Ranking the affinity of aromatic residues for carbon nanotubes by using designed surfactant peptides.

A series of surfactant peptides were created to evaluate the affinity of aromatic AAs for single-walled carbon nanotubes in the absence of complications from peptide folding or self-association. Each surfactant peptide has a lipidlike architecture, with two Lys residues at the C-terminus as a hydrophilic head, five Val residues to form a hydrophobic tail, and the testing AA at the N-terminus. Raman and CD spectroscopic studies reveal that the surfactant peptides have a large unordered structural component which is independent of peptide concentration, suggesting that the peptides undergo minimal association under experimental conditions, thus removing this interference from interpretation of the peptide/carbon nanotube interactions. A lack of peptide self-association is also indicated by sedimentation equilibrium ultracentrifugation results. Optical spectroscopy of the peptide/carbon nanotube dispersions indicate that among the three aromatic AAs, tryptophan has the highest affinity for carbon nanotubes (both bundled and individual states) when incorporated into a surfactant peptide, while the Tyr-containing peptide is more selective for individual carbon nanotubes. Phe has the lowest overall affinity for carbon nanotubes. Raman spectra of dispersions made with SPF, SPY and SPW display similar types of nanotubes dispersed, although differences in the relative nanotube populations are observed by optical spectroscopy.     

Self-assembly of the octapeptide lanreotide and lanreotide-based derivatives: the role of the aromatic residues.

We investigated the spectroscopic properties of the aromatic residues in a set of octapeptides with various self-assembly properties. These octapeptides are based on lanreotide, a cyclic peptide analogue of somatostatin-14 that spontaneously self-assembles into very long and monodisperse hollow nanotubes. A previous study on these lanreotide-based derivatives has shown that the disulfide bridge, the peptide hairpin conformation and the aromatic residues are involved in the self-assembly process and that modification of these properties either decreases the self-assembly propensity or modifies the molecular packing resulting in different self-assembled architectures. In this study we probed the local environment of the aromatic residues, naphthyl-alanine, tryptophan and tyrosine, by Raman and fluorescence spectroscopy, comparing nonassembled peptides at low concentrations with the self-assembled ones at high concentrations. As expected, the spectroscopic characteristics of the aromatic residues were found to be sensitive to the peptide-peptide interactions. Among the most remarkable features we could record a very unusual Raman spectrum for the tyrosine of lanreotide in relation to its propensity to form H-bonds within the assemblies. In Lanreotide nanotubes, and also in the supramolecular architectures formed by its derivatives, the tryptophan side chain is water-exposed. Finally, the low fluorescence polarization of the peptide aggregates suggests that fluorescence energy transfer occurs within the nanotubes.     

Molecular origin of the self-assembly of lanreotide into nanotubes: a mutational approach

Lanreotide, a synthetic, therapeutic octapeptide analog of somatostatin, self-assembles in water into perfectly hollow and monodisperse (24-nm wide) nanotubes. Lanreotide is a cyclic octapeptide that contains three aromatic residues. The molecular packing of the peptide in the walls of a nanotube has recently been characterized, indicating four hierarchical levels of organization. This is a fascinating example of spontaneous self-organization, very similar to the formation of the gas vesicle walls of Halobacterium halobium. However, this unique peptide self-assembly raises important questions about its molecular origin. We adopted a directed mutation approach to determine the molecular parameters driving the formation of such a remarkable peptide architecture. We have modified the conformation by opening the cycle and by changing the conformation of a Lys residue, and we have also mutated the aromatic side chains of the peptide. We show that three parameters are essential for the formation of lanreotide nanotubes: i), the specificity of two of the three aromatic side chains, ii), the spatial arrangement of the hydrophilic and hydrophobic residues, and iii), the aromatic side chain in the β-turn of the molecule. When these molecular characteristics are modified, either the peptides lose their self-assembling capability or they form less-ordered architectures, such as amyloid fibers and curved lamellae. Thus we have determined key elements of the molecular origins of lanreotide nanotube formation.     

