Topological Probes Research Articles

Recognition of the folding consensus in RNA secondary structures by the topological‐filtering method

Functionally homologous RNA sequences can substantially diverge in their primary sequences but it can be reasonably assumed that they are related in their higher-degree structures. The problem to find such structures and simultaneously satisfy as far as possible the free-energy-minimization criterion, is considered here in two aspects. Firstly a quantitative measure of the folding consensus among secondary structures is defined, translating each structure into a linear representation and using the correlation theorem to compare them. Secondly an algorithm for the parallel search for secondary structures according to the free-energy-minimization criterion, but with a filtering action on the basis of the folding consensus measure is presented. The method is tested on groups of RNA sequences different in origin and in functions, for which proposals of homologous secondary structures based on experimental data exist. A comparison of the results with a blank consisting of a search on the basis of the free energy minimization alone is always performed. In these tests the method shows its ability in obtaining, from different sequences, secondary structures characterized by a high-folding consensus measure also when lower free energy but not homologous structures are possible. Two applications are also shown. The first demonstrates the transfer of experimental data available for one sequence, to a functionally related and therefore homologous one. The second application is the possibility of using a topological probe in the search for precise structural motifs.

Peptide-specific antibodies and proteases as probes of the transmembrane topology of the bovine heart mitochondrial porin

We have investigated the transmembrane topology of the bovine heart mitochondrial porin by means of proteases and antibodies raised against the amino-terminal region of the protein. The antisera against the human N-terminus reacted with porin in Western blots of NaDodSO4-solubilized bovine heart mitochondria and with the membrane-bound porin in enzyme-linked immunosorbent assay (ELISA). The immunoreaction with mitochondria coated on microtiter wells showed that the amino-terminal region of the protein is not embedded in the lipid bilayer but is exposed to the cytosol. Back-titration of unreacted anti-N-terminal antibodies after their incubation with intact mitochondria demonstrated that the porin N-terminus is also exposed in "noncoated" mitochondria. No difference in antisera reactivity was observed between intact and broken mitochondria. Intact and broken mitochondria were subjected to proteolysis by specific proteases. The membrane-bound bovine heart porin was strongly resistant to proteolysis, but a few specific cleavage sites were observed. Staphylococcus aureus V8 protease gave a large 24K N-terminal peptide, trypsin produced a 12K N-terminal and an 18K C-terminal peptide, and chymotrypsin gave two peptides of Mr 19.5K and 12.5K, which were both recognized by the antiserum against the human N-terminus. Carboxypeptidase A was ineffective in cleaving the membrane-bound porin in both intact and broken mitochondria. Thus, the carboxy-terminal part of the protein is probably not exposed to the water phase. The cleavage patterns of membrane-bound porin, obtained with S. aureus V8 protease, trypsin, and chymotrypsin, showed no difference between intact and broken mitochondria, thus indicating that all porin molecules have the same orientation in the membrane. The computer analysis of the sequence of human B-lymphocyte porin suggested that 16 beta-strands can span the phospholipid bilayer. This result, together with the overall information presented, allowed us to draw a possible scheme of the transmembrane arrangement of mammalian mitochondrial porin.

Macromolecular conjugates of transport inhibitors: new tools for probing topography of anion transport proteins

Macromolecular-conjugated, water-soluble, membrane-impermeant compounds were designed and assessed as topological probes for chloride-transporting agencies. The novel compounds were derivatives of either disulfonic stilbene (DS) and benzylaminoethylsulfonate (BS), "classical" inhibitors of erythrocyte chloride-bicarbonate exchange, or of phenylanthranilates (PA), high-affinity blockers of epithelial chloride channels. Covalent reactive derivatives of various DS, BS, and PA were synthesized and coupled either directly to polyethylene glycol or via spacer arms of different lengths to dextrans. The macromolecular conjugates were demonstrably inhibitory to red blood cell anion exchange when the ligands were appropriately coupled: inhibitory efficacy strongly depended on the chemical structure of the coupled ligand and the spacer length between the inhibitory moiety and the macromolecule. Mechanistic studies indicated that impermeant DS and PA derivatives acted exofacially on sites, which although different in their affinity for chloride, shared geographical proximity. BS derivatives were unique in that they affected transport from either surface. The results suggest asymmetric aqueous access routes leading to the functional domain of the anion transporter from either membrane surface.

