C.d. studies have shown that mouse SAA2 (serum amyloid A2) protein has about one-half of the alpha-helix content of the SAA1 (serum amyloid A1) analogue (15 as against 32%), although secondary-structure prediction analyses based on sequence data do not suggest such a large difference between the forms. The decreased helical content may be a reflection or indication of a stronger propensity to aggregation of the SAA2 form compared with SAA1. The main elements of secondary structure in both proteins are beta-sheets/turns. Interactions with Ca2+ are accompanied by small losses in alpha-helix content, whereas binding to chondroitin-6-sulphate in the presence of millimolar Ca2+ also decreases the amount of secondary structure. However, SAA2 binding to heparan sulphate increases its beta-sheet structure, whereas with SAA1 secondary structure is not apparently altered by its interaction with heparan sulphate. Computer-generated surface profiles show slight differences in accessibility, hydrophilicity and flexibility between the proteins. Understanding these differences may help to explain why SAA2 is found in amyloid fibrils whereas SAA1 is not. In particular, a stronger tendency to aggregation might be the reason why SAA2 is deposited exclusively in these fibrils.

The structure of a 39 amino acid proteolytic fragment of rabbit skeletal troponin C containing the fourth Ca(2+)-binding site has been determined by an approach involving nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations. Hydrodynamic and NMR evidence establishes unambiguously that the fragment forms a stable dimer in solution in the presence of excess Ca2+. The calculation of the dimeric structure is based on a total of 1056 experimental restraints comprising 422 interproton distances, 35 phi, 28 psi, and 28 chi 1 torsion angle restraints within each subunit, 30 intermonomer distance restraints, and 6 Ca2+ restraints per subunit. A total of 48 final structures were calculated having an rms deviation about the mean atomic backbone coordinate positions of 1.0 A for residues Asp128-Glu156. The solution structure consists of a dimer of helix-loop-helix motifs related by a 2-fold axis of symmetry. The overall architecture of the dimer is very similar to the C-terminal domain in the crystal structure of chicken skeletal troponin C.

Biochemical and physiological studies of Synechococcus cyanobacteria have indicated the presence of a low-Mr heavy-metal-binding protein with marked similarity to eukaryotic metallothioneins (MTs). We report here the characterization of a Synechococcus prokaryotic MT isolated by gel-permeation and reverse-phase chromatography. The large number of variants of this molecule found during chromatographic separation could not be attributed to the presence of major isoproteins as assessed by amino acid analysis and amino acid sequencing of isoforms. Two of the latter were shown to have identical primary structures that differed substantially from the well-described eukaryotic MTs. In addition to six long-chain aliphatic residues, two aromatic residues were found adjacent to one another near the centre of the molecule, making this the most hydrophobic MT to be described. Other unusual features included a pair of histidine residues located in repeating Gly-His-Thr-Gly sequences near the C-terminus and a complete lack of association of hydroxylated residues with cysteine residues, as is commonly found in eukaryotes. Similarly, aside from a single lysine residue, no basic amino acid residues were found adjacent to cysteine residues in the sequence. Most importantly, sequence alignment analyses with mammalian, invertebrate and fungal MT sequences showed no statistically significant homology aside from the presence of Cys-Xaa-Cys structures common to all MTs. On the other hand, like other MTs, the prokaryotic molecule appears to be free of alpha-helical structure but has a considerable amount of beta-structure, as predicted by both c.d. measurements and the Chou & Fasman empirical relations. Considered together, these data suggested that some similarity between the metal-thiolate clusters of the prokaryote and eukaryote MTs may exist.

