Secondary Structural Studies of Bovine Caseins: Structure and Temperature Dependence of β-Casein Phosphopeptide (1-25) as Analyzed by Circular Dichroism, FTIR Spectroscopy, and Analytical Ultracentrifugation

The defining structural feature of all of the caseins is their common phosphorylation sequence. In milk, these phosphoserine residues combine with inorganic calcium and phosphate to form colloidal complexes. In addition, nutritional benefits have been ascribed to the phosphopeptides from casein. To obtain a molecular basis for the functional, chemical, and biochemical properties of these casein peptides, the secondary structure of the phosphopeptide of bovine β-casein (1–25) was reexamined using Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies. Both methods predict secondary structures for the peptide which include polyproline II elements as well as β-extended sheet and turn-like elements. These structural elements were highly stable from 5° to 70°C. Reexamination of previously published ¹H NMR data using chemical shift indices suggests structures in accord with the CD and FTIR data. Dephosphorylation showed little or no secondary structural changes, as monitored by CD and FTIR, but the modified peptide demonstrated pronounced self-association. The polymers formed were not highly temperature sensitive, but were pressure sensitive as judged by analytical ultracentrifugation at selected rotor speeds. Molecular dynamics (MD) simulations demonstrated relatively large volume changes for the dephosphorylated peptide, in accord with the pressure dependent aggregation observed in the analytical ultracentrifuge data. In contrast the native peptide in MD remained relatively rigid. The physical properties of the peptide suggest how phosphorylation can alter its biochemical and physiological properties.     

Solution Structures of Casein Peptides: NMR, FTIR, CD, and Molecular Modeling Studies of αs1-Casein, 1–23

To determine its potential for interacting with other components of the casein micelle, the N-terminal section of bovine _s1-casein-B, residues 1-23, was investigated with nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies, and molecular modeling. NMR data were not consistent with conventional -helical or -sheet structures, but changes in N-H proton chemical shifts suggested thermostable structures. Both CD and FTIR predicted a range of secondary structures for the peptide (30–40% turns, 25–30% extended) that were highly stable from 5°C to 25°C. Other conformational elements, such as loops and polyproline II helix, were indicated by FTIR only. Molecular dynamics simulation of the peptide predicted 32% turns and 27% extended, in agreement with FTIR and CD predictions and consistent with NMR data. This information is interpreted in accord with recent spectroscopic evidence regarding the nature of unordered conformations, leading to a possible role of _s1-casein (1–23) in facilitating casein-casein interactions.     

Secondary structure of ShK toxin, a potassium-channel-blocking peptide

Summary Sea anemones possess small K-channel-blocking peptides about the same size as the scorpion K-channel toxins. We have estimated the secondary structure content (33% helix, 26% -sheet) of one of these toxins, ShK toxin, using CD, Raman, and FTIR spectroscopy. A hypothetical 3D structure of the peptide core has been constructed using secondary structure and disulfide-linkage constraints; a single helical segment running from Ala¹⁴ through Leu²⁵ is predicted.     

Involvement of protein kinase A and casein kinase II in thein vivo protein kinase activities in prophase arrested Xenopus oocytes

In vivo casein phosphorylation was analysed in Xenopus full-grown oocytes arrested in the prophase of the meiotic cell division. The phosphorylation was inhibited by the protein kinase inhibitor (PKI) and also by heparin (3 g/ml; final concentration). casein phosphorylation was increased by spermine (2 mM). Therefore, protein kinase A and casein kinase II are both activein vivo in full-grown oocytes and may be involved in the prophase arrest of meiotic cell division.     