Self-association of collagen triple helic peptides into higher order structures

Interest in self-association of peptides and proteins is motivated by an interest in the mechanism of physiologically higher order assembly of proteins such as collagen as well as the mechanism of pathological aggregation such as beta-amyloid formation. The triple helical form of (Pro-Hyp-Gly)(10), a peptide that has proved a useful model for molecular features of collagen, was found to self-associate, and its association properties are reported here. Turbidity experiments indicate that the triple helical peptide self-assembles at neutral pH via a nucleation-growth mechanism, with a critical concentration near 1 mM. The associated form is more stable than individual molecules by about 25 degrees C, and the association is reversible. The rate of self-association increases with temperature, supporting an entropically favored process. After self-association, (Pro-Hyp-Gly)(10) forms branched filamentous structures, in contrast with the highly ordered axially periodic structure of collagen fibrils. Yet a number of characteristics of triple helix assembly for the peptide resemble those of collagen fibril formation. These include promotion of fibril formation by neutral pH and increasing temperature; inhibition by sugars; and a requirement for hydroxyproline. It is suggested that these similar features for peptide and collagen self-association are based on common lateral underlying interactions between triple helical molecules mediated by hydrogen-bonded hydration networks involving hydroxyproline.     

Fabrication and application of enzyme-incorporated peptide nanotubes

Enzyme engineering is a fast-growing field in the pharmaceutical and food markets. For those applications, various substrates have been examined to immobilize and stabilize enzymes. In this report, we examined peptide nanotubes as supports for enzymes. When a model enzyme, Candida rugosa lipase, was encapsulated in peptide nanotubes, the catalytic activity of nanotube-bound lipases was increased 33% as compared to free-standing lipases at room temperature. At an elevated temperature, 65 degrees C, the activity of lipases inside the nanotubes was 70% higher than free-standing lipases. The activity enhancement of lipases in the peptide nanotubes is likely induced by the conformation change of lipases to the open form (the enzymatically active structure) as lipases are adsorbed on the inner surfaces of peptide nanotubes.     

Influence of transmembrane peptides on bilayers of phosphatidylcholines with different acyl chain lengths studied by solid-state NMR

The molecular orientation in a lipid membrane of the peptide fragment VEYAGIALFFVAAVLTLWSMLQYLSAAR (phosphatidylglycerophosphate synthase (Pgs) peptide E) of an integral membrane protein, Pgs, in Escherichia coli has been investigated by solid-state 15N nuclear magnetic resonance (NMR) on macroscopically aligned lipid bilayers. The secondary structure of the peptide in lipid vesicles was determined by circular dichroism spectroscopy. Furthermore, the phase behaviour of the Pgs peptide E/dierucoylphosphatidylcholine (DEruPC)/water system was determined by (2)H, (31)P and 15N solid-state NMR spectroscopy. The phase behaviour obtained was then compared to that of the Pgs peptide E solubilised in dioleoylphosphatidylcholine and water that was previously studied by Morein et al. [Biophys. J. 73 (1997) 3078-3088]. This was aimed to answer the question whether a difference in the length of the hydrophobic part of this peptide and the hydrophobic thickness of the lipid bilayer (hydrophobic mismatch) will affect the phase behaviour. The peptide mostly has a transmembrane orientation and is in an alpha-helical conformation. An isotropic phase is formed in DEruPC with high peptide content (peptide/lipid molar ratio (p/l) > or =1:15) and high water content (> or =50%, w/w) at 35 degrees C. At 55 and 65 degrees C an isotropic phase is induced at high water content (> or =50%, w/w) at all peptide contents studied (no isotropic phase forms in the lipid/water system under the conditions in this study). At high peptide contents (p/l> or =1:15) an isotropic phase forms at 20 and 40% (w/w) of water at 55 and 65 degrees C. A comparison of the phase behaviour of the two homologous lipid systems reveals striking similarities, although the thicknesses of the two lipid bilayers differ by 7 A. This suggests that the rationalisation of the phase behaviour in terms of the hydrophobic mismatch is not applicable to these systems. The C-terminus of Pgs peptide E is amphiphilic and a considerable part of the peptide is situated outside the hydrophobic part of the bilayer, a property of the peptide that to a large extent will affect the lipid/peptide phase behaviour.     