Transport domain of the erythrocyte anion exchange protein

The anion transport domain of the anion exchange protein (AEP) of human erythrocyte membranes (band 3, 95 kD mol wt) was probed with the substrate and affinity label pyridoxal-5'-phosphate (PLP). Acting from outside, this probe labels two chymotryptic fragments of 65 and 35 kD of AEP but only the 35-kD fragment is protected from labeling by reversibly acting disulfonic stilbenes (DS). It is shown here by functional studies and by immunoblotting with anti-PLP antibodies that transmembrane gradients of anions determine the availability of a 35-kD fragment lys residue to surface labeling by PLP, in analogy with their effects on labeling of 65-kD fragment by DS. On this basis, it is suggested that both fragments contribute to the formation of the transport domain. However, unlike DS, PLP blocks transport when reacted from within released membranes, indicating that the 35-kD fragment might contain components of the mobile unit of the AEP. Using impermeant fluorescence quenchers of PLP of both complexation type (anti-PLP antibodies) or collisional type (acrylamide) as topological probes for PLP-labeled sites, it is deduced that the 65-kD PLP-labeled and the 35-kD PLP-labeled lys groups are inaccessible to macromolecules from either surface, but the 65-kD PLP-lys is accessible to low molecular weight molecules from without while the 35-kD PLP-labeled lys shows accessibility primarily from within the cell surface. The studies indicate that the accommodation of a wide class of anions by AEP might be associated with the flexibility of the transport domain of the protein and its capacity to undergo transport-related conformational changes.

A native cruciform DNA structure probed in bacteria by recombinant T7 endonuclease.

T7 endonuclease preferentially cleaves purified supercoiled pBR322 and colE1 plasmids at the single-stranded regions exposed when palindromic sequences assume cruciform structures (Panayotatos, N., and Wells, R.D. (1981) Nature 289, 466-470). In vivo, however, induction of nuclease synthesis off a cloned gene caused complete degradation of the bacterial DNA but not of the plasmid vector; presumably, single-stranded regions (cruciforms?) on the genome effectively complete for the nuclease with similar sites on the plasmid (Panayotatos, N., and Fontaine, A. (1985) J. Biol. Chem. 260, 3173-3177). To overcome this competition, we introduced on the plasmid the naturally occurring colE1 palindrome which forms a more stable cruciform in vitro. In addition, we increased the target size (and the T7 endonuclease gene dosage) by raising the copy number of the plasmid 5-fold. Induction of the endonuclease encoded by this new plasmid (pLAT75) resulted not only in degradation of genomic DNA but also in intracellular nicking and linearization of the plasmid. The cleavage site in vivo was mapped at the colE1 palindrome and coincided with the site cleaved specifically in vitro by either T7 or S1 endonuclease only when this palindrome assumes the cruciform structure. These results indicate that cruciform structures exist intracellularly and demonstrate the usefulness of endonucleases as probes of DNA topology in vivo.

Dynamics of molecule-surface interactions

A multidimensional potential energy surface for the interaction of a diatomic molecule with a metal surface is presented. The potential includes the effects of an activation barrier to the adsorption process which has a value changing with position within the surface unit cell. The model accounts for recent experimental data for the diffraction of H 2 beams with energy in excess of the measured activation barrier. Wavepacket calculations for the diffracted intensities show features which should make H 2 diffraction experiments a detailed probe of the reactive-potential topology.

Dynamics of molecule-surface interactions

A multidimensional potential energy surface for the interaction of a diatomic molecule with a metal surface is presented. The potential includes the effects of an activation barrier to the adsorption process which has a value changing with position within the surface unit cell. The model accounts for recent experimental data for the diffraction of H 2 beams with energy in excess of the measured activation barrier. Wavepacket calculations for the diffracted intensities show features which should make H 2 diffraction experiments a detailed probe of the reactive-potential topology.

