Structure characterization of the central repetitive domain of high molecular weight gluten proteins. II. Characterization in solution and in the dry state.

The structure of the central repetitive domain of high molecular weight HMW) wheat gluten proteins was characterized in solution and in the dry state using HMW proteins Bx6 and Bx7 and a subcloned, bacterially expressed part of the repetitive domain of HMW Dx5. Model studies of the HMW consensus peptides PGQGQQ and GYYPTSPQQ formed the basis for the data analysis (van Dijk AA et al., 1997, Protein Sci 6:637-648). In solution, the repetitive domain contained a continuous nonoverlapping series of both type I and type II II beta-turns at positions predicted from the model studies; type II beta-turns occurred at QPGQ and QQGY sequences and type I beta-turns at YPTS and SPQQ. The subcloned part of the HMW Dx5 repetitive domain sometimes migrated as two bands on SDS-PAGE; we present evidence that this may be caused by a single amino acid insertion that disturbs the regular structure of beta-turns. The type I beta-turns are lost when the protein is dried on a solid surface, probably by conversion to type II beta-turns. The homogeneous type II beta-turn distribution is compatible with the formation of a beta-spiral structure, which provides the protein with elastic properties. The beta-turns and thus the beta-spiral are stabilized by hydrogen bonds within and between turns. Reformation of this hydrogen bonding network after, e.g., mechanical disruption may be important for the elastic properties of gluten proteins.     

Identification of Influenza C Virus Phosphoproteins

The HMV-II cells infected with influenza C virus were labeled with inorganic [³²P]phosphate to identify phosphorylated proteins. Analysis by radioimmunoprecipitation with antiviral serum or monoclonal antibodies revealed that three major structural proteins of the virus, hemagglutinin-esterase (HE), nucleoprotein (NP), and matrix protein (M1) are all phosphorylated in both infected cells and virions. It was also observed that, in the presence of trypsin (10 μg/ml), the unphosphorylated form of the HE glycoprotein was cleaved efficiently whereas the phosphorylated form was not, raising the possibility that phosphorylation of HE may influence its susceptibility to degradation by proteolytic enzymes.     

Multiple molecular recognition properties of the lipocalin protein family

The lipocalins, a diverse family of small extracellular ligand proteins, display a remarkable range of different molecular properties. While their binding of small hydrophobic molecules, and to a lesser extent their binding to cell surface receptors, is well known, it is shown here that formation of macromolecular complexes is also a common feature of this family. Analysis of known crystallographic structures reveals that the lipocalins process a conserved common structure: an antiparallel β-barrel with a repeated +1 topology. Comparisons show that within this overall similarity the structure of individual proteins is specifically adapted to bind their particular ligands, forming a binding site from an internal cavity (within the barrel) and/or an external loop scaffold, which gives rise to different binding modes that reflects the need to accommodate ligands of different shape, size, and chemical structure. The architecture of the lipocalin fold suggests that the both the ends and sides of this barrel are topologically distinct, differences also apparent in analyses of structural and sequence variation within the family. These different can be linked to experimental evidence suggesting a possible functional dichotomy between the two ends of the lipocalin fold. The structurally invariant end of the molecule may be implicated in general binding small ligands and forming macromolecular complexes via an exposed binding surface.     

A procedure for detecting structural domains in proteins.

A procedure is described for detecting domains in proteins of known structure. The method is based on the intuitively simple idea that each domain should contain an identifiable hydrophobic core. By applying the algorithm described in the companion paper (Swindells MB, 1995, Protein Sci 4:93-102) to identify distinct cores in multi-domain proteins, one can use this information to determine both the number and the location of the constituent domains. Tests have shown the procedure to be effective on a number of examples, even when the domains are discontinuous along the sequence. However, deficiencies also occur when hydrophobic cores from different domains continue through the interface region and join one another.     

Twisted β-pleated sheet: the molecular conformation which possibly dictates the formation of the helicoidal architecture of several proteinaceous eggshells

Evidence from X-ray diffraction, laser-Raman spectroscopy, secondary structure prediction, freeze-fracturing, conventional electron microscopy and Fourier analysis suggests that the helicoidal structure of the silkmoth eggshell (chorion) is created by protein molecules, most probably in a twisted β-pleated sheet conformation. It is proposed that this conformation also dictates the formation of the helicoidal architecture of other proteinaceous eggshells; apparently, it may also play an important role in the formation of the helicoidal architecture in other biological systems with protein components.     