A Hot Aldehyde-Peroxide Fixation Method for Electron Microscopy of the Free-Living Nematode Caenorhabditis Elegans

An improved method for the fixation of the third and fourth larval stages and adults of Caenorhabditis clegans has been developed. Worms are placed in a mixture of 1.5% paraformaldehyde and 1.0% glutaraldehyde at pH 7.0 and 70 C and the suspension promptly cooled in a water bath at 20 C for 1 hr. The fixed worms are then immersed in a mixture of 5% glutaraldehyde and hydrogen peroxide at 4 C for 1 hr, stained en bloc in uranyl acetate, and embedded in resin for electron microscopy. The procedure results in superior fixation, particularly of micro filaments and micro tubules. The high temperature of the initial fixation straightens the worms and thus facilitates serial sectioning.     

Aspects of the molecular structure and dynamics of neuropeptide Y.

Human neuropeptide Y (hNPY) and the Q34-->P34 mutant (P34-hNPY) have been characterized by CD spectroscopy. hNPY self-associates in aqueous solution with a dimerization constant in the micromolar range. The self-association correlates with an increase in secondary-structure content which was studied as a function of concentration, temperature and pH. The effects of temperature were measured in water (5-84 degrees C) and in ethanediol/water (2 : 1) (-90 degrees to +90 degrees C). A single-residue mutation, Q34-->P34, affects the pH, thermal and self-association properties of NPY. The CD results are correlated with photochemically induced dynamic nuclear polarization NMR experiments which show that the tyrosines at the interface between two monomer units present limited accessibility to a photoreactive dye. An equilibrium state is described, involving a PP-fold monomer form and a handshake dimer form, that accommodates the physicochemical properties of NPY.     

Helical structure and self-association in a 13 residue neuropeptide Y Y2 receptor agonist: relationship to biological activity

The solution structure and self-association behaviour of a 13 residue peptide analogue of the C-terminal region of human neuropeptide Y (NPY) have been investigated. NMR analysis of Ac[Leu(28,31)]NPY(24-36), a potent Y2 receptor agonist, shows that it is unstructured in aqueous solution at 5-20 degrees C, but forms a well-defined helix (encompassing residues 25-35) in 40% trifluoroethanol/water at 20 degrees C. Sedimentation experiments show that, in contrast to many peptides in aqueous trifluoroethanol, Ac[Leu(28,31)]NPY(24-36) associates to form a trimer or, more likely, a tetramer in 40% trifluoroethanol, even though it is monomeric in water. This is consistent with the observation of inter-molecular nuclear Overhauser enhancements in trifluoroethanol. Possible models of the associated form that are consistent with the NMR data are described. The relevance of the helical structure observed in trifluoroethanol to the structure of this peptide bound to the NPY Y2 receptor is discussed.     

Coexistence of a two-states organization for a cell-penetrating peptide in lipid bilayer

Primary amphipathic cell-penetrating peptides transport cargoes across cell membranes with high efficiency and low lytic activity. These primary amphipathic peptides were previously shown to form aggregates or supramolecular structures in mixed lipid-peptide monolayers, but their behavior in lipid bilayers remains to be characterized. Using atomic force microscopy, we have examined the interactions of P(alpha), a primary amphipathic cell-penetrating peptide which remains alpha-helical whatever the environment, with dipalmitoylphosphatidylcholine (DPPC) bilayers. Addition of P(alpha) at concentrations up to 5 mol % markedly modified the supported bilayers topography. Long and thin filaments lying flat at the membrane surface coexisted with deeply embedded peptides which induced a local thinning of the bilayer. On the other hand, addition of P(alpha) only exerted very limited effects on the corresponding liposome's bilayer physical state, as estimated from differential scanning calorimetry and diphenylhexatriene fluorescence anisotropy experiments. The use of a gel-fluid phase separated supported bilayers made of a dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine mixture confirmed both the existence of long filaments, which at low peptide concentration were preferentially localized in the fluid phase domains and the membrane disorganizing effects of 5 mol % P(alpha). The simultaneous two-states organization of P(alpha), at the membrane surface and deeply embedded in the bilayer, may be involved in the transmembrane carrier function of this primary amphipathic peptide.     

