The organization of bacteriorhodopsin (bR) within reconstituted purple membranes (RPM) was examined using atomic force microscopy (AFM). Five reconstituted species were examined: RPM 3 (bR/native ...polar lipids/dimyristoylphosphatidylcholine (DMPC) in a 1:9:14 molar ratio), RPM 4 (bR/native polar lipids in a 1:7 molar ratio), RPM 5 (bR/native polar lipids/1,2-di-
O-phytanyl-
sn-glycerol in a 1:3.5:6.1 molar ratio), RPM 6 (bR/native polar lipids/1,2-di-
O-phytanyl-
sn-glycero-3-phosphocholine in a 1:3.5:4.9 molar ratio), and RPM 7 (bR/native polar lipids/1,2-diphytanoyl-
sn-glycero-3-phospho-
l-serine in a 1:3.5:4.6 molar ratio). RPM 3 patches adsorbed onto mica exhibit domains of crystallized bR trimers arranged in a hexagonal packing structure, similar to those found in native purple membrane (NPM). These domains are enclosed by DMPC-rich regions. RPM 4 patches were observed to have larger domains of crystallized bR, with trimer orientation 30° different from that found in NPM. The bR-rich domains are enclosed by a large, protein-free, lipid-rich region. The topography of RPM 5 was difficult to resolve as the surface had no discernable patterns or structure. The topographies of RPM 6 and 7 were similar to that found in RPM 3 in that higher domains were formed within the patch adsorbed onto mica. They may contain protein-rich regions, but clear images of protein arrangement could not be obtained using AFM. This may be a result of imaging limitations or of the lack of organization of bR within these domains.
The mouse X-chromosomal amelogenin gene promoter was used to drive the expression of mutated amelogenin proteins in vivo. Two different transgenic mouse lines based on deletions to either the ...amino-terminal (A-domain deletions) or to the carboxyl-region (B-domain deletions) were bred. In the molars of newborn A-domain deleted transgenic mice the formation of the initial layer of aprismatic enamel was delayed. There were severe structural alterations in the enamel of incisors of newborn mice bearing the A-domain deletion which were not apparent in animals bearing the B-domain deletion. In the A-domain-deleted animals, stippled material accumulated throughout the entire thickness of the forming enamel apparently causing a disruption of the normal rod-to-inter-rod relationship. This stippled material was likened to and interpreted as being groupings of amelogenin nanospheres. In the B-domain-deleted animals the stippled material was detected only in minute defects of the forming enamel. These data suggest significant differences in nanosphere assembly properties for animals bearing either the A-domain or the B-domain-deleted transgene. The present in vivo experimental approach suggests that at early stages of enamel formation, the A-domain plays a greater role than does the B-domain in amelogenin self-assembly, and consequently in enamel architecture and structure.
A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling ...protein complex to direct mineral habit. The principle protein of enamel is amelogenin that self-assembles to form nanospheres. In mice, the principal amelogenin product is a 180 amino acid hydrophobic protein. The yeast two-hybrid assay has been used to demonstrate the importance of amelogenin self-assembly domains. We have generated specific variants of amelogenin to analyze contributions of individual amino acids to the self-assembly process. These amelogenin variants have been produced either by deleting carboxyl-terminal amino acids (to generate proteins that relate to the documented proteolytic products of mouse amelogenin) or by a site-directed mutagenesis approach. Assessment of variant amelogenins truncated at the carboxyl-terminal imply that the proline at position 169 of mouse amelogenin (M180) plays a significant role in amelogenin self-assembly. Site-directed mutagenesis of this particular proline, however, failed to disrupt the amelogenin self-assembly property. These conflicting data add to the complexity of protein-protein assembly mechanisms as they relate to the enamel matrix. Available data suggest a robustness of this enamel protein (amelogenin) that ensures a functional, even though mechanically less than optimal, enamel results despite either minor or major genetic errors to the amelogenin gene locus.
A model of a rheologically relevant protein, ω-gliadin, is proposed and studied in this work by means of molecular dynamics techniques. The model is based on an octapeptide repeat motif that is ...experimentally described as characteristic of that protein and as constituting it almost entirely. The initial molecular structure consisted of 20 such repeats. It was optimized and the dynamics developed along 980 ps, at dielectric constant ε=80. Remarkable structural features were observed for the model built, such as an elongated, twisted tubular overall structure with a peculiar interpenetrating folding pattern, of a very regular character, organized strand formation, topologically segregated sites on the outer surface with an alternate hydrophilic/hydrophobic character and a hydrophilic inner cavity. Dynamics produced significantly more relaxed structures, but was not able to change the main geometric features presented by the original structure. Preliminary attempts of correlating some structural/dynamic aspects observed for the model with features of gliadin rheological behavior are presented.
