Hydrophobic Core Domain Research Articles

Chemically induced supramolecular reorganization of triblock copolymer assemblies: trapping of intermediate states via a shell-crosslinking methodology.

The mechanism of morphological phase transitions was studied for rod-shaped supramolecular assemblies comprised of a poly(acrylic acid)-block-poly(methyl acrylate)-block-polystyrene (PAA(90)-b-PMA(80)-b-PS(100)) triblock copolymer in 33% tetrahydrofuran/water after perturbation by reaction with a positively charged water-soluble carbodiimide. Tetrahydrofuran solvation of the hydrophobic core domain provided the dynamic nature required for the rod-to-sphere phase transition to be complete within 30 min. The intermediate morphologies such as fragmenting rods and pearl-necklace structures were trapped kinetically by the subsequent addition of a diamino crosslinking agent, which underwent covalent crosslinking of the shell layer. Alternatively, shell-crosslinked rod-shaped nanostructures with preserved morphology were obtained by the addition of the crosslinking agent before the addition of the carbodiimide, which allowed for the shell crosslinking to be performed at a faster rate than the morphological reorganization. The formation of robust shell-crosslinked nanostructures provides a methodology by which the morphological evolution processes can be observed, and it allows access to otherwise thermodynamically unstable nanostructures.

Lipoprotein–proteoglycan interactions at a silica surface membrane

Abstract Proteoheparan sulfate can be adsorbed to a methylated silica surface in a monomolecular layer via its transmembrane hydrophobic protein core domain. Due to electrostatic repulsion, its anionic polysugar side chains are stretched out into the blood substitute solution representing a receptor site for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. The binding process was studied by ellipsometric techniques showing that low density lipoprotein (LDL) strongly deposited at the proteoheparan sulfate, particularly in the presence of Ca2+. On the other hand, high density lipoprotein (HDL) bound to heparan sulfate proteoglycan (HS-PG) protected against LDL docking even at high LDL and Ca2+ concentrations and completely suppressed calcification of the proteoglycan–lipo–protein complex. These findings correlate well with those obtained from clinical investigations on risk factors for arteriosclerosis. The most important patient-oriented finding was the at least four times higher affinity constant of HDL binding to the proteoglycan receptor as compared to LDL docking. Therefore, under normal in vivo conditions most of the proteoglycan receptors are occupied by HDL and in this way shielded against LDL binding. Since competition is the dominant rule at the receptor site, the ternary complex HS-PG/LDL/Ca2+ aggregation can be reversed by HDL. Altogether, the proteoglycan coated hydrophobic silica surface used in these experiments, mimics the endothelial cell membrane in quite a perfect manner with respect to lipoprotein interactions at the blood–endothelium–matrix interface.

A receptor-based biosensor for lipoprotein docking at the endothelial surface and vascular matrix

Proteoheparan sulfate can be adsorbed to a methylated silica surface in a monomolecular layer via its transmembrane hydrophobic protein core domain. Due to electrostatic repulsion, its anionic glycosaminoglycan side chains are stretched out into the blood substitute solution, representing a receptor site for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. The binding process was studied by ellipsometric techniques showing that HDL has a high binding affinity to the receptor and a protective effect on interfacial heparan sulfate proteoglycan layers, with respect to LDL and Ca 2+ complexation. LDL was found to deposit strongly at the proteoheparan sulfate, particularly in the presence of Ca 2+, thus creating the complex formation ‘proteoglycan–low density lipoprotein–calcium’. This ternary complex build-up may be interpreted as arteriosclerotic nanoplaque formation on the molecular level responsible for the arteriosclerotic primary lesion. On the other hand, HDL bound to heparan sulfate proteoglycan protected against LDL docking and completely suppressed calcification of the proteoglycan–lipoprotein complex. In addition, HDL and aqueous garlic extract were able to reduce the ternary complex deposition and to disintegrate HS-PG/LDL/Ca 2+ aggregates. Although much remains unclear regarding the mechanism of lipoprotein depositions at proteoglycan-coated surfaces, it seems clear that the use of such systems offers possibilities for investigating lipoprotein deposition at a ‘nanoscopic’ level under close to physiological conditions. In particular, Ca 2+-promoted LDL deposition and the protective effect of HDL, even at high Ca 2+ and LDL concentrations, agree well with previous clinical observations regarding risk and beneficial factors for early stages of atherosclerosis. Therefore, we believe that the system can be of some use in investigations, e.g. of the interplay between different lipoproteins in arteriosclerotic plaque formation, as well as in high throughput screening of candidate drugs to atherosclerosis in a biosensor application.

