Circular dichroism and proteolysis of human beta-endorphin in surfactant and lipid solutions.

The influence of micelles of sodium dodecyl sulfate, cetyltrimethylammonium bromide, lysophosphatidylcholine and dodecylphosphorylcholine on the content and stability of the ordered structure of human beta-endorphin and its 12-26 fragment has been investigated. The structure was determined by far-ultraviolet circular dichroism and the stability by the resistance of the polypeptide to proteolysis with trypsin and chymotrypsin, monitored by HPLC. The alpha-helix inducing effects of the amphipathic compounds were in the order anionic greater than zwitterionic greater than cationic. The protection against proteolysis was very marked, especially for trypsin, and it was proportional to the alpha-helix inducing potential of amphipathic compounds. However, the lower resistance to proteolysis of the highly structured 12-26 fragment suggests that factors other than secondary structure may be responsible for the resistance to proteolysis.

Quantitative analysis of anaerobic metabolism in resting anoxic muscle by 31P and 1H MRS.

31P and 1H MRS high resolution measurements at 4.7 T were carried out in isolated frog (Rana esculenta) gastrocnemius muscle during anoxia to assess, using reference compounds, the concentration of high energy phosphates (approximately P = phosphocreatine (PC) plus adenosinetriphosphate (ATP)), inorganic phosphate (P(i)), phosphomonoesters (PME) and lactate (La): Two sets of measurements were performed, with (p) and without (up) muscle IAA poisoning and the time course of the metabolite concentration changes was described. The rate of phosphocreatine hydrolysis during the first phase of anaerobiosis, when no lactate is accumulated in either case, appears to be greater in p than in up preparations. This finding can be explained with the sizeable accumulation of phosphomonoesters (PME) in the former. The efficiency of anaerobic glycolysis, i.e. the approximately P/La ratio, recalculated taking into account also PME changes, was found to be 1.48 +/- 0.28, a value higher than that obtained by previous chemical measurements and close to the maximum stoichiometric approximately P/La value. Hence, the in vivo substrate of glycolysis, in the resting anoxic frog gastrocnemius, appears to be almost exclusively glycogen.

H-NMR studies of native and fragmented subtilisin Carlsberg.

NMR studies have been carried out on subtilisin Carlsberg in order to identify the sharp resonances observed in the proton spectra of the enzyme dissolved in aqueous solution. NMR spectra, obtained with the combination of spin-echo and selective excitation sequences, from both the native and inactivated protein, enabled us to assign sharp signals to subtilisin fragments derived from enzymatic autolysis (monitored by high-performance size-exclusion chromatography), rather than to mobile segments of the intact protein.

Unfolding and refolding of bovine beta-lactoglobulin monitored by hydrogen exchange measurements.

Bovine beta-lactoglobulin (beta-LG) is a widely studied protein belonging to the lipocalin family, whose structural characterisation has been reported by X-ray crystallography and NMR studies at physiological and acidic pH, respectively. Bovine beta-LG consists of nine antiparallel beta-sheets and a terminal alpha-helix segment. The beta-sheets form a calyx structure with a hydrophobic buried cluster conferring stability to the protein while a hydrophobic surface patch provides stabilising interactions between the barrel and the flanking terminal helix. Here, the stability and the folding properties of bovine beta-LG in the presence of a chemical denaturant is probed. The analysis of the NMR spectra recorded in aqueous solution with increasing amounts of urea revealed that the intensities of the backbone cross-peaks decrease upon increasing urea concentration, while their secondary shifts do not change significantly on going from 0 to 5 M urea, thus suggesting the presence of slow exchange between native and unfolded protein. Hydrogen exchange measurements at different urea concentrations were performed in order to evaluate the exchange rates of individual backbone amide protons. The opening reactions that determine protein exchange can be computed for the most slowly exchanging hydrogen atoms, and the measured exchange rates and the corresponding free energies can be expressed in terms of the equilibrium energetic for the global transition and the local fluctuations. Most of the residues converge to define a common isotherm identifying a unique cooperative folding unit, encompassing all the strands, except strand betaI, and the terminal region of the helix. The amides that do not join the same global unfolding isotherm are characterised by low DeltaGH20op and especially by low m values, indicating that they are already substantially exposed in the native state. A two-state unfolding model N <==> U is therefore proposed for this rather big protein, in agreement with CD data. Renaturation studies show that the unfolding mechanism is reversible up to 6 M urea and suggest a similar unfolding and refolding pathway. Present results are discussed in light of the hypothesis of an alpha-->beta transition proposed for bovine beta-LG refolding.

