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Biomedical subjects

D M Engelman

Publications and source records attributed to D M Engelman.

At least 91 records · Page 5Linked to original sources
PubMed1987

Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process.

Possible steps in the folding of bacteriorhodopsin are revealed by studying the refolding and interaction of two fragments of the molecule reconstituted in lipid vesicles. (1) Two denatured bacteriorhodopsin fragments have been purified starting from chymotryptically cleaved bacteriorhodopsin. Cleaved bacteriorhodopsin has been renatured from a mixture of the fragments in Halobacterium lipids/retinal/dodecyl sulfate solution following removal of dodecyl sulfate by precipitation with potassium. The renatured molecules have the same absorption spectrum and extinction coefficient as native cleaved bacteriorhodopsin. They are integrated into small lipid vesicles as a mixture of monomers and aggregates. Extended lattices form during the partial dehydration process used to orient samples for X-ray and neutron crystallography. (2) Correct refolding of cleaved bacterioopsin occurs upon renaturation in the absence of retinal. Regeneration of the chromophore and reformation of the purple membrane lattice are observed following subsequent addition of all-trans retinal. (3) The two chymotryptic fragments have been reinserted separately into lipid vesicles and refolded in the absence of retinal. Circular dichroism spectra of the polypeptide backbone transitions indicate that they have regained a highly alpha-helical structure. The kinetics of chromophore regeneration following reassociation have been studied by absorption spectroscopy. Upon vesicle fusion, the refolded fragments first reassociate, then bind retinal and finally regenerate cleaved bacteriorhodopsin. The complex formed in the absence of retinal is kinetically indistinguishable from cleaved bacterioopsin. The refolded fragments in lipid vesicles are stable for months, both as separate entities and after reassociation. These observations provide further evidence that the native folded structure of bacteriorhodopsin lies at a free energy minimum. They are interpreted in terms of a two-stage folding mechanism for membrane proteins in which stable transmembrane helices are first formed. They subsequently pack without major rearrangement to produce the tertiary structure.

Bacteriorhodopsins↗
PubMed1987

A complete mapping of the proteins in the small ribosomal subunit of Escherichia coli.

The relative positions of the centers of mass of the 21 proteins of the 30S ribosomal subunit from Escherichia coli have been determined by triangulation using neutron scattering data. The resulting map of the quaternary structure of the small ribosomal subunit is presented, and comparisons are made with structural data from other sources.

Bacterial Proteins↗
PubMed1987

Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit.

Animals↗
PubMed1986

Reformation of crystalline purple membrane from purified bacteriorhodopsin fragments.

Reconstituted crystalline purple membrane has been prepared starting from denatured bacteriorhodopsin (BR) fragments, native lipids and retinal. The two chymotryptic fragments are thought to contain respectively five and two transmembrane alpha-helices in native BR. The new reconstitution procedure, a modification of that of Huang et al. (1986, J. Biol. Chem., 256, 3802), relies on dodecylsulfate precipitation by potassium ions and yields samples with a high protein-to-lipid ratio (approximately 1:1 w/w). X-ray and neutron diffraction measurements show that in the reconstituted samples BR molecules are arranged in a P3 two-dimensional lattice with the same unit cell dimensions as the native purple membrane lattice. Analysis of reflection intensities indicates that the reconstituted molecules have regained the structure of native BR to 7 A resolution.

Bacteriorhodopsins↗
PubMed1986

Localization of two chymotryptic fragments in the structure of renatured bacteriorhodopsin by neutron diffraction.

The structure of crystalline purple membrane reconstituted from purified bacteriorhodopsin (BR) chymotryptic fragments has been studied by neutron diffraction. In one of the samples studied, the fragment C-2, encompassing the first two predicted transmembrane segments, was prepared from deuterated purple membrane. The diffraction changes when the natural C-2 fragment is substituted by a deuterated one are analysed in terms of a seven-helix model for BR. The assignment of the labelled fragment to one end of the molecule placed new constraints on folding models for the protein.

Bacteriorhodopsins↗
PubMed1985

Calcium-induced increase in the radius of gyration and maximum dimension of calmodulin measured by small-angle X-ray scattering.

We have used solution small-angle X-ray scattering to characterize bovine brain calmodulin in the presence and absence of calcium. In the presence of calcium, calmodulin exists in solution as an elongated molecule with a radius of gyration of 21.5 A and a maximum vector length of approximately 62 A. These values are consistent with the dimensions recently determined for the crystal form of rat testis calmodulin. In the absence of calcium, the calmodulin molecule is shorter, the radius of gyration decreases to 20.6 A, and the maximum vector length decreases to approximately 58 A. This change in dimensions is consistent with an overall contraction of the protein through movement of the two lobes closer to each other upon removal of calcium from calmodulin.

Animals↗
PubMed1985

Stability of transmembrane regions in bacteriorhodopsin studied by progressive proteolysis.

