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Biomedical subjects

A Watts

Publications and source records attributed to A Watts.

229 records · Page 13Linked to original sources
PubMedDate not supplied

Bacteriorhodopsin: the mechanism of 2D-array formation and the structure of retinal in the protein.

Bacteriorhodopsin, the light driven proton pump of the extreme halophilic bacterium H. salinarium, is an integral membrane protein (M(r) ca. 26000) which forms 2D arrays in the purple membrane of the bacterium. It is this feature which has permitted the use of electron diffraction methods to resolve the protein structure to some degree of atomic detail, although the prosthetic group has not been fully resolved. However, the features which induce the protein to form these arrays have not been previously clarified. We have now shown that the protein array formation is driven by specific interaction of the protein with the charged phospholipid, phosphatidyl glycerol phosphate (or the sulphate derivative), a major (ca. 60%) lipid of the bacterial host membrane. In addition, in an effort to provide further structural information about the chromophore, retinal, of this protein, the orientation of the individual methyl groups of retinal have been determined from solid state deuterium NMR studies of the deuterated chromophore when in the protein binding site. This approach to structural resolution of the prosthetic group is ab initio, agrees with other studies on the chromophore and resolves new features of the bound retinal to a high degree (+/- 2 degrees) of precision. Here, these two studies on this integral membrane protein will be reviewed.

Bacteriorhodopsins↗
PubMedDate not supplied

Bacteriorhodopsin: the effect of bilayer thickness on 2D-array formation, and the structural re-alignment of retinal through the photocycle.

From our earlier extensive protein-lipid reconstitution studies, the conditions under which bacteriorhodopsin forms organised 2D arrays in large unilamellar vesicles have been established using freeze-fracture electron microscopy. In a background bilayer matrix of phosphatidylcholine (diC(14:0)), the protein can form arrays only when the anionic purple membrane lipid, phosphatidylglycerol phosphate (or the sulphate derivative) is present. Here we have now extended this work to investigate the effect of bilayer thickness on array formation. Phosphatidylcholines with various chain lengths (diC(12:0), diC(14:0) and diC(16:0)) and which form bilayers of well defined bilayer thickness, have been used as the matrix into which bacteriorhodopsin, together with minimal levels (c. 4-10 lipids per bacteriorhodopsin) of diphytanyl phosphatidyl-glycerol phosphate, has been reconstituted. Arrays are formed in all complexes and bhickness appears only to alter the type of array formed, either as an orthogonal or as an hexagonal array. Secondly, we have previously deduced the entire conformation of retinal within the bacteriorhodopsin binding pocket in oriented purple membrane fragments. Using solid state deuterium NMR of the specifically deutero-methylated retinal labelled at each of the methyl positions in the molecule, the C-CD(3) bond vectors of the chromophore have been resolved to +/- 2 degrees . The ring conformation is 6-S-trans, but the polyene chain is slightly curved when in the protein binding site. Here, we describe studies on the protein in both the ground state and the trapped M(412)-state of the photocycle, to show that the orientation of the central methyl group (C(19)) on the polyene chain, which is at 40 degrees +/- 1 degrees with respect to the membrane normal, only changes its orientation by approximately 4 degrees upon 13-cis-isomerization. Thus, it is the Schiff base end of the chromophore which moves upon light incidence acting as a local switch on the protein in the photocycle, whilst the ring end of the chromophore moves rather less.

Journal Article↗
PubMedDate not supplied

A review of oxygen-17 solid-state NMR of organic materials--towards biological applications.

17O solid state NMR of organic materials is developing rapidly. This article provides a snapshot of the current state of development of this field. The NMR techniques and enrichment protocols that are driving this progress are outlined. The (17)O parameters derived from solid-state NMR experiments are summarized and the structural sensitivity of the approach to effects such as hydrogen bonding highlighted. The prospects and challenges for (17)O solid-state NMR of biomolecules are discussed.

Chemistry, Organic↗
PubMedDate not supplied

Therapeutic agents utilized in urban/rural prehospital care.