Bovine beta-lactoglobulin: interaction studies with palmitic acid.

Bovine beta-lactoglobulin (BLG) in vivo has been found complexed with fatty acids, especially palmitic and oleic acid. To elucidate the still unknown structure-function relationship in this protein, the interactions between 13C enriched palmitic acid (PA) and BLG were investigated by means of one-, two-, and three-dimensional NMR spectroscopy in the pH range 8.4-2.1. The NMR spectra revealed that at neutral pH the ligand is bound within the central cavity of BLG, with the methyl end deeply buried within the protein. The analysis of 13C spectra of the holo protein revealed the presence of conformational variability of bound PA carboxyl end in the pH range 8.4-5.9, related to the Tanford transition. The release of PA starts at pH lower than 6.0, and it is nearly complete at acidic pH. This finding is relevant in relation to the widely reported hypothesis that this protein can act as a transporter through the acidic gastric tract. Ligand binding and release is shown to be completely reversible over the entire pH range examined, differently from other fatty acid binding proteins whose behavior is analyzed throughout the paper. The mode of interaction of BLG is compatible with the proposed function of facilitating the digestion of milk fat during the neonatal period of calves.

Conformational study of a collagen peptide by 1H NMR spectroscopy: observation of the 14N-1H spin-spin coupling of the Arg guanidinium moiety in the triple-helix structure.

CB2, a CNBr peptide of 36 residues from type I collagen alpha1(I) chain has been studied by NMR spectroscopy as a function of temperature. At low temperature, the guanidinium protons of Arg9 showed sharp 1:1:1 NMR triplets around 6.95 ppm, characteristic of 14N coupled protons (1J(NH)=52 Hz) when the quadrupolar relaxation rate is drastically reduced. These spectral characteristics and the low temperature coefficient of the 1:1:1 triplets (delta delta/delta T of -3.6 ppb/degrees C) suggest that the H atoms of the protonated guanidinium moiety of Arg9 in the triple helix are slowly exchanging with bulk water, most likely involved in hydrogen bonds. On the basis of conformational energy computations on a model segment of type I collagen (Vitagliano, L., Némethy, G., Zagari, A. and Scheraga, H.A. (1993) Biochemistry 32, 7354-7359), similar to CB2, our data could indicate that the guanidinium group of Arg9 form hydrogen bonds with a backbone carbonyl of an adjacent chain probably by using the N(epsilon) hydrogen, leaving the four N(eta) hydrogens bound to water molecules that must be in slow exchange with bulk water and that could therefore be considered structural elements of the trimeric alpha1(I) CB2 triple helix. The behaviour of Arg9 has been investigated also in terms of equilibrium between random monomer and helical trimer conformations controlled by temperature. The thermal unfolding process was found to be reversible and the melting point resulted to be 17 degrees C.

Monomeric bovine beta-lactoglobulin adopts a beta-barrel fold at pH 2.

We have determined a crude structure of the apo form of bovine beta-lactoglobulin, a protein of 162 amino acid residues with a molecular mass of 18 kDa, at a low pH on the basis of data collected using only homonuclear 1H NMR spectroscopy. An ensemble of protein conformations was calculated with the distance-geometry algorithm for NMR applications (DYANA). The monomeric protein at low pH adopts a beta-barrel fold, well-superimposable on the structure determined by X-ray crystallography for the dimer at physiological pH. NMR evidence suggests the presence of disordered loop regions and terminal segments. Structural differences between the monomer at pH 2 and the dimer at pH 7, obtained by X-ray crystallography, are discussed, paying particular attention to surface electrostatic properties, in view of the high charge state of the protein at low pH.

Partially folded structure of monomeric bovine beta-lactoglobulin.