Labeling and quality control of 188Re-lanreotide.

Lanreotide was labelled with 188Re obtained from 188W/188Re generator, using stannous ion as reducing agent, ascorbic acid as stabilizers and hydroxy ethylidene bisphosphonate (HEDP) as intermediary ligand at different molar ratios, pH and incubation times. Best yields (>95%) were obtained using molar ratios SnF2/lanreotide, ascorbic/lanreotide and HEDP/lanreotide of 40, 12 and 260, respectively, pH 1-2 with an incubation at 100 degrees C for 30 min. Quality control evaluation and stability of the radiolabel compound was done by the following selected methods: chromatography in Whatman 3 MM with MEK and NaCl 0.15 M as solvents, ITLC-SG with ethanol-HCl 0.01N (90:10); reverse phase extraction cartridge (Sep-pak C18, Waters Associated) and RP-HPLC with radiometric and UV detection (220 nm) using MCH-5 n-capp column with linear gradient from 90% H2O (TFA 0.1%): 10% ACN (TFA 0.1%) up to 10% H2O (TFA 0.1%):90% ACN (TFA 0.1%) in 30 min, at flow 1 ml/min. Biodistribution in normal mice showed that 188Re-lanreotide is excreted mainly through the hepatobiliary system: more than 70% I.D. is present in gallbladder and intestines at 2 hr post injection. The stability of the 188Re-peptide bond by cysteine challenge test at 37 degrees C, during 2 and 24 hr of incubation time, reveals that approximately 300 and 100 molar ratio cys/peptide is required to displace 50% of the 188Re from the complex. In vitro stability of 188Re-lanreotide at room temperature (Rt) was demonstrated during 24 hr Future works must be done in order to investigate its binding capacity to somatostatin receptors.     

Thermodynamic, thermomechanical, and structural properties of a hydrated asymmetric phosphatidylcholine.

1-Behenyl-2-lauryl-sn-glycero-3-phosphocholine (22/12 PC) belongs to a unique group of phospholipids in which the molecule has one acyl chain almost twice as long as the other. The temperature-composition phase diagram for this lipid in the range of 25-65 degrees C, and 0 to 84.3% (w/w) water has been constructed by using the isoplethal method in the heating direction and x-ray diffraction for phase identification and structure characterization. At water contents between 10.3 and 34% (w/w) and at temperatures below 43 degrees C, a single mixed interdigitated lamellar gel phase (Lm beta, [symbol: see text]) of the type described by Hui et al. (1984. Biochemistry. 23:5570-5577) and McIntosh et al. (1984. Biochemistry. 23:4038-4044) was found. A second phase consisting of bulk aqueous solution coexists with the Lm beta phase at hydration levels above 34% (w/w) water in the temperature range between 25 and 43 degrees C. Above 43 degrees C, a partially interdigitated lamellar liquid crystalline (Lp alpha) phase ([symbol: see text]) is seen in the water concentration range extending from 0 to 84.3% (w/w). The pure Lp alpha phase is found below 43% (w/w) water, while coexistence of the Lp alpha phase and the bulk aqueous solution is observed above this water concentration which marks the hydration boundary. Interestingly, the latter boundary for both Lm beta and Lp alpha phases is nearly vertical in the temperature range studied. Furthermore, the lamellar chain-melting transition temperature appears to be relatively insensitive to hydration in the range 0-85% (w/w) water. We have confirmed the identify of the Lm beta phase by constructing a 5.7-A resolution electron density profile on oriented samples by the swelling method. Temperature-induced chain melting effects an increase in lipid bilayer thickness suggesting that the Lp alpha phase has chains packed in the partially as opposed to the mixed interdigitated configuration. Unlike the symmetric phosphatidylcholines a ripple (P beta') phase was not found as an intermediate between the low and high temperature lamellar phases of 22/12 PC. The specific volume of 22/12 PC is 940 (+/- 1) microliter/g and 946 (+/- 1) microliter/g in the hydrated lamellar gel state at 28 (+/- 2) and 40 (+/- 2) degrees C, respectively, from neutral buoyancy experiments. Based on measurements of the temperature dependence of the various lattice parameters of the different phases encountered in this study the corresponding lattice thermal expansion coefficients have been measured. These are discussed and their dependence on lipid hydration is reported.     

