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Biomedical subjects

G Gröbner

Publications and source records attributed to G Gröbner.

11 recordsLinked to original sources
PubMed2000

Observations of light-induced structural changes of retinal within rhodopsin.

Photo-isomerization of the 11-cis retinal chromophore activates the mammalian light-receptor rhodopsin, a representative member of a major superfamily of transmembrane G-protein-coupled receptor proteins (GPCRs) responsible for many cell signal communication pathways. Although low-resolution (5 A) electron microscopy studies confirm a seven transmembrane helix bundle as a principal structural component of rhodopsin, the structure of the retinal within this helical bundle is not known in detail. Such information is essential for any theoretical or functional understanding of one of the fastest occurring photoactivation processes in nature, as well as the general mechanism behind GPCR activation. Here we determine the three-dimensional structure of 11-cis retinal bound to bovine rhodopsin in the ground state at atomic level using a new high-resolution solid-state NMR method. Significant structural changes are observed in the retinal following activation by light to the photo-activated M(I) state of rhodopsin giving the all-trans isomer of the chromophore. These changes are linked directly to the activation of the receptor, providing an insight into the activation mechanism of this class of receptors at a molecular level.

Animals↗
PubMed2000

Structural and orientational information of the membrane embedded M13 coat protein by (13)C-MAS NMR spectroscopy.

Oriented and unoriented M13 coat protein, incorporated into dimyristoyl phosphatidylcholine bilayers, has been studied by (13)C-magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy. Rotational resonance experiments provided two distance constraints between Calpha and C&z.dbnd6;O positions of the labelled residues Val-29/Val-30 (0.4+/-0.5nm) and Val-29/Val-31 (0.45+/-0. 5nm) in its hydrophobic domain. The derived dihedral angles (Phi, Psi) for Val-30 revealed a local alpha-helical conformation. (13)C-CP-MAS experiments on uniformly aligned samples (MAOSS experiments) using the (13)C&z.dbnd6;O labelled site of Val-30 allowed the determination of the helix tilt (20 degrees +/-10 degrees ) in the membrane. It is shown that one uniform MAS high-resolution solid state NMR approach can be used to obtain structural and orientational data.

Amino Acid Sequence↗
PubMed1999

Binding properties of the stilbene disulfonate sites on human erythrocyte AE1: kinetic, thermodynamic, and solid state deuterium NMR analyses.

A novel stilbene disulfonate, 4-trimethylammonium-4'-isothiocyanostilbene-2,2'-disulfonic acid (TIDS), has been chemically synthesized, and the interaction of this probe with human erythrocyte anion exchanger (AE1) was characterized. Covalent labeling of intact erythrocytes by [N(+)((14)CH(3))(3)]TIDS revealed that specific modification of AE1 was achieved only after removal of other ligand binding sites by external trypsinization. Following proteolysis, (1.2 +/- 0.4) x 10(6) TIDS binding sites per erythrocyte could be blocked by prior treatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a highly specific inhibitor of AE1. Inhibition of sulfate equilibrium exchange by TIDS in whole cells was described by a Hill coefficient of 1.10 +/- 0.06, which reduced to 0.51 +/- 0.01 following external trypsinization. The negative cooperativity of TIDS binding following external trypsinization suggests that trypsin-sensitive proteins modulate allosteric coupling between AE1 monomers. Thermodynamic analysis revealed that TIDS binding induces smaller conformational changes in AE1 than is observed following DIDS binding. The similar inhibitory potencies of both TIDS (IC(50) = 0.71 +/- 0.48 microM) and DIDS (IC(50) = 0.2 microM) imply that there is no correlation between the ability of stilbene disulfonates to arrest anion exchange function and the magnitude of ligand-induced conformational changes in AE1. Solid state (2)H NMR analysis of a [N(+)(CD(3))(3)]TIDS-AE1 complex in both unoriented and macroscopically oriented membranes revealed that large amplitude "wobbling" motions describe ligand dynamics. The data are consistent with a model where TIDS bound to AE1 is located exofacially in contact with the bulk aqueous phase.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1999

Probing membrane surfaces and the location of membrane-embedded peptides by (13)C MAS NMR using lanthanide ions.

