Bicyclic peptides: solution conformation and Ca2+ binding of the heterodetic bicyclic decapeptide cyclo(Glu1-Leu2-Pro3-Gly4-Ser5-Ile6-Pro7-Ala8)- cyclo(1 gamma----5 beta)Phe9-Gly10

The conformational behavior of a heterodetic bicyclic decapeptide (BCPLT) in the absence and in the presence of calcium ions has been studied by means of mono and two-dimensional nmr techniques. Free BCPLT possesses a quite compact structure stabilized by intramolecular bonds and turns. In the structure a cluster of carbonyls is located in a cavity that is supposed to be the cation binding site.     

Genetic variation and distances of five Italian native sheep breeds

Genetic distances among five of the most important Italian native sheep breeds were estimated by using gene frequencies of four blood group and seven blood protein loci and by three different computing methods. The values of the three distance matrices were highly correlated. Genetic distances were found within the range values reported in the literature for local breeds. Shorter distances fitted the historical evidence of migrations.     

Chicken liver basic fatty acid-binding protein (pI = 9.0). Purification, crystallization and preliminary X-ray data

Chicken liver basic fatty acid-binding protein (pI = 9.0) has been purified with a high yield by a modification of a method originally applied to rat liver. The final product is highly homogeneous and can be used to grow crystals that belong to two different space groups. The crystals are either tetragonal, space group P4₂2₁2 with a = b = 60.2 Å and c = 138.1 Å or orthorhombic, space group P2₁2₁2₁ with a = 60.7 Å, b = 40.1 Å and c = 66.7 Å. The second form appears to be more suitable for X-ray diffraction studies, it diffracts to at least 2.8 Å resolution and it is believed to contain one protein molecule in the crystallographic asymmetric unit.     

Crystallization of human plasma apo-retinol-binding protein

Crystals of three forms of human plasma apo-retinol-binding protein have been obtained using the procedure described for the holoprotein. The apoprotein was prepared by a novel method, which uses hydrophobic interaction and immobilized dye chromatography. The three forms were separated by fast protein liquid chromatography. All of the crystals are isomorphous and diffract to 2.5 Å resolution. These crystals will be useful for studies of the mechanism of binding of retinol to its carrier using X-ray diffraction techniques.     

Crystallization and preliminary X-ray data of human plasma retinol-binding protein

Crystals of human plasma retinol-binding protein have been obtained from 4.5 m-NaCl buffered at pH 6.8 with 20 mm-cacodylate. The crystals are trigonal with space group R3 and unit cell dimensions, referred to the hexagonal system. $\text{a = b = 104.2}\text{A}\text{?}\text{and}\text{c = 74.5}\text{A}\text{?}$. The crystals diffract to a resolution of 2.0 Å.     

Comparison of AMP and NADH binding to glycogen phosphorylase b

The binding sites for the allosteric activator, AMP, to glycogen phosphorylase b are described in detail utilizing the more precise knowledge of the native structure obtained from crystallographic restrained least-squares refinement than has hitherto been available. Localized conformational changes are seen at the allosteric effector site that include shifts of between 1 and 2 A for residues Tyr75 and Arg309 and very small shifts for the region of residues 42 to 44 from the symmetry-related subunit. Kinetic studies demonstrate that NADH inhibits the AMP activation of glycogen phosphorylase b. Crystallographic binding studies at 3.5 A resolution show that NADH binds to the same sites on the enzyme as AMP, i.e. the allosteric effector site N, which is close to the subunit-subunit interface, and the nucleoside inhibitor site I, which is some 12 A from the catalytic site. The conformations of NADH at the two sites are different but both conformations are "folded" so that the nicotinamide ring is close (approx. 6 A) to the adenine ring. These conformations are compared with those suggested from solution studies and with the extended conformations observed in the single crystal structure of NAD+ and for NAD bound to dehydrogenases. Possible mechanisms for NADH inhibition of phosphorylase activation are discussed.     

Synergistic effect of phosphatidylserine with γ-aminobutyric acid in antagonizing the isoniazid-induced convulsions in mice

The influence of phosphatidylserine (PS) on the isoniazid-induced convulsions has been studied in mice. Sonicated dispersions of this phospholipid given intravenously do not show anticonvulsant activity but they do so when -aminobutyric acid (GABA) is simultaneously injected. GABA alone is inactive. The synergism between PS and GABA is influenced by the structure of the phospholipid liposomes. In contrast to multilamellar vesicles, oligolamellar vesicles are active. Under these conditions the effect shows head group specificity, in that the neutral phosphatidylcholine (PC) or the acidic phosphatidylinositol (PI) are inactive, either in the presence or in the absence of GABA. Lysophosphatidylserine (lysoPS), the deacylated PS derivative, shows increased efficacy as an isoniazid antagonist in the presence of GABA, and has anticonvulsant activity also in the absence of GABA. Other lysophospholipids are inactive. It is suggested that PS, after its metabolic conversion to lysoPS, enhances the anticonvulsant effect of GABA.     

Three-dimensional structure and active site of three hydrophobic molecule-binding proteins with significant amino acid sequence similarity.

We review our work on bovine and human retinol-binding protein (RBP), bovine beta lactoglobulin (BLG), and bovine odorant-binding protein (OBP). These three proteins share a sequence similarity high enough to justify the proposal that their three-dimensional structure ought to be quite similar, and they also share the function of similar or even identical hydrophobic ligand binding, although with a very different degree of specificity. Thus they constitute an ideal system to exhaustively explore the question of three-dimensional structure prediction from sequence similarity and the related question of binding site prediction for similar ligands. We have used x-ray diffraction techniques on single crystals of human and bovine RBP, bovine milk BLG, and bovine nasal mucosa OBP to investigate this problem. The results of these crystallographic studies indicate that to the level of resolution so far attained, the three-dimensional structure of these three proteins is reasonably predicted from the sequence similarity. The fold is the same and structural differences are rather subtle. Finally, we present experimental evidence that the binding sites of RBP, BLG, and OBP are in different regions of the molecules. Thus, it appears that although sequence alignment has correctly predicted the protein fold, it has incorrectly predicted the hydrophobic ligand-binding sites.