A primary lesion model for arteriosclerotic microplaque formation

Proteoheparan sulfate can be adsorbed to a methylated silica surface in a monomolecular layer via its transmembrane hydrophobic protein core domain. Due to electrostatic repulsion, its anionic glycosaminoglycan side chains are stretched out into the blood substitute solution representing a receptor site for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. The binding process was studied by ellipsometric techniques showing that HDL has a high binding affinity to the receptor and a protective effect on interfacial heparan sulfate proteoglycan layers with respect to LDL and Ca2+ complexation. LDL was found to deposit strongly at the proteoheparan sulfate, particularly in the presence of Ca2+ thus creating the ternary complex formation “proteoglycan-LDL-calcium'. This heterotrimeric complex build-up may be interpreted as arteriosclerotic microplaque formation on the molecular level responsible for the primary lesion. On the other hand, HDL bound to heparan sulfate proteoglycan protected against LDL docking and completely suppressed calcification of the proteoglycan-lipoprotein complex. In addition, HDL and garlic extract were able to reduce the ternary complex deposition.

Physicochemical binding properties of the proteoglycan receptor for serum lipoproteins

Proteoheparan sulfate can be adsorbed to a methylated silica surface in a monomolecular layer via its transmembrane hydrophobic protein core domain. Due to electrostatic repulsion, its anionic polysugar side chains are stretched out into the blood substitute solution representing a co-receptor for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. The binding process was studied by ellipsometric techniques showing that oxLDL had a deleterious effect on heparan sulfate proteoglycan binding and conformation. Ca 2+ binding to and storage on the proteoheparan sulfate/LDL compound formed a ‘heterotrimeric’ HS-PG/LDL/Ca 2+ complex of high stability, aggregability and deposit coating. On the other hand, HDL bound to heparan sulfate proteoglycan protected against LDL docking and completely suppressed calcification of the proteoglycan/lipoprotein complex.

Ribosomal protein S7: a new RNA-binding motif with structural similarities to a DNA architectural factor.

The ribosome is a ribonucleoprotein complex which performs the crucial function of protein biosynthesis. Its role is to decode mRNAs within the cell and to synthesize the corresponding proteins. Ribosomal protein S7 is located at the head of the small (30S) subunit of the ribosome and faces into the decoding centre. S7 is one of the primary 16S rRNA-binding proteins responsible for initiating the assembly of the head of the 30S subunit. In addition, S7 has been shown to be the major protein component to cross-link with tRNA molecules bound at both the aminoacyl-tRNA (A) and peptidyl-tRNA (P) sites of the ribosome. The ribosomal protein S7 clearly plays an important role in ribosome function. It was hoped that an atomic-resolution structure of this protein would aid our understanding of ribosomal mechanisms. The structure of ribosomal protein S7 from Bacillus stearothermophilus has been solved at 2.5 A resolution using multiwavelength anomalous diffraction and selenomethionyl-substituted proteins. The molecule consists of a helical hydrophobic core domain and a beta-ribbon arm extending from the hydrophobic core. The helical core domain is composed of a pair of entangled helix-turn-helix motifs; the fold of the core is similar to that of a DNA architectural factor. Highly conserved basic and aromatic residues are clustered on one face of the S7 molecule and create a 16S rRNA contact surface. The molecular structure of S7, together with the results of previous cross-linking experiments, suggest how this ribosomal protein binds to the 3' major domain of 16S rRNA and mediates the folding of 16S rRNA to create the ribosome decoding centre.

P53 functional loss in a colon cancer cell line with two missense mutations (218leu and 248trp) on separate alleles

We have sequenced p53 in three colon cancer cell lines capable of autonomous proliferation. SNU-C1 and SNU-C4 cells, whose autonomous growth is dependent upon autocrine stimulation of epidermal growth factor receptor (EGFR), had wildtype p53 sequence of exons 4-9. In contrast, an EGFR ligand-independent cell line, SNU-C5, had heterozygous missense mutations affecting codons 218 (valine to leucine) and 248 (arginine to tryptophan) of p53. Bacterial cloning of p53 from SNU-C5 cells showed that the 248trp and 218leu mutants were both expressed and on separate alleles. 248trp is a common 'hot spot' mutant of p53 with variable dominant negative activity depending on the celullar context. Valine 218, in contrast, is rarely affected by mutation in cancers and is located in a region of the hydrophobic core domain away from 'hot spot' DNA contact sights. However, valine 218 is completely conserved across species, prompting us to investigate the function of 218leu in SNU-C5 cells. SNU-C5 cells exhibited complete loss of normal p53 function as evidenced by over-expression of p53 protein and by failure to show induction of p53, waf-1, mdm-2 or G1/S arrest in response to the DNA damaging agent, bleomycin. In a yeast p53 functional assay (FASAY), 50% of the clones were unable to transactivate a p53-specific promoter required for yeast colony expansion at 25, 30 or 37 degrees C. Sequencing of the p53 insert from several randomly selected wild-type and mutant yeast clones revealed that 218leu-bearing clones retained their ability to transactivate the p53-specific promoter. As expected, the 248trp-bearing clones lost this function. These data indicate that although 218leu retains normal transactivation activity on a p53 promoter in yeast at physiological temperatures, it is not capable of normal p53 function in the presence of a 248trp allele in SNU-C5 cells. It remains unclear whether the strong dominant negative activity of 248trp in SNU-C5 cells is related to the cellular context or to an unresolved abnormality of 218leu function.