Interaction of alpha-N-Acetyl-beta-endorphin and calmodulin

Acetylation at the -amino terminal is a common post-translational modification of many peptides and proteins. In the case of the potent opiate peptide -endorphin, -N-acetylation is a known physiological modification that abolishes opiate activity. Since there are no known receptors for -N-acetyl--endorphin, we have studied the association of this peptide with calmodulin, a calcium-dependent protein that binds a variety of peptides, phenothiazines, and enzymes, as a model system for studying acetylated endorphin-protein interactions. Association of the acetylated peptide with calmodulin was demonstrated by cross-linking with bis(sulfosuccinimidyl)suberate; like -endorphin, adducts containing 1 mol and 2 mol of acetylated peptide per mole calmodulin were formed. Some of the bound peptides are evidently in relatively close proximity to each other since, in the presence of amidated (i.e., lysine-blocked) calmodulin, cross-linking yielded peptide dimers. The acetylated peptide exhibited no appreciable helicity in aqueous solution, but in trifluoroethanol (TFE) considerable helicity was formed. Also, a mixture of acetylated peptide and calmodulin was characterized by a circular dichroic spectrum indicative of induced helicity. Empirical prediction rules, applied earlier to -endorphin, suggest that residues 14–24 exhibit -helix potential. This segment has the potential of forming an amphipathic helix; this structural unit is believed to be important in calmodulin binding. The acetylated peptide was capable of inhibiting the calmodulin-mediated stimulation of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity with an effective dose for 50% inhibition of about 3 µM; this inhibitory effect was demonstrated using both an enzyme-enriched preparation as well as highly purified enzyme. Thus, acetylation at the -amino terminal of -endorphin, although abolishing opiate activity, does not interfere with the binding to calmodulin. Indeed, -endorphin and the -N-acetylated peptide behave very similarly with respect to calmodulin association.Portions of this work are in partial fulfillment of the requirements for the Ph.D. degree from Vanderbilt University.     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

Structural Changes of β-Lactoglobulin during Thermal Unfolding and Refolding – An FT-IR and Circular Dichroism Study

We have quantitatively characterized by FT-IR spectroscopy the contents of secondary structure of -lactoglobulin during thermal unfolding and subsequent refolding. Our data clearly indicate that considerable amount of secondary structure, particularly -sheet, still remained intact even at 90°C. Noticeable changes in secondary structure of -lactoglobulin were observed only above 70°C. The refolded protein regained, within limits of experimental error, all of the secondary structure lost during thermal unfolding. The data also indicate that the refolding mechanism operating at pH 7.0 and 2.0 are the same. Identical secondary structure of native and refolded -lactoglobulin was also indicated by far-UV circular dichroic spectra of the two forms of protein. Near UV circular dichroic spectra of the same two forms showed considerable differences indicating less tertiary structure of refolded -lactoglobulin. The combined CD and FT-IR data indicated that refolded form of -lactoglobulin could be characterized as a molten globule state as it had native-like secondary structure and compromised tertiary structure.     

Fluorescence and Fourier-transform infrared spectroscopic studies on the role of disulfide bond in the calcium binding in the 33 kDa protein of Photosystem II

The 33 kDa protein of Photosystem II has one intrachain disulfide bond. Fluorescence spectroscopy shows that the major groups in the protein that bind to Ca²⁺ should be the carboxylic side groups of glutamic acid and/or aspartic acid. Fluorescence and Fourier-transform infrared (FTIR) spectroscopic studies indicate that the conformation of the 33 kDa protein is altered upon reduction, while the reduced protein still retains the secondary structure. FTIR spectroscopy also shows that the metal ions induce a relative decrease of unordered structure and -sheet, and a substantial increase of -helix in both the intact and the reduced 33 kDa protein. This indicates that the addition of cations results in a much more compact structure and that both the intact and the reduced 33 kDa proteins have the ability to bind calcium. The above results may suggest that the disulfide bridge is not essential for calcium binding.Abbreviations CD circular dichroism - FTIR Fourier transform infrared - La lanthanum - PS photosystem - Tb terbium     

Interactions of Aβ(1–40) with Glycerophosphocholine and Intact Erythrocyte Membranes: Fluorescence and Circular Dichroism Studies

Deposition of amyloid peptide in human brain in the form of senile plaques is a neuropathological hallmark of Alzheimers disease (AD). Levels of a phospholipid breakdown product, glycerophosphocholine (GPC), also increase in AD brain. The effect of GPC on amyloid (1–40) peptide (A) aggregation in PBS buffer was investigated by circular dichroism and fluoresence spectroscopy; interactions of A and GPC with the intact erythrocyte membrane was examined by fluoresence spectroscopy. Fluorescamine labeled A studies indicate GPC enhances A aggregation. CD spectroscopy reveals that A in the presence of GPC adopts 14% more -sheet structure than does A alone. Fluorescamine anisotropy measurements show that GPC and A interact in the phospholipid head-group region of the erythrocyte membrane. In summary, both soluble A and GPC insert into the phospholipid head-group region of the membrane where they interact leading to -sheet formation in soluble A which enhances A aggregation.     

Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice,Spitzbergen

The diversity of culturable bacteria associated with sea ice from four permanently cold fjords of Spitzbergen, Arctic Ocean, was investigated. A total of 116 psychrophilic and psychrotolerant strains were isolated under aerobic conditions at 4°C. The isolates were grouped using amplified rDNA restriction analysis fingerprinting and identified by partial sequencing of 16S rRNA gene. The bacterial isolates fell in five phylogenetic groups: subclasses and of Proteobacteria, the Bacillus–Clostridium group, the order Actinomycetales, and the Cytophaga–Flexibacter–Bacteroides (CFB) phylum. Over 70% of the isolates were affiliated with the Proteobacteria subclass. Based on phylogenetic analysis (<98% sequence similarity), over 40% of Arctic isolates represent potentially novel species or genera. Most of the isolates were psychrotolerant and grew optimally between 20 and 25°C. Only a few strains were psychrophilic, with an optimal growth at 10–15°C. The majority of the bacterial strains were able to secrete a broad range of cold-active hydrolytic enzymes into the medium at a cultivation temperature of 4°C. The isolates that are able to degrade proteins (skim milk, casein), lipids (olive oil), and polysaccharides (starch, pectin) account for, respectively, 56, 31, and 21% of sea-ice and seawater strains. The temperature dependences for enzyme production during growth and enzymatic activity were determined for two selected enzymes, -amylase and -galactosidase. Interestingly, high levels of enzyme productions were measured at growth temperatures between 4 and 10°C, and almost no production was detected at higher temperatures (20–30°C). Catalytic activity was detected even below the freezing point of water (at –5°C), demonstrating the unique properties of these enzymes.     

Cloning,expression and characterization of a beta-agarase gene from a marine bacterium,Pseudomonas sp. SK38

A gene (pagA) encoding -agarase from Pseudomonas sp. SK38 was cloned and expressed in Escherichia coli. The structural gene consists of 1011 bp encoding 337 amino acids with a predicted molecular weight of 37326 and has a signal peptide of 18 amino acids. The deduced amino acid sequence showed 57% and 58% homology to -agarase from Pseudoalteromonas atalntica and Aeromonas sp., respectively. The recombinant enzyme was purified and biochemically characterized. The enzyme had maximum activity at pH 9 and 30 °C. It was stable at pHs from 8 to 9 and below 37 °C.     

Expression of the human milk protein β-casein in transgenic potato plants

A 1177 bp cDNA fragment encoding the human milk protein -casein was introduced into Solanum tuberosum cells under control of the auxin-inducible, bidirectional mannopine synthase mas12) promoters using Agrobacterium tumefaciens-mediated leaf disc transformation methods. Antibiotic-resistant plants were regenerated and transformants selected based on luciferase activity carried by the expression vector containing the human -casein cDNA. The presence of human -casein cDNA in the plant genome was detected by PCR and DNA hybridization experiments. Human -casein mRNA was identified in leaf tissues of transgenic plants by RT-PCR analysis. Human - casein was identified in auxin-induced leaf and tuber tissues of transformed potato plants by immunoprecipitation and immunoblot analysis. Human -casein produced in transgenic plants migrated in polyacrylamide gels as a single band with an approximate molecular mass of 30 kDa. Immunoblot experiments identified approximately 0.01% of the total soluble protein of transgenic potato leaf tissue as -casein. The above experiments demonstrate the expression of human milk - casein as part of an edible food plant. These findings open the way for reconstitution of human milk inedible plants for replacement of bovine milk in baby foods for general improvement of infant nutrition, and for prevention of gastric and intestinal diseases in children     

NMR studies of Borrelia burgdorferi OspA, a 28 kDa protein containing a single-layer β-sheet

The crystal structure of outer surface protein A (OspA) from Borrelia burgdorferi contains a single-layer -sheet connecting the N- and C-terminal globular domains. The central -sheet consists largely of polar amino acids and it is solvent-exposed on both faces, which so far appears to be unique among known protein structures. We have accomplished nearly complete backbone H, C and N and C^;/H assignments of OspA (28 kDa) using standard triple resonance techniques without perdeuteration. This was made possible by recording spectra at a high temperature (45 °C ). The chemical shift index and ¹⁵N T₁/T₂ ratios show that both the secondary structure and the global conformation of OspA in solution are similar to the crystal structure, suggesting that the unique central -sheet is fairly rigid.     

