the presence of excess Ca2+. The calculation of the dimeric structure is based on a total of 1056 experimental restraints comprising 422 interproton distances, 35 phi, 28 psi, and 28 chi 1 torsion angle restraints within each subunit, 30 intermonomer distance restraints, and 6 Ca2+ restraints per subunit.

Nonrecirculating hydroponic system suitable for uptake studies at very low nutrient concentrations.

We describe the mechanical, electronic, hydraulic, and structural design of a nonrecirculating hydroponic system. The system is particularly suited to studies at very low nutrient concentrations, for which on-line concentration monitoring methods either do not exist or are costly and limited to monitoring relatively few individual plants. Solutions are mixed automatically to chosen concentrations, which can be set differently for every pump fed from a master supply of deionized water and nutrient concentrates.

Four-dimensional 13C/13C-edited nuclear Overhauser enhancement spectroscopy of a protein in solution: application to interleukin 1 beta.

A four-dimensional 13C/13C-edited NOESY experiment is described which dramatically improves the resolution of protein NMR spectra and enables the straightforward assignment of nuclear Overhauser effects involving aliphatic and/or aromatic protons in larger proteins. The experiment is demonstrated for uniformly (greater than 95%) 13C-labeled interleukin 1 beta, a protein of 153 residues and 17.4 kDa, which plays a key role in the immune response.

Multidimensional triple resonance NMR spectroscopy of isotopically uniformly enriched proteins: a powerful new strategy for structure determination.

A procedure is described that affords complete 1H, 13C and 15N resonance assignment in proteins of up to about 25 kDa. The new approach requires uniform isotopic enrichment of the protein with 13C and 15N and correlates resonances of adjacent nuclei using the relatively large and well-resolved one-bond J couplings. Spectral overlap, a common problem in the application of two-dimensional NMR, is removed by increasing the dimensionality of the new methods to three or four, without increasing the number of observed resonances.

Four-dimensional heteronuclear triple-resonance NMR spectroscopy of interleukin-1 beta in solution.

A method is presented that dramatically improves the resolution of protein nuclear magnetic resonance (NMR) spectra by increasing their dimensionality to four. The power of this technique is demonstrated by the application of four-dimensional carbon-13--nitrogen-15 (13C-15N)--edited nuclear Overhauser effect (NOE) spectroscopy to interleukin-1 beta, a protein of 153 residues. The NOEs between NH and aliphatic protons are first spread out into a third dimension by the 15N chemical shift of the amide 15N atom and subsequently into a fourth dimension by the 13C chemical shift of the directly bonded 13C atoms.

Heteronuclear 3D NMR and isotopic labeling of calmodulin. Towards the complete assignment of the 1H NMR spectrum.

New methods are described that permit detailed analysis of the NMR spectra of calmodulin, an alpha-helical protein with a molecular weight of 16.7 kD. Two complementary approaches have been used: uniform labeling with 15N and labeling of specific amino acids with either 15N or 13C.

A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin.

A novel approach is described for obtaining sequential assignment of the backbone 1H, 13C, and 15N resonances of larger proteins. The approach is demonstrated for the protein calmodulin (16.7 kDa), uniformly (approximately 95%) labeled with 15N and 13C.

Spontaneous intra-uterine total rupture of a velamentous umbilical cord; a case report.

A case of spontaneous intra-uterine total rupture of a velamentous umbilical cord is reported. Shortly after delivery of an exsanguinated stillborn, a completely separated cord was spontaneously delivered. At its insertion the umbilical cord was velamentous, and it consisted of only three vessels at the site of rupture.

The epidemiology of gallstones in a 70-year-old Danish population.

A random sample of 540 men and women aged 70 years who lived in the western part of Copenhagen County was drawn from the National Central Person Register. The response rate was 74.8% among men and 63.

Studies on the solution conformation of human thioredoxin using heteronuclear 15N-1H nuclear magnetic resonance spectroscopy.

The solution conformation of uniformly labeled 15N human thioredoxin has been studied by two-dimensional heteronuclear 15N-1H nuclear magnetic resonance spectroscopy. Assignments of the 15N resonances of the protein are obtained in a sequential manner using heteronuclear multiple quantum coherence (HMQC), relayed HMQC-correlated (COSY), and relayed HMQC-nuclear Overhauser (NOESY) spectroscopy. Values of the 3JHN alpha splittings for 87 of the 105 residues of thioredoxin are extracted from a variant of the HMQC-COSY experiment, known as HMQC-J, and analyzed to give accurate 3JHN alpha coupling constants.

Systematic pre-deposit autologous blood provision for elective surgery: an important contribution to hospital blood supply.

From the 8th of September 1987 to the 31st of July 1988 all patients scheduled for major elective orthopaedic surgery were systematically offered the facility of pre-deposit autologous blood storage. Of 251 patients, 204 (81%) were eligible to pre-donate, and of these only 9 declined to do so. 72% of the participants required only autologous blood at operation, 23% required some additional homologous blood.

Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease.

This paper describes the use of novel two-dimensional nuclear magnetic resonance (NMR) pulse sequences to provide insight into protein dynamics. The sequences developed permit the measurement of the relaxation properties of individual nuclei in macromolecules, thereby providing a powerful experimental approach to the study of local protein mobility. For isotopically labeled macromolecules, the sequences enable measurements of heteronuclear nuclear Overhauser effects (NOE) and spin-lattice (T1) and spin-spin (T2) 15N or 13C relaxation times with a sensitivity similar to those of many homonuclear 1H experiments.

Methods of autologous blood transfusion.

Fear of AIDS has been a major factor in the re-examination of the methods of autologous blood transfusion. Four techniques are currently available, one of which, the pre-operative donation scheme, has supplied 70 per cent of participants' total blood requirements at operation.

Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta.

The application of three-dimensional (3D) heteronuclear NMR spectroscopy to the sequential assignment of the 1H NMR spectra of larger proteins is presented, using uniformly labeled (approximately 95%) [15N]interleukin 1 beta, a protein of 153 residues and molecular mass of 17.4 kDa, as an example. The two-dimensional (2D) 600-MHz spectra of interleukin 1 beta are too complex for complete analysis, owing to extensive cross-peak overlap and chemical shift degeneracy.