Overall signal sequence hydrophobicity determines the in vivo translocation efficiency of a herpesvirus glycoprotein

We have described three mutant strains of Pseudorabies virus that contain mutations in the signal sequence coding region of a nonessential envelope glycoprotein, gIII. The alterations disrupt, truncate, or eliminate the hydrophobic core domain of the signal sequence. Each mutant was assayed for its ability to promote the translocation of gIII across the endoplasmic reticulum membrane and the subsequent localization of the mature form of the glycoprotein to the infected cell surface or the virus envelope. Our results confirm and extend findings in other systems that the overall hydrophobicity of the signal sequence core region is a major determinant of translocation efficiency. We were unable to correlate simply the length of the core or the average hydrophobicity of core residues with export efficiency. Because our work involved the use of infectious virus mutants, we were able to identify a virus defect associated with a complete block in gIII export. This defect will facilitate a pseudo-reversion analysis of gIII signal sequence function.

Eukaryotic signal peptide structure/function relationships: Identification of conformational features which influence the site and efficiency of co-translational proteolytic processing by site-directed mutagenesis of human pre(Δpro)apolipoprotein A-II

Abstract The structural features which influence the efficiency and site of cleavage by eukaryotic signal peptidase have not been fully defined. Human pre(delta pro) apolipoprotein A-II is a useful model system for such an analysis. We have recently shown that a panel of mutants which have amino acids of varying physical-chemical properties substituted for the -1 residue (Ala20) of its signal peptide exhibit cleavage at one of three potential sites or, in some cases, bidirected cleavage (Folz, R. J., Nothwehr, S. F., and Gordon, J. I. (1988) J. Biol. Chem. 263, 2070-2078). In this present study, a subset of the pre(delta pro)Xaa20apoA-II mutants were used to identify conformational features, located COOH-terminal to the hydrophobic core domain, which regulate its cotranslational translocation and proteolytic processing. Proline residues were substituted at positions 13, 14, or 15 in ten of the original position 20 mutant preproteins in order to induce formation of a beta-turn structure at various positions upstream of the cleavage site. The effects of these mutations were assessed using an in vitro transcription/translation/microsome processing assay. Substitution of proline at position 13 resulted in a dramatic decrease in processing efficiency in all Xaa20 sequence contexts, while processing progressively increased when the proline was moved downstream to positions 14 and 15. Elongating the hydrophobic core of the Pro13 mutants by 1 residue reversed this effect. NH2-terminal sequence analysis of 34 co-translationally processed mutants revealed that the optimum distance between the site of proline introduction and the site of co-translational cleavage was 4-5 residues. However, this distance could be altered by the presence of certain amino acids at positions -1 and -3. The data suggest that several structural motifs NH2-terminal to residues -1 and -3 influence the location, site, and efficiency of cleavage by eukaryotic signal peptidase. These include specifically the length of the hydrophobic core, the location of beta-turns relative to the COOH terminus of this core domain, as well as the physical-chemical properties of amino acids at potential cleavage sites.

Effects of prolipoprotein signal peptide mutations on secretion of hybrid prolipo-beta-lactamase in Escherichia coli.

Hybrid proteins were constructed by coupling beta-lactamase to the signal sequence (plus nine amino acids) of selected mutant prolipoproteins of Escherichia coli. The mutant prolipoprotein signal peptides contained lesions in two structural domains of the signal peptide, the basic amino-terminal domain and the hydrophobic core domain. We then compared the processing and localization of the mutant prolipo-beta-lactamases to the processing and localization of the comparable mutant prolipoproteins. We show that a mutant signal sequence with an anionic amino terminus exhibits similar limitations in the processing of prolipo-beta-lactamase as previously observed in prolipoprotein. Deletion of four hydrophobic residues from hydrophobic core results in a signal peptide which slowly translocates a fraction of the total mutant hybrid protein synthesized. This signal peptide was previously shown to translocate lipoprotein efficiently. Alteration of this hydrophobic core, which stimulated synthesis of mutant prolipoproteins, does not stimulate synthesis of prolipo-beta-lactamase. Finally mutations that slowed processing of prolipoprotein by affecting the proposed helical structure of the signal peptide had no significant effect on the processing of prolipo-beta-lactamase. These results suggest that the positively charged amino-terminal domain of the signal peptide has a common role in protein secretion regardless of the secretory protein. On the other hand, other domains of the signal peptide exhibit different phenotypes when the secretory protein is changed.