Paola Spadon

Crystal structure of the trigonal form of bovine beta-lactoglobulin and of its complex with retinol at 2.5 A resolution.

The structure of the trigonal crystal form of bovine beta-lactoglobulin has been determined by X-ray diffraction methods. An electron density map, calculated with phases obtained by the multiple isomorphous replacement method, served as a starting point for alternate cycles of model building and restrained least-squares refinement. The model of the molecule fitted to the initial Fourier map was the one built for the orthorhombic crystal form of beta-lactoglobulin, solved at 2.8 A resolution (1 A = 0.1 nm). The final R factor for 1456 atoms (1276 non-hydrogen protein atoms and 180 solvent atoms) is 0.22, including 5245 reflections from 6.0 to 2.5 A. The molecule shows significant differences in the two crystal forms mentioned, mainly due to different packing. In the trigonal form, the species crystallized does not appear to be dimeric, but a linear polymer with tight intermolecular contacts. A difference electron density map between the complex of beta-lactoglobulin with retinol and the native protein shows no significant peaks in the cavity which, in the similar retinol-binding protein, binds the chromophore. Instead, differences are found at a surface pocket, which is limited almost completely by hydrophobic residues.

Crystal structure of chicken liver basic fatty acid-binding protein at 2.7 Å resolution

The three-dimensional structure of chicken liver basic fatty acid-binding protein has been determined at 2.7 Å resolution by X-ray crystallography. Phases were calculated using the multiple isomorphous replacement procedure and a preliminary model was built. This model, with an initial R-factor of 0.57, was then improved by a cycle of refinement by simulated annealing which brought the R factor down to 0.32. The protein is structured as a compact 10-stranded-β-barrel which encapsulates a residual electron density that can be interpreted as a fatty acid molecule. The NH2-terminus portion of the molecule contains two short α-helices. The structure of this liver protein appears very similar to that of the Escherichia coli derived rat intestinal FABP recently determined by X-ray diffraction methods.

The primary structure of a basic (pI 9.0) fatty acid-binding protein from liver of Gallus domesticus

The complete amino acid sequence of a basic (pI 9.0) fatty acid-binding protein purified from liver of Gallus domesticus was determined by automated Edman degradation of tryptic, CNBr/HFBA and Staphylococcus aureus protease peptides. The protein contains 125 amino acid residues which correspond to a molecular mass of 14094. The identification of the blocked N-terminus Ac-Ala required digestion of a SV-8 peptide with the acylamino acid-releasing enzyme prior to sequence analysis. Sequence comparison shows that chicken liver basic-FABP has a significant similarity to other proteins belonging to the superfamily of intracellular lipid molecule binding proteins. Moreover, these sequence data confirm that basic-FABP probably binds its substrate in a slightly different way when compared with other FABPs. Basic-FABP was submitted to the EMBL Data Library with an accession number of P80226.

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 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, a = b = 104.2 A and c = 74.5 A. The crystals diffract to a resolution of 2.0 A.

Crystallization of hen eggwhite riboflavin-binding protein

Crystals of hen eggwhite riboflavin-binding protein have been grown by equilibrium dialysis in solutions buffered with 0.05 M-Tris X HCl (pH 8.5) using ammonium sulphate as the precipitant. The crystals belong to the space group P3121 (or enantiomorph) with a = b = 112.5 A and c = 72.0 A, and diffract to a resolution of 2.8 A.

Crystallographic Studies on Retinol-Binding Protein and Beta Lactoglobulin

It is not difficult to understand the need for a carrier for retinol in plasma since the molecule is not soluble in an aqueous medium. The protein that effects this function is called plasma Retinol-Binding Protein (RBP) (for a recent review see reference 1). Two other additional functions are performed by this protein: protection of the labile retinol molecule and selective delivery, since the vitamin is only transferred from the carrier to the cells that have a specific membrane receptor (2,3). Retinol-Binding Proteins have been characterized in the plasma of many species including man. The retinol transport system in human plasma includes another well known protein, transthyretin, formerly known as prealbumin. The two proteins form, under physiological conditions, a one to one complex that has an enhanced tendency to dissociate upon delivery of the vitamin to the receptors. Other conditions that favor dissociation are low ionic strength (4) and perhaps disruption of hydrophobic contacts (5).