The size and shape of the Em protein from wheat embryos have been examined by gel filtration, densitometry, ultracentrifugation, and viscosity. Circular dichroism and fluorescence measureFents have also been made. Em has an intrinsic sedimentation coefficient, s&, , , of 1.1 1 S and a Stokes radius, RS, of 28.2 A ( 1 A = 0.1 nm) as determined by high performance liquid chromatography gel filtration on a TSK 2000SW column. The partial specific volume i from density measurements is 0.683 mL/g, a much lower than typical value. The molecular weight from sedimentation equilibrium is 1 1 200, with no indication for protein aggregation. The intrinsic viscosity [TI of Em is 6.02 mL/g. Circular dichroism shows the molecule to be about 70% random coil. The fluorescence emission spectrum is typical for a tyrosine-containing protein. The hydrodynamic data indicates a poor fit to either a prolate or oblate ellipsoid model; excess hydration or flexibility of the polypeptide chain caused by the rather unusual amino acid composition may be a possible cause. The implications that the low value of i , the high value of Rs, and the random-coil configuration of the Em protein may have on its ability to bind water and to contribute to the maintenance of a minimal level of hydration compatible with the sustained viability of the "dry" organism are subjects of an extended discussion. Briefly, it is suggested that Em may provide a matrix of bound water which opposes denaturation of proteins in the "desiccated" cytoplasm of dry plant embryos. McCubbin, W. D., Kay, C. M. & Lane, B. G. (1 985) Hydrodynamic and optical properties of the wheat germ Em protein. Can. J . Biochem. Cell Biol. 63, 803-81 1 Nous avons examine les dimensions et la forme de la proteine Em des embryons de bl6 par filtration sur gel, densitometrie, ultracentrifugation et viscositk. Nous avons aussi fait des mesures de dichroi'sme circulaire et de fluorescence. Tel que determine par chromatographie liquide de filtration sur gel a haute performance sur une colonne TSK 2000SW, la proteine Em a un coefficient de sedimentation intrinseque, $",,, de 1,l IS et un rayon de Stokes, Rs, de 28,2 A (1 A = 0 , l nm).Le volume specifique partiel, i , d'apres les mesures de densite, est de 0,683 mL/g, une valeur beaucoup plus basse que la valeur typique. D'apres I'equilibre de sedimentation, le poids mol6culaire est de 11 200 sans aucune indication d'agregation proteique. La viscositk intrinseque, [TI, de Em est de 6,02 mL/g. Le dichroi'sme circulaire montre que la mol6cule prksente environ 70% de repartition au hasard. Le spectre d'imission de fluorescence est typique d'une protkine contenant de la tyrosine. Les donnCes hydroxydynamiques s'accordent peu avec un modele ellipsoi'dal allongk ou aplati; l'exces d'hydratation ou la flexibilitk de la chai'ne polypeptidique causee par une composition en acides amines plut6t inhabituelle peuvent etre des causes possibles. Les implications que peuvent avoir la faible valeur de F, la valeur elevee de Rs et la configuration de repartition au ...

Calponin interacts with several Ca2+ binding proteins in a Ca(2+)-dependent manner. In order to determine the possible biological relevance of these interactions in smooth muscle function, it is necessary to characterize the strength and stoichiometry of the complexes formed. The interaction between calponin and calmodulin can be monitored through an acrylodan label on a cysteine of calponin. The fluorescently labeled calponin possesses the same biological function and physical behavior in binding to calmodulin as the native calponin. This probe is very environment-sensitive and responds to the calponin-calmodulin interaction by the emission peak blue-shifting 20 nm and by the fluorescent quantum yield increasing 3.5 times at 460 nm. The stoichiometric nature of this complex has been determined using analytical ultracentrifugation and is two calmodulins to one calponin, and the interaction is Ca(2+)-sensitive with a Kd1 of < or = 0.22 microM and a Kd2 of 2.5-3.4 microM. Calmodulin is not the only protein which interacts with calponin in this manner, but rather this interaction seems to be a general feature attributable to all hydrophobic patch exposing proteins, suggesting that it may be nonspecific, occurring because of a generalized mode of interaction. Two other proteins, S-100b from bovine brain and SMCaBP-11 from smooth muscle, had stronger affinities for calponin, and in particular interaction of SMCaBP-11 with calponin may be biologically relevant. In determining the nature of calponin's interaction with these Ca2+ binding proteins, it was apparent there was no effect of Ca2+ upon calponin itself and physical studies could find no evidence that calponin interacts with calcium.