INTRODUCTION: Objectives of this study were to determine the number of prehospital emergency patients who were given advanced life support (ALS) drugs and to compare utilization rates for ALS drugs in urban and rural environments. Certified ALS emergency medical technicians (Arizona) have 29 therapeutic agents authorized for prehospital administration. These agents may be administered only under direction of a medical control authority or by following standing orders. METHODS: A retrospective review was made of prehospital emergency encounter records. They were acquired by the Arizona Office of Emergency Medical Services (OEMS) from rural EMS providers who used optically scannable forms and from a metropolitan fire department's medical emergency response records. RESULTS: In 1989 and 1990, 273,611 emergency patient encounter records were entered into the EMS database; 197,260 were urban responses and 76,351 were rural responses. Drugs (ALS) were administered to 16,730 (8.5%) urban emergency patients and to 5,359 (7%) rural emergency patients at the incident site or during transport to a medical care facility. Nitrostat, 0.4 mg sublingual tablet, was the drug most frequently administered to emergency patients in the prehospital setting. Utilization rates found in the urban and the rural data sets were consistent for the individual agents. Variations in use frequency between urban and rural setting were noted for some drugs. Of the 29 approved ALS drugs, seven (24%) were administered to 10% or more urban patients who received drugs. In the rural areas, eight (27.6%) were administered to 10% or more patients who received drugs. There were nine (31%) agents administered to less than 1% of all patients who received drugs. A majority of the approved drugs, 17 (59%) were administered at a rate below 5% of all patients receiving medications. CONCLUSION: Severity of illness or injury prompted administration of ALS drugs to 8.1% of patients receiving prehospital emergency care. The most frequently utilized medication in the urban/rural areas was for treatment of cardiac symptoms. Variations between urban/rural drug utilization reflected the drugs of choice which are compatible with long transport times to a medical facility.

Arizona↗
PubMedDate not supplied

Structural investigations of pneumolysin/lipid complexes.

Pneumolysin, a virulence factor from the human pathogen Streptococcus pneumoniae, is a water-soluble protein which forms ring-shaped oligomeric structures upon binding to cholesterol-containing lipid membranes. It induces vesicle aggregation, membrane pore formation and withdrawal of lipid material into non-bilayer proteolipid complexes. Solid-state magic angle spinning and wideline static NMR, together with freeze-fracture electron microscopy, are used to characterize the phase changes in fully hydrated cholesterol-containing lipid membranes induced by the addition of pneumolysin. A structural model for the proteolipid complexes is proposed where a 30-50-meric pneumolysin ring lines the inside of a lipid torus. Cholesterol is found to be essential to the fusogenic action of pneumolysin.

Bacterial Proteins↗
PubMedDate not supplied

Structural and dynamic studies of the gamma-M4 trans-membrane domain of the nicotinic acetylcholine receptor.

A structural characterization of a synthetic peptide corresponding to the fourth transmembrane domain (M4-TMD) of the gamma-subunit of the nicotinic acetylcholine receptor from Torpedo californica has been undertaken. Solid-state NMR and CD spectroscopy studies indicate that upon reconstitution into lipid vesicles or magnetically aligned lipid bilayers, the synthetic M4-TMD adopts a linear alpha-helical conformation with the helix aligned within 15 degrees of the membrane normal. Furthermore, analysis of the motional averaging of anisotropic interactions present in the solid-state NMR spectra of the reconstituted peptide, indicate that the dynamics of the peptide within the bilayer are highly sensitive to the phase adopted by the lipid bilayer, providing an insight into how the interaction of lipids with this domain may play a important role in the modulation of this receptor by its lipid environment.

Animals↗
PubMedDate not supplied

A combined X-ray and neutron diffraction study of selectively deuterated melittin in phospholipid bilayers: effect of pH.

In order to study consequences of protonation of the N-terminus upon the interaction of the bee venom melittin with phospholipid bilayers, analogues of melittin, some of which were specifically deuterated at either Ala-12 or 15, were synthesized. These peptides were incorporated into bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine at either low pH (N-terminus protonated) or high pH (N-terminus unprotonated). X-ray and neutron diffraction data were collected from ordered stacks of these bilayers and from peptide-free controls. Phase determination was carried out using the swelling series (X-ray) and isomorphous derivative (neutron) methods. The water distribution between adjacent bilayers in the stacks may be described by a pair of Gaussians whose position and width change with the protonation state of the melittin. Difference Fourier profiles reveal that the melittin largely incorporates into the phospholipid bilayers. Changes in the water, melittin and deuterium label distributions fit a model in which the melittin lies both at the surface and close to the centre of the bilayer, the distribution of peptide between these locations being pH-dependent, with a larger population of surface melittin when the N-terminus is unprotonated.

Amino Acid Sequence↗
PubMedDate not supplied

Membrane protein structure: the contribution and potential of novel solid state NMR approaches.

Alternative methods for describing molecular detail for large integral membrane proteins are required in the absence of routine crystallographic approaches. Novel solid state NMR methods, devised for the study of large molecular assemblies, are now finding applications in biological systems, including integral membrane proteins. Wild-type and genetically engineered proteins can be investigated and detailed information about side chains, prosthetic groups, ligands (e.g. drugs) and binding sites can be deduced. The molecular structure and dynamics of selected parts of the proteins are accessible by a range of different solid state NMR approaches. Inter- and intra-atomic distances can be determined rather accurately (within ångströms) and the orientation of molecular bonds (within 2 degrees) can be measured in ideal cases. Here, a brief description of the methods is given and then some specific examples described with an indication of the future potential for the approaches in studying membrane proteins. It is anticipated that this emerging NMR methodology will be more widely used in the future, not only for resolving local structure, but also for more expansive descriptions of membrane protein structure at atomic resolution.

Magnetic Resonance Spectroscopy↗