Phosphorylation of membrane proteins by cytosolic casein kinases in human erythrocytes. Effect of monovalent ions, 2,3-bisphosphoglycerate and spermine

Summary Membrane proteins of human erythrocytes can be phosphorylated not only by membrane casein kinase (MS) but also by cytosolic casein kinases CS and CTS, resembling casein kinase I and II, respectively.Casein kinase CS, like membrane casein kinase MS, preferentially phosphorylates membrane proteins such as band 2 (spectrin, -subunit) and band 3, which are the major phosphate-acceptor proteins in the endogenous phosphorylation of isolated ghosts in the presence of [-³²P]ATP.By contrast, cytosolic casein kinase CTS phosphorylates, in addition to band 2, some membrane proteins, whose endogenous phosphorylation in isolated ghosts under the same conditions is negligible, if any.The CS- and CTS-catalyzed phosphorylations exhibit different response to increasing NaCl (or KCI) concentrations up to physiological levels (140 mM KCI, 20 mM NaCI); i.e. CS-and MS-catalyzed phosphorylations are strongly inhibited by 75–150 mM KCI (or NaCl), while CTS-catalyzed phosphorylation is practically unaffected.In the absence of added NaCl, CS- and MS-catalyzed phosphorylations are markedly inhibited by 1.5-3 mM 2,3-bisphosphoglycerate, whereas CTS-catalyzed phosphorylation appears to be practically unaffected.Finally, CS- and MS-catalyzed phosphorylations are slightly inhibited also by 1 mM spermine, while CTS-catalyzed phosphorylation is enhanced by this polycation concentration.     

Transforming growth factor-β1 immobilises dendritic cells within skin tumours and facilitates tumour escape from the immune system

Human skin tumours often regress spontaneously due to immune rejection. Murine skin tumours model this behaviour; some regress and others progress in syngeneic immunocompetent hosts. Previous studies have shown that progressor but not regressor skin tumours inhibit dendritic cell (DC) migration from the tumour to draining lymph nodes, and transforming growth factor-₁ (TGF-₁) has been identified as a responsible factor. To determine whether increased production of TGF-₁ in the absence of other differences inhibits DC migration from the tumour and enables it to evade immune destruction, a murine regressor squamous cell carcinoma clone was transfected with the gene for TGF-₁. This enhanced growth in vitro and in vivo, causing it to become a progressor. TGF-₁ transfection reduced the number of infiltrating DCs by about 25%. Quantitation of CD11c⁺ E-cadherin⁺ (epidermally derived) DCs in lymph nodes determined that TGF-₁ reduced the number of DCs that migrated from the tumour to undetectable levels. This was supported by showing that TGF-₁ reduced DC migration from cultured tumour explants by greater than tenfold. TGF-₁ transfection also reduced the number of infiltrating CD4 and CD8 T cells. Thus, TGF-₁ production by skin tumours is sufficient to immobilise DCs within the tumour, preventing their migration to lymph nodes. This reduces the number of T cells that infiltrate the tumour, preventing regression. Thus, TGF-₁ is a key regulator of whether skin tumours regress or progress.     