Multiple gene action determining a mouse salivary protein phenotype: Identification of the structural gene for androgen binding protein (Abp)

A novel variation in electrophoretic phenotype is described for a mouse salivary androgen binding protein (Abp). Crosses show that the variation is inherited in an autosomal codominant manner and protein characterization studies show that the variant Abp differs in isoelectric point from the common form of the protein. Those observations suggest that the variation involves the structural gene for the mouse salivary Abp. The genetic studies also show that the electrophoretic mobility of the variant Abp can be influenced by the sex-limited saliva pattern (Ssp) gene. The Ssp ^S allele alters the electrophoretic mobility of Abp in males at puberty or in females which have received exogenous testosterone [Karn, R. C., Dlouhy, S. R., Springer, K. R., Hjorth, J. P., and Nielsen, J. T. (1982). Biochem Genet. 20:493]. This study shows that Abp and Ssp are distinct genes which are not closely linked and that Ssp ^S is trans active in F₁ (Abp ^a /Abp ^b , Ssp ^S /Ssp ^F ) males.SRD was supported in part by PHS General Medical Training Grant T32 GMO7468 and the Indiana University School of Medicine Research Program in Academic Medicine. RCK was supported in part by PHS Career Development Award 1 KO4 AMOO284.     

How stable are genomes of tropical woody plants? Heterozygosity in C-banded karyotypes ofPorcelia as compared withAnnona (Annonaceae) andDrimys (Winteraceae)

InPorcelia goyazensis (2n = 18) Giemsa C-banding patterns differ from those ofAnnona reticulata (2n = 14) and reveal structural heterozygosity. The amplitude of karyological variation in theAnnonaceae is greater than expected for a primitive woody family. In a comparison with other tropical angiosperm groups, the highly differentiated karyotype ofDrimys brasiliensis (2n = 86) is interpreted as being the end-point of numerous karyological changes.     

Characterization of thermotropic structural transitions of the erythrocyte membrane: a biochemical and electron-paramagnetic resonance approach

The relationship between membrane structural properties and functions has been generally inferred from observed thermotropic phenomena. By the use of 16-dinyloxyl stearic acid spin probe we investigated the red blood cell membrane components involved in three characteristic thermotropic structural transitions occurring at 8, 20, and 40 degrees C. The transition at 8 degrees C is removed by chymotrypsin treatment at the cytoplasmic membrane layer. The 20 degrees C phase transition is unmodified after chymotrypsin treatment and occurs at 15 degrees C after complete proteolysis of intramembrane chymotrypsin-insensitive peptides. Liposomes from the total lipid extract of RBC show only one thermotropic transition at 15 degrees C. The 40 degrees C phase transition is absent in vesicles free of skeletal proteins, in vesicles obtained after RBC storage, and in low-ionic-strength resealed ghosts. Transitions at 8 degrees C and 40 degrees C appear to be due to the interactions of cytoplasmic exposed proteins with membrane, whereas the 20 degrees C transition is intrinsic to the lipid component.     

Human β-glucuronidase: Assignment of the structural gene to chromosome 7 using somatic cell hybrids

-Glucuronidase (GUS) has become an important enzyme model for the genetic study of molecular disease, enzyme realization, and therapy, and for the biogenesis and function of the lysosome and lysosomal enzymes. The genetics of human -glucuronidase was investigated utilizing 188 primary man-mouse and man-Chinese hamster somatic cell hybrids segregating human chromosomes. Cell hybrids were derived from 16 different fusion experiments involving cells from ten different and unrelated individuals and six different rodent cell lines. The genetic relationship of GUS to 28 enzyme markers representing 19 linkage groups was determined, and chromosome studies on selected cell hybrids were performed. The evidence indicates that the -glucuronidase gene is assigned to chromosome 7 in man. Comparative linkage data in man and mouse indicate that the structural gene GUS is located in a region on chromosome 7 that has remained conserved during evolution. Involvement of other chromosomes whose genes may be important in the final expression of GUS was not observed. A tetrameric structure of human -glucuronidase was demonstrated by the formation of three heteropolymers migrating between the human and mouse molecular forms in chromosome 7 positive cell hybrids. Linkage of GUS to other lysosomal enzyme genes was investigated. -Hexosaminidase HEX _B) was assigned to chromosome 5; acid phosphatase₂ (ACP ₂) and esterase A₄ (ES-A ₄) were assigned to chromosome 11; HEX _A was not linked to GUS; and -galactosidase (-GAL) was localized on the X chromosome. These assignments are consistent with previous reports. Evidence was not obtained for a cluster of lysosomal enzyme structural genes. In demonstrating that GUS was not assigned to chromosome 9 utilizing an X/9 translocation segregating in cell hybrids, the gene coding for human adenylate kinase₁ was confirmed to be located on chromosome 9.Supported by NIH Grants HD 05196, GM 20454, and GM 06321, by NSF Grant BMS 73-07072, and by HEW Maternal and Child Health Service, Project 417.     

Basic steroid saponins from aerial parts of Fritillaria Thunbergii

From the aerial parts of Fritillaria thunbergii, three glycosidal Solanum alkaloids (basic steroid saponins) were isolated together with minor