A simple but efficient (13)C MAS NMR method is presented for the determination of the location of embedded molecules such as peptides relative to biological membrane surfaces by exploiting the interaction with paramagnetic lanthanide ions. Using various aqueous Dy(3+) concentrations a distance-dependent differential paramagnetic quenching of NMR lipid resonance intensities for specific carbon sites was observed, with residues at the bilayer surface quenched effectively and hydrophobic sites unaffected by Dy(3+). Tested on the membrane-embedded 50 residue long M13 coat protein, (13)C labeled at its Val-29 and Val-31 residues, no paramagnetic quenching was observed for the peptide resonances by Dy(3+), suggesting that Val-29 and Val-31 are not in close proximity to the bilayer interface, but buried deeply inside the hydrophobic region of the lipid bilayer.

Carbon Isotopes↗
PubMed1998

Probing the agonist binding pocket in the nicotinic acetylcholine receptor: a high-resolution solid-state NMR approach.

Acetylcholine, the agonist for the nicotinic acetylcholine receptor, has been observed directly when bound specifically to its binding site in the fully functional receptor-enriched membranes from Torpedo nobiliana. High-resolution solid-state, magic angle spinning 13C NMR methods have been used to observe selectively N+(13CH3)3 acetylcholine bound in as few as 20 nmol of receptor binding sites, against a background of natural abundance membrane resonances and excess acetylcholine in free solution. The specificity of the binding has been demonstrated to be pharmacologically significant through the use of the competitive inhibitor alpha bungarotoxin which selectively displaces and prevents binding of acetylcholine to the membrane-bound receptor. The chemical shift assigned to N+(13CH3)3 acetylcholine in solution and crystalline solid is 53.9 +/- 0.04 ppm, and it changes by 1.6 ppm (p < 0.05) for agonist when bound specifically in the receptor binding site. Through the use of computer simulations of chemical shifts carried out on acetylcholine bound to the acetylcholinesterase, we propose that the cause for this change is the presence of aromatic side chains lining the receptor binding site. It is suggested that the binding of acetylcholine to the nicotinic acetylcholine receptor is mediated primarily through the interaction of the quaternary ammonium group of the acetylcholine with the pi bonded systems in the aromatic side chains. Longitudinal relaxation time measurements show that the residency time for the acetylcholine observed in DDCP experiments is long (> 200 ms) with respect to the longitudinal relaxation time of other assignable resonances within the spectrum from the lipid and protein and confirms that the acetylcholine is protein-associated, and not free in solution or nonspecifically bound.

Acetylcholine↗
PubMed1998

Interaction of a type II myosin with biological membranes studied by 2H solid state NMR.

Deuterium nuclear magnetic resonance spectroscopy (2H NMR) has been employed to investigate the interaction of lung type II myosin protein with neutral bilayers containing dimyristoylphosphatidylcholine (DMPC) as the only constituent and mixed bilayers containing the negatively charged lipid dimyristoylphosphatidylglycerol (DMPG). DMPC was deuterated at its headgroup by substituting the four protons at the alpha- and beta-positions (DMPC-d4) and the nine protons at the gamma-position (DMPC-d9). DMPG was perdeuterated at its headgroup (DMPG-d5). No changes were observed in the quadrupole splittings or spin-lattice relaxation times for the deuterated DMPC headgroup segments when increasing amounts of myosin were added to liposomes, made exclusively of DMPC-d9 or of DMPC-d4. However, upon the insertion of the negatively charged lipid DMPG at 1:1 molar ratio into the DMPC bilayers, myosin was found to interact electrostatically with the liposomes, thereby affecting significantly both the quadrupole splittings and spin-lattice relaxation rates of the alpha-, beta-, and gamma-deuterons in labeled DMPC. Monitoring DMPG-d5 in mixed DMPC/DMPG bilayers revealed a direct electrostatic interaction of DMPG with the protein, where positively charged lysine residues located at the tail domain of myosin provide the necessary sites for the interaction to occur. When ATP and Mg2+ were complexed to the head domain of myosin, a reduced interaction with the negatively charged bilayers was observed. The results clearly indicate that a type II myosin can interact with membranes without the need for a specific hydrophobic domain or an anchor in the protein molecule, provided that negatively charged lipids are present in the bilayer.