This report describes the complete translated gene sequence, predicted secondary structure and lipid bilayer association of a novel kinetoplastid membrane protein (KMP-11) from Leishmania donovani promastigotes. KMP-11 was previously referred to as the lipophosphoglycan-associated protein (LPGAP). The isolation, species distribution and chemical characterization, including a partial protein sequence analysis and post-translational modifications, of this major membrane component have been described [Jardim, Funk, Caprioli and Olafson (1995) Biochem. J. 305, 307-313]. C.d. measurements of KMP-11 indicated a very high helical content estimated to be approximately 86% in trifluoroethanol. This was in agreement with computer-based secondary-structure analyses which predicted KMP-11 to be almost exclusively alpha-helical, with the protein adopting a helix-loop-helix motif. Arrangement of the residues located in the putative helical regions on an Edmundson helical wheel showed that this molecule could have a strongly amphipathic conformation and provided an explanation for how such a highly charged protein might be inserted into the plasma membrane. Evidence in support of KMP-11 association with lipid bilayers was provided by showing that KMP-11 could mediate carboxyfluorescein release from liposomes. These findings suggested that KMP-11 may function in part to increase bilayer pressure, stabilizing molecules such as lipophosphoglycan within the parasite pellicular membrane.

The polar flagellar filament of Campylobacter coli VC167 is composed of two highly related (98%) flagellin subunit proteins, FlaA and FlaB, whose trifluorethanol showed that the secondary structure of T2 FlaA flagellin was altered, with oa-helix structure being increased to 25% in the nonpolar environment. The molecule also contained 35 to 48% D-sheet and 11 to 29%o j-turn structure. Mimeotope analysis of octapeptides representing the sequence of FlaA together with immunoelectron microscopy and enzyme-linked immunosorbent assay with a panel of antisera indicated that many residues in presumed linear epitopes were inaccessible or nonepitopic in the assembled filament, with the majority being in the N-terminal 337 residues of the 572-residue flagellin. Residues at the carboxy-terminal end of the T2 FlaA subunit also become inaccessible upon assembly. Digestion with trypsin, chymotrypsin, and endoproteinase Glu-C revealed a protease-resistant domain with an approximate Mr of 18,700 between residues 193 and 375. Digestion with endoproteinase Arg-C and endoproteinase Lys-C allowed the mapping of a segment of surface-exposed FlaA sequence which contributes serospecificity to the VC167 T2 flagellar filament at residues between 421 and 480.

Electron microscopic examination of ultrathin sections and freeze-etched and shadow cast preparations of a bovine prepuce isolate of Campylobacter fetus VC119 showed an S layer with subunits in an apparent linear arrangement. Surface radioiodination, edzyme digestion, low-pH extraction, and Western immunoblotting showed that the layer was composed mainly of one protein which is the predominant protein antigen of C. fetus. This protein was purified to homogeneity by gel filtration, ion-exchange chromatography, and highperformance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed an apparent molecular weight of 131,000 for this protein with a pI of 6.35, and no carbohydrate could be detected by a variety of techniques. Anmino acid composition analysis showed that the protein contained approximately 1,304 residues per molecule, 41.2% of which were hydrophobic and approximately 22% of which were acidic. Cysteine and histidine were absent. Circular dichroisnm spectra showed that the prominent structure of the S layer protein was a "-pleated sheet (36%) with aperiodic foldings (31%); a moderate amount of a-helix (28%) and a low amount of ,8-turn (5%) were also present. The N-terminal amino acid sequence was determined for the first 18 residues. No sequence homology with other S layer proteins was found.