The role of proteins in a dipole model for steady-state ionic transport through biological membranes.

The steady-state current-voltage characteristics of biological membranes are analyzed for means of an application of the electrodiffusion theory to the passage of ions through "dielectric pores", with orientable dipoles at the pore-water interfaces. A detailed evaluation of the electrostatic potential barrier shows, indeed, that the ions have practically no chance to penetrate into the phospholipid bilayer, but that they can cross the membrane through local protein inclusions, of high dielectric constant. A "gating mechanism" can be provided, moreover, by a change of the potential barrier, resulting from a dipole reorientation at the pore-water interface. Dipole-dipole interactions are opposed to the orienting effect of an applied field, but they can be neglected when the separation between the dipoles exceeds a certain critical value. The high polarizability of the pore material leads to an amplification of the effect of an applied field on the orientable dipoles. It is therefore possible to achieve a satisfactory agreement with the experimental results of Gilbert and Ehrenstein (Biophys. J., 9: 447, 1969) for the squid axon, and, in particular, to account for the width of the negative resistance regions with a relatively small value for the length of the orientable dipoles.     

Multiple topogenic sequences determine the transmembrane orientation of the hepatitis B surface antigen.

To investigate the mechanism by which complex membrane proteins achieve their correct transmembrane orientation, we examined in detail the hepatitis B surface antigen for sequences which determine its membrane topology. The results demonstrated the presence of at least two kinds of topogenic elements: an N-terminal uncleaved signal sequence and an internal element containing both signal and stop-transfer function. Fusion of reporter groups to either end of the protein suggested that both termini are translocated across the membrane bilayer. We propose that this topology is generated by the conjoint action of both elements and involves a specifically oriented membrane insertion event mediated by the internal sequence. The functional properties of each element can be instructively compared with those of simpler membrane proteins and may provide insight into the generation of other complex protein topologies.     

Towards a comparative anatomy of N-terminal topogenic protein sequences

A comparative study of three kinds of eukaryotic N-terminal topogenic sequences, viz signal peptides, N-terminal transmembrane anchors, and mitochondrial targeting sequences, suggests: that the sign of the N-terminal charge might influence the orientation of an N-terminal hydrophobic segment relative to the membrane and give rise to N-terminally anchored proteins with their main mass exposed either on the cytosolic or extra-cytosolic side of the membrane; and that N-terminal transmembrane segments in mitochondrial targeting sequences have a relatively low overall hydrophobicity, probably in order to avoid being recognized by the endoplasmic reticulum export machinery.     

Membrane topology and insertion of membrane proteins: search for topogenic signals.

Integral membrane proteins are found in all cellular membranes and carry out many of the functions that are essential to life. The membrane-embedded domains of integral membrane proteins are structurally quite simple, allowing the use of various prediction methods and biochemical methods to obtain structural information about membrane proteins. A critical step in the biosynthetic pathway leading to the folded protein in the membrane is its insertion into the lipid bilayer. Understanding of the fundamentals of the insertion and folding processes will significantly improve the methods used to predict the three-dimensional membrane protein structure from the amino acid sequence. In the first part of this review, biochemical approaches to elucidate membrane protein topology are reviewed and evaluated, and in the second part, the use of similar techniques to study membrane protein insertion is discussed. The latter studies search for signals in the polypeptide chain that direct the insertion process. Knowledge of the topogenic signals in the nascent chain of a membrane protein is essential for the evaluation of membrane topology studies.     

The movement of tRNA through the ribosome.

The interaction between tRNA and the ribosome during translation, specifically during elongation, constitutes an example of the motion and adaptability of living molecules. Recent results obtained by cryoelectron microscopy of "naked" ribosomes and ribosomes in functional binding states shine some light on this fundamental life-sustaining process. Inspection of the surface contour of our reconstruction reveals a precise "lock-and-key" fit between the intersubunit space and the tRNA molecule.     

The role of hydrophobic interactions in positioning of peripheral proteins in membranes

Background  

Three-dimensional (3D) structures of numerous peripheral membrane proteins have been determined. Biological activity, stability, and conformations of these proteins depend on their spatial positions with respect to the lipid bilayer. However, these positions are usually undetermined.     

The role of membrane proteins and phospholipids in the interaction of ribosomes with endoplasmic reticulum membranes.

Hydrolysis of the membrane proteins and phospholipid headgroups of rat liver rough endoplasmic reticulum membranes showed that the ribosomal binding sites involve membrane proteins susceptible to low concentrations of trypsin, chymotrypsin, and papain. Three membrane proteins having molecular weights of 120 000, 93 000 and 36 000 are found to be altered by trypsin and chymotrypsin treatment. Also the polar headgroup of phosphatidylinositol appears to play a role in the binding process.     

Targeting of proteins to membranes through hedgehog auto-processing

Hedgehog proteins use an auto-processing strategy to generate cholesterol-conjugated peptide products that act as extracellular ligands in a number of developmental signaling pathways. We describe an approach that takes advantage of the hedgehog auto-processing reaction to carry out intracellular modification of heterologous proteins, resulting in their localization to cell membranes. Such processing occurs spontaneously, without accessory proteins or modification by other enzymes. Using the green fluorescent protein (GFP) and the product of the Hras as model proteins, we demonstrate the use of hedgehog auto-processing to process heterologous N-terminal domains and direct the resulting biologically active products to cell membranes. This system represents a tool for targeting functional peptides and proteins to cell membranes, and may also offer a means of directing peptides or other small molecules to components of cholesterol metabolism or regulation.     

Separation of MBP fusion proteins through affinity membranes

Cellulose microporous membranes have been modified in order to obtain a stationary phase specific for the recovery of a class of fusion proteins containing the maltose binding protein domain, through affinity chromatography separations. The feasibility of a single step separation process for the recovery of large amounts of the desired product has been considered. To that purpose, a preparative scale module has been realized, suitable for flat sheet membranes. The affinity matrix used proved to be highly selective toward the fusion proteins examined. The binding capacity determined is comparable with the nominal binding capacity of commercially available supports. The influence of the relevant working parameters, such as flow rate, on the performances of the recovery process has been studied.     

The movement of ion pairs across bilayer lipid membranes.

If a polyhalide concentration gradient exists across a bilayer lipid membrane (BLM), ion pair movement occurs. The term ion pair indicates a lipid soluble complex of cation and anion with stoichiometry dictated by the respective charges. In a mixture of metal halide (MX_n, X = I, Cl, Br) and iodine, the ion pair is of the form M(I₂X)_n. The flux of ion pairs was monitored by measuring the flow of metal ions or polyhalide ions across the BLM. The flux of ion pairs across the BLM depended on cation crystal radius, fluidity of the membrane, strength of the ion pair complex and on the osmotic gradient (i.e., there exists a coupling between water and ion pair fluxes). The relationship between ion pairing and the electrical conductivity of BLM is briefly discussed.