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Biomedical subjects

H Saibil

Publications and source records attributed to H Saibil.

18 recordsLinked to original sources
PubMed2000

Molecular chaperones: containers and surfaces for folding, stabilising or unfolding proteins.

Newly solved chaperone structures include the thermosome, a group II chaperonin, and a small heat-shock protein. Novel ideas on chaperone mechanism are presented in the forced unfolding hypothesis of GroEL action. Structures of chaperone-pilin complexes reveal the mechanism of chaperone interaction in bacterial pilus assembly and there have been major advances in understanding the structure and function of Hsp100 unfoldases.

Adenosine Diphosphate↗
PubMed1999

Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation.

The outer membrane protein PulD (secretin) of Klebsiella oxytoca is required for transport of pullulanase across this membrane. We have purified a multimeric PulD complex from an Escherichia coli strain expressing all the proteins involved in pullulanase secretion. The outer membrane-anchored lipoprotein PulS was found to copurify with PulD. The molar ratio of the two proteins is close to 1:1, and the size of the complex is approximately 1 MDa. Scanning transmission electron and cryo-electron microscopy analyses showed that the purified complex is a cylindrical structure having a central cavity of approximately 7.6 nm and peripheral radial spokes. Fusion of proteoliposomes containing the purified complex with a planar lipid bilayer resulted in the appearance of small, voltage-activated, ion-conducting channels. We conclude that the central cavity seen in the electron microscope is part of a large gated channel and propose that the observed current fluctuations correspond to voltage-induced, relatively minor displacements of domains in the purified complex rather than to a complete opening of the secretin channel.

Bacterial Outer Membrane Proteins↗
PubMed1999

Reversible conversion of monomeric human prion protein between native and fibrilogenic conformations.

Prion propagation involves the conversion of cellular prion protein (PrPC) into a disease-specific isomer, PrPSc, shifting from a predominantly alpha-helical to beta-sheet structure. Here, conditions were established in which recombinant human PrP could switch between the native alpha conformation, characteristic of PrPC, and a compact, highly soluble, monomeric form rich in beta structure. The soluble beta form (beta-PrP) exhibited partial resistance to proteinase K digestion, characteristic of PrPSc, and was a direct precursor of fibrillar structures closely similar to those isolated from diseased brains. The conversion of PrPC to beta-PrP in suitable cellular compartments, and its subsequent stabilization by intermolecular association, provide a molecular mechanism for prion propagation.

Circular Dichroism↗
PubMed1996

The lid that shapes the pot: structure and function of the chaperonin GroES.

The structure of GroES reveals a potential for instability at odds with the idea of a fixed ring whose only flexible regions are at the outer edges. The importance of GroES in chaperoned protein folding is highlighted by evidence that folding substrates are transiently enclosed under the GroES cap.

Adenosine Triphosphate↗
PubMed1995

Effect of the C-terminal proline repeats on ordered packing of squid rhodopsin and its mobility in membranes.

Negative stain electron microscopy and saturation transfer electron spin resonance spectroscopy have been used to compare the lattice ordering and in-plane membrane mobility of full-length and C-terminally cleaved squid rhodopsin. The C-terminus of squid rhodopsin contains a negatively charged region followed by 9-10 repeats of a proline-rich sequence, not found in rhodopsins other than those of cephalopod invertebrates, but similar proline repeats are found in other, unrelated membrane proteins. We find that the proline repeats cluster the rhodopsins into small groups, interfering with two-dimensional crystallization and maintaining their mobility in the membrane.

Animals↗
PubMed1992

Activation of the GTP-binding protein Gq by rhodopsin in squid photoreceptors.

Photoaffinity labelling by a GTP analogue has been used to identify a 42 kDa band as the major G alpha subunit in squid photoreceptor membranes, recently identified by partial sequence analysis to be a member of the Gq sub-group of GTP-binding proteins [Pottinger, Ryba, Keen & Findlay (1991) Biochem. J. 279, 323-326]. Guanine-nucleotide-binding displacement analysis gave a stoichiometry of 1 G-protein per 12.5 rhodopsin molecules, the same as in vertebrate rod photoreceptors. Binding was not detected above background in the dark, but was rapidly activated by light. Unlike vertebrate transducin, this G-protein is very temperature-sensitive. GTP binding is maximal at temperatures less than 10 degrees C and is much decreased after several minutes above 18 degrees C. The light-stimulated GTPase rate is maximal around 10 degrees C, above which the loss of binding sites counteracts the increase in hydrolytic rate per site. Earlier studies described light-sensitive G alpha components of 40 and 45 kDa, by ADP-ribosylation in the presence of cholera and pertussis toxins. These are now shown to be very minor components, as the prolonged treatment at elevated temperature required for ADP-ribosylation is sufficient to inactivate the major G alpha totally. Unlike the minor G alpha components, the 42 kDa G alpha is not inhibited by Ca2+.

Affinity Labels↗
PubMed1992

T-complex polypeptide-1 is a subunit of a heteromeric particle in the eukaryotic cytosol.