A differential scanning calorimetric study of chymotrypsin in the presence of added polymers

Scanning calorimetry measurements of different amounts of chymotrypsin in water alone gave a temperature of denaturation (T(d)) value of 54 degrees C. However, when high-molecular-weight poly(ethylene glycol) was added to aqueous solutions of chymotrypsin, the thermostability of the enzyme was enhanced. For example, the addition of 20% (w/w) of poly(ethylene glycol) of molecular weight of 100,000 increased the T(d/) value to 66 degrees C. In toluene containing various amounts of added water, ethyl cellulose was used to improve the thermostability of chymotrypsin. For this system, a T(d) value of 82 degrees C was obtained with a 20% (w/w/) concentration of ethyl cellulose and 2% (v/v) of added water. Polymers in these solvents interact with water, which could otherwise denature the enzyme; polymers also from complexes with enzyme molecules to produce a more stable structure. (c) 1994 John Wiley & Sons, Inc.     

Extruding foams from corn starch acetate and native corn starch

Because of the hydrophilic characteristics of native starch foams and the cost of modifying starch, the uses of starch and modified starch foams are hindered. To decrease hydrophilicity and cost of starch foams, native corn starch was blended with starch acetate and extruded. A twin-screw mixing extruder was used to produce the foams. Native starch content, screw speed, and barrel temperature had significant effects on molecular degradation of starches during extrusion. The melting temperature of extruded starch acetate/native starch foam was higher (216 degrees C) than that for starch acetate (193.4 degrees C). Strong peaks in the X-ray diffractograms of extruded starch acetate/native starch foam suggested new crystalline regions were formed. Optimum conditions for high radial expansion ratio, high compressibility, low specific mechanical energy requirement, and low water absorption index were 46.0% native starch content, 163 rpm screw speed, and 148 degrees C barrel temperature.     

Influence of membrane-spanning alpha-helical peptides on the phase behavior of the dioleoylphosphatidylcholine/water system

The effect of solubilized hydrophobic peptides on the phase behavior of dioleoylphosphatidylcholine (DOPC)/water system was studied by 2H- and 31P-NMR spectroscopy and by x-ray diffraction, and partial phase diagrams were constructed. The utilized peptides were HCO-AWW(LA)5WWA-NHCH2CH2OH (WALP16), which is an artificial peptide designed to resemble a transmembrane part of a membrane protein; and VEYAGIALFFVAAVLTLWSMLQYLSAAR (Pgs peptide E), a peptide that is identical to one of the putative transmembrane segments of the membrane-associated protein phosphatidylglycerophosphate synthase (Pgs) in Escherichia coli. Circular dichroism spectroscopy suggests that both peptides are mostly alpha-helical in DOPC vesicles. The most striking features in the phase diagram of the WALP16/DOPC/water system are 1) a single lamellar liquid crystalline (L alpha) phase forms only at very low peptide concentrations. 2) At low water content and above a peptide/lipid molar ratio of approximately 1:75 a reversed hexagonal liquid crystalline (H[II]) phase coexists with an L alpha phase, while in excess water this phase forms at a peptide/lipid molar ratio of approximately 1:25. 3) At peptide/lipid ratios > or =1:6 a single H(II) phase is stable. Also, the Pgs peptide E strongly affects the phase behavior, and a single L alpha phase is only found at low peptide concentrations (peptide/lipid molar ratios <1:50), and water concentrations <45% (w/w). Higher peptide content results in coexistence of L alpha and isotropic phases. Generally, the fraction of the isotropic phase increases with increasing temperature and water concentration, and at 80% (w/w) water content only a single isotropic phase is stable at 55 degrees C. Thus, both peptides were found to be able to induce nonlamellar phases, although different in structure, in the DOPC/water system. The phase transitions, the extensions of the one-phase regions, and the phase structures observed for the two systems are discussed in terms of the molecular structure of the two peptides and the matching between the hydrophobic lengths of the peptides and the bilayer thickness of DOPC.     