Proteinase K digestions of bacteriorhodopsin were carried out with the aim of characterizing the membrane-embedded regions of the protein. Products of digestions for two, eight or 24 hours were separated by high-pressure liquid chromotography. A computerized search procedure was used to compare the amino acid analyses of peptide-containing peaks with segments of the bacteriorhodopsin sequence. Molecular weight distributions of the products were determined by sodium dodecylsulfate-urea polyacrylamide gel electrophoresis. The structural integrity of the protein after digestion was monitored through the visible absorption spectrum, by X-ray diffraction of partially dried membranes, and by following release of biosynthetically-incorporated 3H leucine from the digested membranes. During mild proteolysis, bacteriorhodopsin was cleaved near the amino and carboxyl termini and at two internal regions previously identified as being accessible to the aqueous medium. Longer digestion resulted in cleavage at new sites. Under conditions where no fragments of bacteriorhodopsin larger than 9000 mol wt were observed, a significant proportion of the digested membranes retained diffraction patterns similar to those of native purple membranes. The harshest digestion conditions led to complete loss of the X-ray diffraction patterns and optical absorption and to release of half the hydrophobic segments of the protein from the membrane in the form of small soluble peptides. Upon cleavage of aqueous loop regions of the protein, isolated transmembrane segments may experience motion in a direction perpendicular to the plane of the membrane, allowing them access to protease.

Amino Acid Sequence↗
PubMed1984

Neutron scattering shows that cytochrome b5 penetrates deeply into the lipid bilayer.

Cytochrome b5 was asymmetrically reconstituted into small lipid vesicles made of a highly deuterated phospholipid. Small-angle neutron diffraction patterns were collected in a series of H2O-D2O mixtures from vesicles consisting of lipid and native or trypsinized cytochrome b5. The second moment of the radial distribution of scattering density in the vesicles was derived from these data and was compared to values calculated from three proposed models, which differ by the degree that cytochrome b5 penetrates the lipid bilayer. The model in which the hydrophobic domain of the protein is distributed across the bilayer agreed most closely with the data.

Animals↗
PubMed1983

Bacteriorhodopsin remains dispersed in fluid phospholipid bilayers over a wide range of bilayer thicknesses.

We have used vesicles made from delipidated bacteriorhodopsin and synthetic lecithins to address the following questions. If the transmembrane dimension of a protein hydrophobic surface differs from the equilibrium thickness of its lipid bilayer environment, will protein monomers aggregate to decrease the protein-lipid contact surface area? If so, how large must the difference be to induce aggregation? Using lecithins with acyl chains from di-10:0 to di-24:1, the thickness of the bilayer hydrocarbon region above the lipid phase transition temperature (tm) was varied from 14.5 A less than to 7.5 A more than the transmembrane dimension of the bacteriorhodopsin hydrophobic region. Bacteriorhodopsin remains dispersed when the surrounding bilayer hydrophobic region is 4 A thicker or 10 A thinner than the bacteriorhodopsin hydrophobic surface. Only the thin- (10:0) and thick- (24:1) bilayer samples showed any bacteriorhodopsin aggregation above tm. Thus a surprisingly large difference between protein and lipid hydrophobic thicknesses can be accommodated without protein aggregation. The lipid bilayer can evidently sustain large local distortions with a small change in free energy.

Bacteriorhodopsins↗
PubMed1983

Lipid bilayer thickness varies linearly with acyl chain length in fluid phosphatidylcholine vesicles.

The thickness of the lipid bilayer in vesicles made of pure phosphatidylcholines, with acyl chain lengths ranging from 10 to 24 carbons, has been determined by analysis of continuous X-ray scattering data from vesicle pellets at temperatures above the lipid phase transition temperature. Bilayer thickness was found to vary linearly with the number of carbons per acyl chain. The lines for saturated and monounsaturated acyl chains were slightly displaced but had similar slopes. For the saturated species di-12:0, di-14:0, di-16:0, and di-18:0 phosphatidylcholine the surface areas per molecule were all 65.7 to 66.5 A2, while the monounsaturated species and di-10:0 phosphatidylcholine all occupied 67.7 to 70.1 A2 per molecule.

Lipid Bilayers↗
PubMed1983

Pair distribution functions of bacteriorhodopsin and rhodopsin in model bilayers.

The pair distribution functions have been measured from freeze-fracture pictures of bacteriorhodopsin and rhodopsin recombinants with diacyl phosphatidylcholines (PC) of various hydrocarbon chain lengths. Pictures were used of samples that had been frozen from above the phase transition temperature of the lipid. Measured functions were compared with those calculated from two model interparticle potential energy functions, (a) a hard-disk repulsion only, and (b) a hard-disk repulsion plus electrostatic repulsion for a point charge buried in the membrane. The measured functions for bacteriorhodopsin di 12:0 PC, di 14:0 PC, and di 16:0 PC recombinants can be simulated using an interparticle hard-disk repulsion only. Bleached rhodopsin di 12:0 PC and di 18:1 trans-PC recombinants, and dark-adapted rhodopsin di 10:0 PC recombinants yield functions that are better simulated by assuming an additional repulsive interaction. The measured functions resemble those calculated using the hard-disk plus electrostatic repulsion model. The picture of dark-adapted rhodopsin in di 18:1 trans-PC frozen from 20 degrees C shows partial aggregation that is apparent in the measured pair distribution function. This attractive interaction persists even at 37 degrees C, where the measured function shows deviations from the hard-disk repulsive model, indicative of an attractive interparticle interaction. Implications of these results are discussed in terms of protein-lipid interactions.

Bacteriorhodopsins↗
PubMed1983

Neutron diffraction analysis of cytochrome b5 reconstituted in deuterated lipid multilayers.

Cytochrome b5 was reconstituted with a highly deuterated phospholipid to form ordered multilayers consisting of repeated centrosymmetric pairs of asymmetric lipid-protein bilayers. Lamellar neutron diffraction data were collected to approximately 29 A resolution, and have been interpreted using models for the interaction of the membrane-binding domain of cytochrome b5 with the lipid bilayer. A range of different models was examined, and those in which the protein penetrates well into the bilayer, possibly spanning it, are favored.

Animals↗