Bovine beta-LG (beta-lactoglobulin) has been studied under a variety of solution conditions by one- and two-dimensional NMR spectroscopy. At highly acidic pH (pH=2) and low ionic strength the protein is present in a monomeric form, exhibiting a highly structured beta-sheet core and less ordered regions as evidenced by both CD data and the NOESY spectra. Marginal protection was observed for most of the amide protons as a result of high conformational mobility. This structural state of beta-LG may be considered as an attractive model for a partially folded structure occurring late in the folding process of the protein.

The Sso7d DNA-binding protein from Sulfolobus solfataricus has ribonuclease activity.

Sso7d is a small, basic, abundant protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Previous research has shown that Sso7d can bind double-stranded DNA without sequence specificity by placing its triple-stranded beta-sheet across the minor groove. We previously found RNase activity both in preparations of Sso7d purified from its natural source and in recombinant, purified protein expressed in Escherichia coli. This paper provides conclusive evidence that supports the assignment of RNase activity to Sso7d, shown by the total absence of activity in the single-point mutants E35L and K12L, despite the preservation of their overall structure under the assay conditions. In keeping with our observation that the residues putatively involved in RNase activity and those playing a role in DNA binding are located on different surfaces of the molecule, the activity was not impaired in the presence of DNA. If a small synthetic RNA was used as a substrate, Sso7d attacked both predicted double- and single-stranded RNA stretches, with no evident preference for specific sequences or individual bases. Apparently, the more readily attacked bonds were those intrinsically more unstable.

Solid state and microscopy NMR study of the chemical constituents of Afzelia cuanzensis seeds.

Multi-echo, chemical shift selective, and 3D NMR imaging at microscopy resolution, and CP/MAS 13C NMR spectroscopy have been applied to the chemistry study of oil in oil-rich seeds of Afzelia cuanzensis, a tropical plant belonging to the Leguminosae taxum Caesalpinoideae or Caesalpinaceae.

Magnetic resonance imaging of molecular transport in living morning glory stems.

MRI was applied to investigate the transport pathways in Morning Glory plant stems. The study was carried out on living plants without affecting their integrity. The architecture of a dicotyledonous plant was deeply characterized: the root system structure and the vascular bundle location were identified, the presence of central voids caused by cell maturation and loss were observed in the stem. Molecular transport components were recognized, by observing the concentration profile of a tracer, which changed with time after its absorption by the plant roots. MRI analysis revealed the presence of an axial transport as the progress of the tracer front through the vascular bundles and a radial molecular transport from the vascular bundles toward the surface of the stem. As a result, the tracer molecular transport formed the parabolic tracer front (PTF). A model was built up through the analysis of the PTF that consisted of an axial front at the peak position and a radial front at the width of the parabolic tail. PTF analysis revealed differences between the tracer transport velocities in the axial and the radial directions in the plant stem. The model revealed that the width of the parabolic tail reflected the magnitudes of diffusion and permeation of the tracer in the plant stem.

Application of NMR microscopy to the morphological study of the silkworm, Bombyx mori, during its metamorphosis.

Zero (ZQ) and double (DQ) quantum 2D chemical shift selective and spin-echo 3D NMR imaging at microscopy resolution, has been applied to the morphological study of silkworm, Bombyx mori, during its metamorphosis. Attention had been focused on the evolution of the internal structure of the insect during its postembryonal life occurring through the larval, pupal and adult development. A major objective of this work was the characterization of the silk glands, responsible for the synthesis and secretion of fibroin and sericin, through the changes of distribution and mobility of water, by imaging the water protons during postembryonal life stages. Moreover, alanine deriving from silk gland proteins was imaged during the last life stage of Bombyx mori.

1H NMR spin-spin relaxation and imaging in porous systems: an application to the morphological study of white portland cement during hydration in the presence of organics.

Proton nuclear magnetic resonance (NMR) spin-spin relaxation and imaging have been applied to investigate white Portland cement pastes during hydration in the absence and in the presence of organic solvents. The main organic solvent investigated was methanol, alone or together with the organic waste 2-chloroaniline (2-CA), an aromatic amine representative of an important class of highly toxic compounds. For all the analysed samples, prepared with a solvent-to-cement ratio of 0.4, the decay of the echo magnetization has been fitted by adopting a model that combines an exponential component with a gaussian one. The calculated independent relaxation parameters have been discussed in terms of morphological and dynamical changes that occur during the cement hardening process and pore formation. Three kinds of water molecules: "solid-like" (chemically and physically bound), "liquid-like" (porous trapped) and "free" water, endowed with anisotropic, near isotropic and isotropic motion, respectively, were identified. Spin-echo images collected on the same samples during the hydration kinetics, allowed the changes of water and solvents spatial distribution in the porous network to be monitored, showing percolation phenomena and confirming the multimodal open channels structure of the hardened cement system. Both T(2) relaxation and imaging data indicated that a pronounced delay occurs in the cement hardening when organics are present.