N-Linked glycoprotein assembly. Evidence that oligosaccharide attachment occurs within the lumen of the endoplasmic reticulum.

The transbilayer orientation of the oligosaccharide chain transferred from oligosaccharide-lipid to endogenous protein acceptors in sealed hen oviduct microsomes has been examined using endo-beta-N-acetylglucosaminidase H as a topological probe. The oligosaccharide moiety of these acceptors was released by the enzyme only under conditions where the microsomes were made permeable to macromolecules. The release of the oligosaccharide chain by endo-beta-N-acetylglucosaminidase H was not increased by removal of ribosomes or by mild trypsinization of the sealed microsomes. The endogenous acceptors were shown to be membrane-associated proteins that are not a part of the mRNA . ribosome . tRNA . nascent chain complex. From these results we conclude that the transfer of oligosaccharide from oligosaccharide-lipid occurs at the luminal face of the rough endoplasmic reticulum.

A calculation of the electronic structure of the amorphous Pd80Si20system

A basis of s, p and orbitals on Pd and s and p orbitals on Si sites is used to calculate the electronic density of states per unit cell of the Pd3Si crystal and formula unit densities of states for different regions of two network models of the amorphous Pd80Si20 system. In contrast to the results of previous calculations which involved simpler model Hamiltonians for the system, it has been found that the differences in the electronic structure which reflect the local topological environment are rather modest and are certainly less prominent than those obtained in the study of three- and fourfold-coordinated amorphous semiconductors. The results compare favourably with available photoemission data and it is suggested that the s valence features of silicon may be a sensitive probe of topology in the Pd80Si20 system. The local density of states at the silicon site is spread over a wide range of energies and contributes significantly to the Fermi level density of states, thus providing a clue to the strength and transport properties of these amorphous alloys.

Antibiotics as probes of ribosome structure: binding of chloramphenicol and erythromycin to polyribosomes; effect of other antibiotics.

Antibiotics were used as probes of ribosome topology and function. Studies of [(14)C]chloramphenicol and [(14)C]erythromycin binding to ribosomes and polyribosomes revealed the following features. The requirement of high K(+) concentration (150 mM) for [(14)C]chloramphenicol binding to NH(4)Cl-washed ribosomes resulted from the washing procedure. Neither native 70S ribosomes nor polyribosomes require K(+) greater than 30 mM for [(14)C]chloramphenicol binding. Whereas [(14)C]chloramphenicol binds to both ribosomes and polyribosomes, [(14)C]erythromycin binds essentially only to ribosomes. After removal of peptidyl-transfer ribonucleic acid (tRNA) from polyribosomes, [(14)C]erythromycin could then be bound. The effects of a number of antibiotics on [(14)C]chloramphenicol binding to ribosomes and polyribosomes was assessed. It was found that most of the macrolides (erythromycin, carbomycin, spiramycin III, niddamycin, oleandomycin, and tylosin) and streptogramins A and B (vernamycin A, PA114A, vernamycin Balpha, and PA114B) inhibited chloramphenicol binding to NH(4)Cl-washed and native 70S ribosomes, but not to polyribosomes. After removal of peptidyl-tRNA from polyribosomes, [(14)C]chloramphenicol binding was then inhibited. In contrast, sparsomycin and althiomycin inhibited chloram-phenicol binding to polyribosomes, but not to ribosomes. After removal of peptidyl-tRNA from polyribosomes, sparsomycin and althiomycin were then ineffective. The presence of peptidyl-tRNA on polyribosomes apparently is required for binding of sparsomycin and althiomycin, but prevents binding of most macrolides and streptogramins. The lincosaminides (lincomycin and celesticetin) and methymycin (a small macrolide) inhibited [(14)C]chloramphenicol binding to NH(4)Cl-washed and native 70S ribosomes best, but also inhibited the binding to polyribosomes. The amino nucleosides and other antibiotics tested do not seem to interact strongly with the major chloramphenicol-binding site. These results provide knowledge of the interrelationships between antibiotic and substrate ribosome binding sites which should eventually contribute to a map of ribosomal topology.