Mammalian enamel is the unique hierarchically organized bioceramic material that owes its existence to a transient precursor, the enamel organic extracellular matrix. The organic matrix is ...biosynthesized by epithelial derived cells called ameloblasts and the selection of genes expressed, the timing and amount of proteins expressed serve to impose constraints on the matrix. The protein matrix components undergo self-assembly to form a microenvironment that regulates the mineral phase, serving to control crystal habit and spacing between crystallites. We have focused on amelogenin, the most abundant protein of the enamel organic matrix. Amelogenin self-assembles into nanospheres that participate in control over enamel organization. Ameloblasts also interact with the matrix and these interactions are important to the organization of hydroxyapatite crystallites into woven bundles. Changes to conserved domains within amelogenin alter protein-to-protein interactions as well as cell to matrix interactions. Changes to enamel organization observed during evolution may be accounted for, in part, by changes to critical domains within enamel proteins that form the matrix.
The influence of the composition of a mixed binary protein emulsifier composed of αs1-casein + β-casein on the rheology of concentrated oil-in-water emulsions (45 vol% oil, 5 wt% protein, pH 7) has ...been investigated over the temperature range 0–40°C. Controlled stress viscometric data are reported over the shear stress range 0.1–30 Pa for systems with αs1-casein/β-casein ratios of 100:0, 98:2, 95:5, 90:10, 75:25, 50:50, and 0:100. The pure casein emulsions showed substantially different temperature-dependent rheology, and there was observed to be a pronounced maximum in the small-deformation complex modulus of the pure αs1-casein emulsion in the range 30–40°C. In the emulsions containing ≥90% αs1-casein in the emulsifier mixture, all of the β-casein present was found to be associated with the surface of the droplets. Average droplet sizes and protein surface coverages were higher in the mixed casein systems than in the equivalent pure casein systems. The strongly pseudoplastic character of the emulsions is consistent with extensive reversible flocculation caused probably by a depletion mechanism involving unadsorbed protein. The degree of flocculation is sensitive to temperature and to the αs1-casein/β-casein ratio. The results can be interpreted in terms of changes in protein self-assembly and adsorbed layer structure which influence the strength of the interdroplet interactions and hence the rheological behavior of the emulsions. There is some evidence of a specific role for αs1-casein–β-casein complexes in these systems.
Kinetics of Streptolysin O Self-Assembly Palmer, Michael; Valeva, Angela; Kehoe, Michael ...
European journal of biochemistry,
07/1995, Letnik:
231, Številka:
2
Journal Article
Kinetics of Streptolysin O Self‐Assembly Palmer, Michael; Valeva, Angela; Kehoe, Michael ...
European journal of biochemistry,
1995-Jul-15, Letnik:
231, Številka:
2
Journal Article
Recenzirano
Streptolysin O is a member of a family of membrane‐damaging toxins that bind to cell membranes containing cholesterol and then polymerize to form large pores. We have examined the kinetics of toxin ...action using 125I‐labelled streptolysin O. Binding of toxin monomers to membranes displays first‐order kinetics and is reversible; the rate of desorption from red cells shows a marked dependence on temperature. To study oligomerization, toxin was bound to erythrocytes at 0°C. Oligomer formation was then triggered by a sudden temperature shift and stopped by solubilization of membranes with deoxycholate. While at moderately high streptolysin O concentrations oligomerization behaves as a reaction of second order, the kinetic pattern changes with increasing toxin concentration. We show that this can be accounted for by the assumption of a two‐step reaction mechanism: two membrane‐bound monomers first associate into a start complex, which then is rapidly extended by the sequential addition of further monomers up to the final oligomer size.
Dissociated bovine brain microtubule protein has been shown to reassemble at 0 degrees C in the presence of the drug taxol. Tubulin polymerization was monitored both by electron microscopy of the ...polymeric structures and by incorporation of tritiated GTP into filterable polymeric structures. Most of the labeled guanine nucleotide uptake into tubulin polymeric structures occurred in the first 30 minutes of incubation with the drug. The initial polymerization event results in the formation of protofilamentous tubulin ribbons. The first microtubules were noted after 1 hour of incubation with the drug. After 20 hours of incubation at 0 degrees C with taxol, the bulk of the polymerized tubulin appeared to be in the form of microtubules. Cold-stable tubulin rings with a mean diameter of 34 nm were present in the reaction mixture before the addition of taxol and throughout the 20-hour incubation. Most of the rings were apparently not involved in the taxol-induced microtubule assembly. The results are consistent with a model whereby taxol induces an initial formation of protofilamentous ribbon structures, mostly from free tubulin dimers, and a slower subsequent folding of the ribbon structures into microtubules.
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