1H-NMR of reverse micelles. II: Conformational studies of peptides and proteins in the AOT/water/isooctane system.

The interaction of AOT reverse micelles with Met-enkephalin, the pancreatic secretory trypsin inhibitor (PSTI) and the epidermal growth factor (EGF) is examined by NMR methods and the three systems are compared. While Met-enkephalin adopts a folded conformation, PSTI appears to become highly flexible, suggestive of a non-specific interaction with the micelles. On the other hand, the EGF spectrum shows that, although the main globular features of the protein are retained in the presence of AOT, the C-terminal fragment has to rearrange its conformation when put in contact with the micelle wall.

Reaction of 2-amino-2-deoxy-D-glucose and lysine: isolation and characterization of 2,5-bis(tetrahydroxybutyl)pyrazine.

The reaction of 2-amino-2-deoxy-D-glucose with lysine in water under simulated physiological conditions gave several browning products, with characteristic optical (lambda max 340 nm) and fluorescent properties (emission at 430 nm for excitation at 362 nm). The major product was isolated and characterized by mass spectrometry and n.m.r. spectroscopy as 2,5-bis(tetrahydroxybutyl)pyrazine derived by the condensation of two molecules of 2-amino-2-deoxy-D-glucose.

Characterization of the polymer formed from methylglyoxal in the presence of L(+)-lysine.

L(+)--lysine reacts with methylglyoxal in aqueous solution to give a yellow polymer. This polymer has been subjected to some chemical and physico-chemical characterizations. The gross structure of the polymer is probably formed by 3-hydroxypyrrolic nuclei, bridged together by vinylenic groups or -CH(OH)-CH2-CO-CH(OH)-groups.

Fibroblast growth factor-2 antagonist and antiangiogenic activity of long-pentraxin 3-derived synthetic peptides.

Angiogenesis and inflammation are closely integrated processes. Fibroblast growth factor-2 (FGF2) is a prototypic angiogenesis inducer belonging to the family of the heparin-binding FGF growth factors. FGF2 exerts its pro-angiogenic activity by interacting with various endothelial cell surface receptors, including tyrosine kinase receptors, heparan-sulfate proteoglycans, and integrins. A tight cross-talk exists between FGF2 and the inflammatory response in the modulation of blood vessel growth. Pentraxins act as soluble pattern recognition receptors with a wide range of functions in various pathophysiological conditions. The long-pentraxin PTX3 shares the C-terminal pentraxin-domain with short-pentraxins and possesses a unique N-terminal domain. These structural features indicate that PTX3 may have distinct biological/ligand recognition properties when compared to short-pentraxins. Co-expression of PTX3 and FGF2 has been observed in different inflammation/angiogenesis-dependent diseases. PTX3 binds FGF2 with high affinity and specificity. The interaction prevents the binding of FGF2 to its cognate tyrosine kinase receptors, leading to inhibition of the angiogenic activity of the growth factor. This suggests that PTX3 may exert a modulatory function by limiting the angiogenic activity of FGF2. An integrated approach that utilized PTX3 fragments, monoclonal antibodies, and surface plasmon resonance analysis has identified the FGF2-binding domain in the unique N-terminal extension of PTX3. On this basis, PTX3-derived synthetic peptides have been designed endowed with a significant antiangiogenic activity in vitro and in vivo. They may provide the basis for the development of novel antiangiogenic FGF2 antagonists.

Fast ternary and quaternary breakup of the 197Au + 197Au system in collisions at 15 MeV/nucleon.

A new reaction mechanism of violent reseparation of a heavy nucleus-nucleus system, 197Au + 197Au, into three or four massive fragments in collisions at 15 MeV/nucleon has been observed. After reseparation, the fragments are almost exactly aligned, thus showing a very short time scale of the reseparation process, of about 70-80 fm/c.