Animals↗
PubMed1998

Photoreceptor rhodopsin: structural and conformational study of its chromophore 11-cis retinal in oriented membranes by deuterium solid state NMR.

Rhodopsin is the retinal photoreceptor responsible for visual signal transduction. To determine the orientation and conformation of retinal within the binding pocket of this membrane bound receptor, an ab initio solid state 2H NMR approach was used. Bovine rhodopsin containing 11-cis retinal, specifically deuterated at its methyl groups at the C19 or C20 position, was uniaxially oriented in DMPC bilayers. Integrity of the membranes and quality of alignment were monitored by 31P NMR. Analysis of the obtained 2H NMR spectra provided angles for the individual labelled chemical bond vectors leading to an overall picture for the three dimensional structure of the polyene chain of the chromophore in the protein binding pocket around the Schiff base attachment site.

Animals↗
PubMed1997

Macroscopic orientation of natural and model membranes for structural studies.

One approach for obtaining high-resolution structural and functional information for biomembranes and their proteins is by static solid-state NMR of oriented systems. Here, a general procedure to align fully functional biological membranes containing large membrane proteins (Mr >30,000) is described. The method, based on the isopotential spin-dry ultracentrifugation technique, relies on the centrifugation of membrane fragments onto a support with simultaneous, or subsequent, partial evaporation of the solvent which aids alignment. The quality of orientation, as shown by the mosaic spread of the samples, was monitored by static solid-state 31P NMR for the phospholipids and by 2H NMR for a deuterated retinal in bovine rhodopsin. The generality of this method is demonstrated with three different membranes containing bovine rhodopsin in reconstituted bilayers, natural membranes with the red cell anion exchange transport protein in erythrocytes, band 3, and the nicotinic acetylcholine receptor.

Animals↗
PubMed1992

Deuteron nuclear magnetic resonance study of the dynamic organization of phospholipid/cholesterol bilayer membranes: molecular properties and viscoelastic behavior.

The influence of cholesterol on the dynamic organization of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers was studied by deuteron nuclear magnetic resonance (2H NMR) using unoriented and macroscopically aligned samples. Analysis of the various temperature- and orientation-dependent experiments were performed using a comprehensive NMR model based on the stochastic Liouville equation. Computer simulations of the relaxation data obtained from phospholipids deuterated at the 6-, 13- and 14-position of the sn-2 chain and cholesterol labeled at the 3 alpha-position of the rigid steroid ring system allowed the unambiguous assignment of the various motional modes and types of molecular order present in the system. Above the phospholipid gel-to-liquid-crystalline phase transition, TM, 40 mol % cholesterol was found to significantly increase the orientational and conformational order of the phospholipid with substantially increased trans populations even at the terminal sn-2 acyl chain segments. Lowering the temperature continuously increases both inter- and intramolecular ordering, yet indicates less ordered chains than found for the pure phospholipid in its paracrystalline gel phase. Trans-gauche isomerization rates on all phospholipid alkyl chain segments are slowed down by incorporated cholesterol to values characteristic of gel-state lipid. However, intermolecular dynamics remain fast on the NMR time scale up to 30 K below TM, with rotational correlation times tau R parallel for DMPC ranging from 10 to 100 ns and an activation energy of ER = 35 kJ/mol. Below 273 K a continuous noncooperative condensation of both phospholipid and cholesterol is observed in the mixed membranes, and at about 253 K only a motionally restricted component is left, exhibiting slow fluctuations with correlation times of tau R perpendicular greater than 1 microsecond. In the high-temperature region (T greater than TM), order director fluctuations are found to constitute the dominant transverse relaxation process. Analysis of these collective lipid motions provides the viscoelastic parameters of the membranes. The results (T = 318 K) show that cholesterol significantly reduces the density of the cooperative motions by increasing the average elastic constant of the membrane from K = 1 x 10(-11) N for the pure phospholipid bilayers to K = 3.5 x 10(-11) N for the mixed system.

Cholesterol↗