The genetic defect in the various types of human β-galactosidase deficiency

Summary Gene localization studies revealed the presence of two structural -galactosidase (GAL) loci on the human chromosomes 3 and 22 (de Wit et al., 1979). To determine the function of these genes, proliferating hybrid cell lines were isolated following fusion of fibroblasts from two different patients with a GAL deficiency and Chinese hamster cells. The hybrids were analyzed electrophoretically and immunologically.Fibroblasts from a patient with an adult type of GAL deficiency associated with a neuraminidase deficiency were used for the first fusion. No evidence for a structural GAL mutation was found in these hybrids. The absence of a structural GAL mutation is consistent with a primary defect in neuraminidase in this adult patient.Fibroblasts from a patient with the infantile type 1 G_M1-gangliosidosis were used for the second fusion. It is concluded that the human determinants present in the isolated hybrid lines occur in heteropolymeric man-Chinese hamster molecules. The heteropolymeric isoenzyme in (+3–22) hybrids is very labile and is sensitive to neuraminidase treatment. Therefore it is concluded that the infantile type 1 patient is mutated in the structural GAL gene on chromosome 3. Because this patient has a primary defect in G_M1-GAL, the GAL gene on chromosome 3 is apparently a G _M1-GAL gene. Interaction of the two GAL loci results in an additional band of GAL activity on electrophoresis. This suggests that the gene on chromosome 22 is also a structural G _M1-GAL gene.     

Production of a high activity of an extremely thermostable β-mannanase by the thermophilic eubacterium Rhodothermus marinus, grown on locust bean gum

The production of a highly thermostable mannanase by Rhodothermus marinus was increased 16.5-fold by optimising the concentrations of locust bean gum and yeast extract using central composite designs. The optimised medium and culture conditions yielded mannanase activity at 495 nkat ml–1 (248 nkat mg–1 protein). In addition, -L-arabinofuranosidase, -xylanase, -xylosidase, -glucosidase, -mannosidase, -galactosidase, -galactosidase and endoglucanase activities were detected at 32 nkat ml–1, 30 nkat ml–1, 16 nkat ml–1, 15 nkat ml–1, 0.1 nkat ml–1, 1 nkat ml–1, 0.5 nkat ml–1 and 8 nkat ml–1, respectively. No filter paper cellulase activity could be detected. The optimum pH of the mannanase was 5.0–6.5 and it showed high stability from pH 5 to 10 after 16 h incubation at 50 °C. The enzyme activity was maximum at 85 °C, with half lives of 45.3 h at 85 °C and 4.2 h at 90 °C. This is the first report on the production of such a high activity of extremely thermostable mannanase by an extreme thermophilic bacterium. © Rapid Science Ltd. 1998     

Evidence that lipolytic peptide B occurs in the ACTH/MSH-cells of the pituitary and in the brain

Summary Several lipid-mobilizing peptides occur in the pituitary, among them -lipotropin and lipolytic peptide A and peptide B. The latter two peptides are distinct from -lipotropin and appear to be chemically related to the neurophysins. Immunohistochemistry has now revealed that the lipolytic peptide B of the pituitary is localized in the ACTH- and MSH-cells. In addition, immunoreactive peptide B was found in axons of the posterior lobe of the pituitary. Immunoreactive peptide B was found also in nerve fibers and nerve cell bodies in the hypothalamus, particularly in the hypothalamo-hypophyseal tract and in the magnocellular neuronal system. Immunoreactive nerve fibers were numerous also in the periventricular nucleus of the thalamus. The antiserum against peptide B cross-reacts with neurophysin I, and hence, it cannot be excluded that at least part of the immunostaining in the brain reflects the presence of the latter component. However, the regional distribution of immunoreactive peptide B and neurophysin was not identical. Therefore, it is possible that authentic peptide B occurs not only in the pituitary but also in the brain.     

Binding of kynurenine to catecholamine

Summary Papiliochrome II is a pale yellow pigment of butterflies and consists of one molecule each ofL-kynurenine and N--alanyldopamine (NBAD). The aromatic amino nitrogen of kynurenine is bonded to the-carbon of NBAD. There are isomers IIa and IIb which show opposite circular dichroism. The-alanine contents of IIa and IIb were determined and the molar ratio of IIa to IIb has proved to be 1.17. The IIa and IIb were decomposed toL-kynurenine and N--alanylnorepinephrine (NBANE) by being heated in water at 80°C for 30 min. In both IIa and IIb, circular dichroism of the NBANE showed the same positive peak at 280 nm. The NBANE were further decomposed to-alanine and norepinephrine (NE) by being heated in 1 N HC1 at 100°C for 2 hr. The NE was submitted to enantioseparation and has proved to be a racemic mixture in both cases of IIa and IIb. These results are discussed in the light of the enzymic synthesis of IIa and IIb.