The murine t-complex encodes t-complex polypeptide-1 (TCP1), which is constitutively expressed in almost all cells, and upregulated during spermatogenesis. Mammalian sequences have greater than 96% identity with each other, and greater than 60% identity with Drosophila melanogaster and yeast orthologues. TCP1 is essential in yeast, and is postulated to be the cytosolic mammalian equivalent of groEL. We report here that, in the native state, murine and human TCP1 is distributed throughout the cytosol as an 800K-950K hetero-oligomeric particle in association with four to six unidentified proteins and two Hsp70 heat-shock proteins. Negative-stain electron microscopy indicates that the structure is two stacked rings, 12-16 nm in diameter. Therefore, despite similarities with the chaperonin 60 proteins, these data indicate that TCP1 is biochemically and structurally unique. We suggest that TCP1 may represent one of a family of molecules in the eukaryotic cytosol involved in protein folding and regulated in part by their heteromeric associations.

Amino Acid Sequence↗
PubMed1991

Signal transduction in photoreceptors.

The biochemical role of the visual-pigment protein, rhodopsin, is reviewed, with reference to vertebrate rods and cones and the microvillar photoreceptors of invertebrates. New results are presented on the structure of squid rhodopsin, which possesses an extensive proline-rich repeat at its C-terminus, using negative-stain electron microscopy.

Amino Acid Sequence↗
PubMed1989

Recent advances in biological imaging.

Recent innovations in microscopy and digital image processing have greatly enhanced the power of biological imaging. Basic principles of several new methods in light and electron microscopy will be discussed, and examples presented of their application to cell and molecular biology.

Freezing↗
PubMed1989

Visualization of cyclic nucleotide binding sites in the vertebrate retina by fluorescence microscopy.

Cyclic nucleotides play a major role in cell signaling, especially in the nervous system. They act as cytoplasmic messengers in a wide range of physiological responses, but the spatial distribution of their sites of action within cells and tissues is not well-known. In the vertebrate retina, there is a class of well-characterized cGMP binding sites which control the permeability of cation channels in the rod outer segments (ROS), while cAMP is involved in several other systems in the inner retina. Biochemical studies of the cGMP-activated permeability in ROS have not distinguished between the subcellular compartments of disk and plasma membrane. By a new method using fluorescein-conjugated cyclic nucleotides, we have found strong cyclic GMP binding to the plasma membrane of the ROS, both on frozen sections of retina and in freshly isolated, leaky ROS. We also found a high density of cGMP binding sites on structures resembling the inner segment calycal processes. Little specific binding could be detected on the disk membranes or on any other retinal layer. In contrast, fluorescent cAMP did not label ROS, but gave a striking pattern of labeling on several deeper layers of the retina. These results suggest that the ROS plasma membrane has a much higher density of cGMP-controlled cation channels than the disk membranes, and point to other retinal layers where cAMP is likely to shape cellular responses. This method opens up novel morphological approaches to the study of cyclic nucleotide regulation.

Animals↗
PubMed1987

Ordered transmembrane and extracellular structure in squid photoreceptor microvilli.

Invertebrate retinas contain hexagonal arrays of microvilli that form the honeycomb structure of rhabdome photoreceptors. The largest and most crystalline rhabdomes are found in the squid retina, and we have taken advantage of their unique properties to derive a model for the electron density distribution in microvillar membranes using low angle X-ray diffraction combined with correlation averaging of electron microscope images. The model electron density map, calculated to a resolution of approximately 35 A, shows an unusually high protein content in the membranes. This may be associated with a dense meshwork of membrane junctions between neighboring microvilli as revealed by electron microscope image analysis. Membrane pair contacts are resolved as two or more strands of density crossing the membranes. The microvilli are also linked together by Y-shaped junctions at their three-way contacts. These two sorts of junctions link the membranes into a three-dimensional array and partition them into a mosaic of deformable and rigid domains. This arrangement maintains a remarkable degree of long-range order in squid rhabdomes, and may be responsible for the alignment of rhodopsin molecules. The structural order observed is necessary for these photoreceptors to achieve their high sensitivity to the plane of polarized light. Rhodopsin constitutes about one-half the microvillar protein. The remaining proteins, which can be divided into approximately equal detergent-soluble and insoluble fractions, could account for the composition of the new structures described.

Animals↗
PubMed1984

Physical analysis of light-scattering changes in bovine photoreceptor membrane suspensions.

We have used electron microscopy and model calculations to analyze the physical basis of light-scattering signals from suspensions of photoreceptor membranes. These signals have previously been used to probe interactions between photoactivated rhodopsin (R*) and the peripheral membrane enzyme, GTP-binding protein (G) (Kühn et al., 1981, Proc. Natl. Acad. Sci. USA., 78:6873-6877). Although there is no unique physical interpretation of these signals, we have shown in this study that they were qualitatively unchanged when the rod outer segment fragments (containing stacked disks) were fragmented by sonication or osmotic shock to produce spherical disk membrane vesicles. An exact treatment of the scattering process for spherical vesicles enabled us to evaluate the effects of changing membrane thickness, refractive index, or vesicle diameter. We present a particular redistribution of mass upon R*-G interaction that fits the experimental data.

Animals↗
PubMed1976

Neutron diffraction studies of retinal rod outer segment membranes.

Neutron diffraction measurements on isolated retinal rod outer segments show that most of the visual pigment protein, rhodopsin, is embedded in the hydrophobic core of the disk membrane. A very slight outward shift of protein at the cytoplasmic side of the membrane is associated with pigment bleaching.

Animals↗