Molecular packing in type I collagen fibrils

Previous studies of the X-ray diffraction pattern of the crystalline regions of type I collagen fibrils yielded information on the unit cell parameters and also the orientation of the pseudo-hexagonally packed molecular segments in the overlap region. The absence of Bragg reflections at high angles attributable to the molecular segments in the gap region led to the suggestion that these segments were more mobile than those in the overlap region. We report a study of the low-angle Bragg reflections in a search for information about the nature of the orientation and packing of the molecular segments in the gap region. We conclude that the (m = 0, n = 0) helix layer plane of the molecular segments in the overlap region makes little or no contribution to the Bragg reflections at low angles, and identify three possible origins for the observed low-angle reflections in the electron density contrast associated with: (1) the "hole" created by the missing molecular segment in the gap region; (2) the telopeptides; or (3) the axial regularities in amino acid residues of a particular type, with periodicities of D/5 or D/6. Sufficient information is available to investigate the first two of these possibilities, and the results obtained suggest specific arrangements for the molecular segments in the overlap and gap regions, and specific connectivities between the molecular segments in successive overlap regions. In addition, we have examined the amino acid sequence and identified features related to the mobility of the molecular segments in the gap region and to the regions where it is thought that molecules are kinked.     

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.     

Characterization of caulobacters isolated from wastewater treatment systems.

Caulobacters are generally assumed to be found only in environments of low organic content; however, we readily isolated strains from a variety of sewage treatment system designs and locations, and 33 distinct strains were characterized. Most were morphologically similar, having the crescent-shaped cell body, short stalk, and hexagonally packed, paracrystalline surface (S) layer characteristic of several Caulobacter crescentus laboratory strains. Upon closer examination, they were distinguishable on the basis of protein band profiles on polyacrylamide gel electrophoresis, gross colony characteristics, or holdfast composition or by DNA restriction fragment length polymorphism analysis with flagellin and S-layer gene probes. Most of the isolates contained one or more high-molecular-weight plasmids and were resistant to a number of antibiotics, characteristics generally not shared with caulobacters isolated from other sources. Six of the 33 strains were retained because they did not fit the typical isolate profile; these strains are overrepresented in our collection compared with their relative proportion in wastewater treatment systems. By colony hybridization and restriction fragment length polymorphism analysis, all of these and one typical isolate showed less homology than the others to the surface array gene of a laboratory strain (C. crescentus CB15), and three hybridized less strongly with the flagellin gene from the same strain. In sum, although the strains were distinguishable, caulobacters from the wastewater treatment systems we examined were relatively homogenous, were similar to characterized laboratory strains, and, with exceptions, could probably be reliably detected as a group by gene probes derived from C. crescentus strains.     

Growth of lipolytic psychrotrophic pseudomonads in raw and ultra-heat-treated milk

The lipolytic floras of 36 raw milk samples showing lipolytic defects were dominated by pseudomonads. Representative lipolytic isolates were selected and tested for growth, lipase activity and lipolysis in ultra-heat-treated milk at temperatures ranging from 5 degrees to 30 degrees C. Pseudomonas fluorescens was the most frequently encountered species but Ps. fragi was found to cause more severe lipolytic defects in both single and mixed strain milk cultures. A representative strain of Ps. fragi multiplied faster in cold-stored milk than did three representative strains of Ps. fluorescens. The lipases produced by Ps. fragi strains were more heat-stable than those produced by Ps. fluorescens strains.     

Intermediate filament structure

In a previous communication (Biosci. Rep. 3, 517–525, 1993) we described quantitative X-ray diffraction studies of -keratin which were shown to be consistent with the presence of finite arrays of repeating units, successive arrays being set down at axial intervals of 470 Å. In addition the axial interval between repeating units in an array was shown to be 197.9 Å. It was suggested that this could most readily be explained by supposing that a surfacelattice was present which contained a dislocation along a helical path with unit heighth = 470 Å and unit twist |t| = 49.1° . The number of repeating units was shown to be in the range 7–9. With 7 repeats the mismatch of the lattice along the dislocation is small and this choice was used to develop a detailed model for the filament. Subsequent studies of molecular interactions have shown however that the coiled-coil rope segments in the rod domain of the molecule are most probably oriented parallel to the dislocation, and so minimization of lattice mismatch may be less important than originally supposed. In the present communication it is shown that the choice of 8, rather than 7, for the number of repeating units yields a model which is more compatible with estimates of the linear density and also provides the basis for a general model for polymorphism in intermediate filament lattices.     

Tubulin isoforms in the brine shrimp, Artemia: primary gene products and their posttranslational modification

The brine shrimp, Artemia, contains 3 alpha- and 2 beta-tubulins as shown by Coomassie Blue staining of two-dimensional gels. In order to study the biosynthetic origins of the isotubulins, we hybridized cloned Drosophila tubulin genes, under stringent conditions, to blots of Artemia DNA and RNA. Southern blot analyses indicate a tubulin gene family of limited complexity. One size class of alpha- and beta-tubulin mRNA at 1800 bases was observed on Northern blots. Fluorograms of Artemia tubulin synthesized in vitro, revealed one alpha- and one beta-tubulin on two-dimensional gels, indicating that each mRNA is translated into one polypeptide and that additional tubulin spots observed on Coomassie-stained two-dimensional gels may arise posttranslationally. Artemia tubulin, which was either purified to homogeneity, or in crude cell-free extracts, was analyzed with a panel of tubulin-specific antibodies. The presence of acetylated tubulin, restricted to one of the three major alpha-tubulin spots on two-dimensional gels, demonstrated that Artemia tubulin diversity is partially generated by posttranslational mechanisms. Artemia tubulin reacted very well with an antibody to tyrosinated tubulin, but there was no, or very little, detectable detyrosinated tubulin unless the purified Artemia tubulin was exposed to carboxypeptidase. The results suggest that all microtubule-dependent events in Artemia, a complex metazoan animal, are accomplished with microtubules composed from a limited repertoire of tubulins and that none of these events require appreciable amounts of detyrosinated tubulin.