Mass transfer and temperature effects on substrate utilization in brewery granules

Liquid film and diffusional resistances of brewery granules during acetate, propionate, and ethanol utilization were investigated. Substrate utilization rate increased with decreased granule size. Effectiveness factors for acetate, propionate, and ethanol were calculated by comparing the maximum rates of substrate utilization of whole granules (1.8 to 3.0 mm) and fine flocs (20 to 75 mum) derived by disrupting whole granules. For acetate, propionate, and ethanol, maximum specific substrate utilization rates (k(m') g/g VS . d) for the flocs, were 5.11, 6.25, and 5.49, respectively, and half-velocity coefficients (K(g') mM) were 0.45, 0.40, and 3.37, respectively. Calculated effectiveness factors were 0.32, 0.41, and 0.75 for acetate, propionate, and ethanol, respectively. The effect of temperature on substrate utilization was examined at 26 degrees C, 31 degrees C, and 37 degrees C using acetate as sole carbon source. Utilization rates increased with temperature. Flocs were most sensitive to temperature, and whole granules were least affected. The behavior of flocs was well described by the Van't Hoff-Arrhenius equation. Effectiveness factors for acetate utilization by the granules were 0.36, 0.35, and 0.32 at 26 degrees C, 31 degrees C, and 37 degrees C, respectively, indicating little effect of temperature. Based on these results, we conclude that both liquid film and diffusional resistances influenced the rate of substrate utilization in a UASB reactor with granular sludge. Temperature effects were much less important than diffusional limitations within the granules. (c) 1995 John Wiley & Sons, Inc.     

Role of lipids in the Neurospora crassa membrane. II. Membrane potential and resistance studies; the effect of altered fatty acid composition on the electrical properties of the cell membrane.

The effect of doubling the saturated fatty acid content on the electrophysiology of Neurospora crassa membranes was studied. Intracellular membrane input resistance (Rm) and potential (Em) were measured for wild-type (w/t) and cel- (Tween 40) organisms as a function of temperature. Over the 0 to 40 degrees C temperature range studied, mean Em values of both w/t and cel- (Tw 40) organisms increased from -160 to -210 mV. This difference is greater than that expected from Nernst potential considerations, indicating an active component of Em. This active component is insensitive to a doubling of the saturated fatty acid content. Rm exhibits a temperature dependence and hysteresis. Averaged data indicate an increase in Rm with decreased temperature. The slope of the temperature dependence varies among individual hyphae. Above 17.5 degrees C cel- (Tw 40) hyphae averaged greater than 70% higher values of Rm than w/t. Below 17.5 degrees C w/t Rm data divided into low and high temperature dependence groups, while cel- data exhibited a low temperature dependence. The results are discussed in relation to gel-liquid crystal phase transitions, membrane fluidity, and the contribution of fatty acid structure to membrane electrical properties.     

Motility inOscillatoria salina as affected by different factors

All 3-10-d-old Oscillatoria salina filaments glide with the speed of 323-330 microm/min (BG 11 medium, pH 7.5, 21 +/- 2 degrees C, continuous light intensity of approximately 30 micromol m(-2) s(-1)) in a culture chamber. However, a time bound progressive decrease in gliding speed and in percentage of gliding filaments occurred, depending upon the severity of different stress factors studied, viz. water stress (2-8% agarized media, liquid media with 0.2-1 mol/L NaCl, blot-dryness of filaments for > or = 5 min), temperature shock (5, 40 degrees C for > or = 5 min; 35 degrees C for > or = 15 min), darkness and low light intensity (2, 10 micromol m(-2) s(-1)), UV exposure (0.96-3.84 kJ/m2), pH extremes (< or = 6.5 and > or = 9.5), lack of all nutrients from liquid medium (double distilled water), presence of 'heavy' metals (1, 25 ppm Fe, Cu, Zn, Ni, Co, Hg) or organic substances in liquid medium (25, 250 ppm 2,4-D, captan, urea, DDT, thiourea). This feature of the alga (i.e. reduction in speed and percentage of gliding filaments depending upon severity of stress conditions) may thus be suggested to be used in assessing water quality.     

Water, temperature and gas composition interactions affect growth and ochratoxin A production by isolates of Penicillium verrucosum on wheat grain

AIMS: To examine the effect of interactions between water, temperature and gas composition on growth and ochratoxin A (OTA) production by isolates of Penicillium verrucosum in vitro and in situ on grain-based media and wheat grain. METHODS AND RESULTS: Three isolates of P. verrucosum were examined in relation to radial growth rate and OTA production, and to interacting conditions of water activity (a(w)), temperature and gas composition on a milled wheat medium. Subsequently, detailed temporal studies were carried out on gamma irradiated wheat grain over the range 0.75-0.995 a(w), 10-25 degrees C and air, 25 or 50% CO(2). This showed that optimum growth of P. verrucosum was at 0.98 a(w) in vitro at 25 degrees C, but at 0.95 a(w) and 25 degrees C on wheat grain. The a(w) minimum for growth was about 0.80 a(w), although no OTA was produced under this condition even after 56 days. Significant inhibition of growth and OTA production occurred with 50% CO(2), and 0.90-0.995 a(w) at 25 degrees C. CONCLUSIONS: The optimum and marginal conditions for growth and OTA production on wheat grain have been identified. At least 50% CO(2) is needed to inhibit growth and OTA production by >75% in moist grain (0.90-0.995 a(w)). SIGNIFICANCE AND IMPACT OF THE STUDY: First detailed identification of optimal and marginal interacting conditions of water/temperature and gas composition on growth and OTA production by P. verrucosum on wheat grain. This is a critical component of the postharvest management strategy for minimizing contamination by this important mycotoxin and predicting risk, based on environmental conditions, during drying and storage.     

Conformational analysis of the hydrophobic peptide alphas1-casein(136-196).

Hydrophobic interactions are important in the self-association of milk proteins, including alphas1-casein. The extent to which casein interaction sites are influenced by local secondary structure is not widely known. Both primary amino acid sequence and local secondary structure are shown to affect the self-association of the hydrophobic peptide alphas1-casein(136-196). The peptide is aggregated at low concentrations (7 microM and above), as determined by 1H nuclear magnetic resonance (NMR) measurements at pH 6.0 in phosphate buffer. Increase in temperature is shown to induce side chain mobility (melting) as indicated by both 1H NMR and near-UV circular dichroism (CD) measurements. As determined by far-UV CD, there is also a loss in the global amount of extended structure with increasing temperature, while beta-turn structures and some aromatic dichroism are conserved at temperatures as high as 70 degrees C. Similar retention of structure occurs at pH 2 and in 6 M guanidine HCl. The observed stability of beta-turns and some side chains in alphas1-casein(136-196) supports previous assumptions that hydrophobic, proline-based turns are important interaction sites in the self-association of alphas1-casein, and possibly in the formation of the calcium transport complexes, the casein micelles. It may be speculated that these areas of the peptide represent a 'molten globule-like', heat stable, core structure for alphas1-casein.