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.

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.

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.

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 P4(2)2(1)2 with a = b = 60.2 A and c = 138.1 A or orthorhombic, space group P2(1)2(1)2(1) with a = 60.7 A, b = 40.1 A and c = 66.7 A. The second form appears to be more suitable for X-ray diffraction studies, it diffracts to at least 2.8 A resolution and it is believed to contain one protein molecule in the crystallographic asymmetric unit.

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.

A possible route for the release of fatty acid from fatty acid-binding protein.

A simulation of the release of fatty acid from intestinal fatty acid-binding protein was attempted, starting with the crystallographic model and using molecular-dynamic processes at different temperatures. The release of the ligand was observed only at high temperature, which perhaps makes the process unreliable in detail. Nevertheless, the overall behaviour of the protein, also confirmed by the simulation performed at room temperature, strongly supports the idea that the fatty acid leaves the protein through an opening formed by alpha-helix II and turns beta C-beta D and beta E-beta F. Additionally, it suggests a role for the lack of hydrogen bonds between the main chains of beta-strands D and E: this feature, observed in all the protein structures of this family which have currently been determined, seems to provide the structure with great flexibility, allowing the barrel to open and close without disruption of the hydrogen-bond network.

X-ray study on homo-oligopeptides t-Boc(L-Nva)6OMe and HCl.H(L-Nva)6OMe.

Observations of extended peptide chains, whose direction is perpendicular to the fiber axis (cross-beta-structure) have hitherto been confined to fibrous proteins and to some synthetic polydisperse polypeptides of rather low molecular weight. This structure has now been found in some monodisperse linear homo-oligopeptides with aliphatic hydrocarbon side chains. X-ray fiber diagrams of t-Boc(L-Nva)6OMe and HCl.H(L-Nva)6OMe show the characteristic reflections of this form. In addition, the good orientation of suitably prepared specimens has enabled a fairly complete determination of the hexapeptides unit cell to be made. Both molecules are packed in a pseudomonoclinic lattice whose dimensions are: a = 4.80, b = 59.0, c = 14.86 A, beta = 85 degrees and a = 4.80, b = 58.50, c = 14.84 and beta = 86 degrees for t-Boc(L-Nva)6-OMe and HCl.H(L-Nva)6OMe respectively. It has not been possible from the available experimental data to establish whether the arrangement of the chains within the sheets is parallel or antiparallel.

Conformational and binding properties of chicken liver basic fatty acid binding protein in solution.

The conformation of basic fatty acid binding protein from chicken liver and the binding properties of the apo protein toward 11-dansylamino-undecanoic acid were investigated by CD and fluorescence spectroscopy. In one set of experiments the binding process was followed by the appearance of induced optical activity in the absorption region of the dansyl chromophore. In a second set of experiments the binding process was followed by the large enhancement of emission fluorescence of the dansyl fluorophore. From the saturation curves, the stoichiometry of the complex and the binding constant of the fatty acid to the protein were precisely determined. The values of the dissociation constant determined with the two methods were in excellent agreement: we obtained KD = (1.0 +/- 0.1) x 10(-6) M in a 0.9: 1 stoichiometry. The native conformation of the protein is remarkably stable in a variety of solvent systems, including acetonitrile-water, ethylene glycol-water, and dioxane-water of various compositions. The CD results also showed that the binding of the fatty acid does not induce any appreciable change in the protein conformation. In a mixture of water and 2,2,2-trifluoroethanol 1:9 (v/v), the native conformation collapses and a new ordered structure is formed, characterized by a high amount of alpha-helix.

Three-dimensional structure of recombinant human muscle fatty acid-binding protein.

The three-dimensional structure of recombinant human muscle fatty acid-binding protein with a bound fatty acid has been solved and refined with x-ray diffraction data to 2.1 A resolution. The refined model has a crystallographic R factor of 19.5% for data between 9.0 and 2.1 A (7243 unique reflections) and root-mean-square deviations in bond length and bond angle of 0.013 A and 2.7 degrees. The protein contains 10 antiparallel beta-strands and two short alpha-helices which are arranged into two approximately orthogonal beta-sheets. Difference electron density maps and a multiple isomorphous derivative electron density map showed the presence of a single bound molecule of a long chain fatty acid within the interior core of the protein. The hydrocarbon tail of the fatty acid was found to be in a "U-shaped" conformation. Seven ordered water molecules were also identified within the interior of the protein in a pocket on the pseudo-si face of the fatty acid's bent hydrocarbon tail. The methylene tail of the fatty acid forms van der Waals interactions with atoms from 13 residues and three ordered waters. The carboxylate of the fatty acid is located in the interior of the protein where it forms hydrogen bonds with the side chains of Tyr128 and Arg126 and two ordered water molecules. A comparison of the three-dimensional structure of human muscle fatty acid-binding protein and rat intestinal fatty acid-binding protein shows strong similarity. Both proteins bind a single fatty acid within their interior cores, but the bound fatty acids are very different in their conformations and interactions. These findings suggest that the intestinal and muscle fatty acid-binding proteins have evolved distinct binding sites in order to satisfy different requirements within the tissues where they are expressed.

X-ray study on homo-oligopeptides t-BOC-(D-Ala)7-OMe and HCl.H(D-Ala)7-OMe.

Observations of extended peptide chains, whose direction is perpendicular to the fiber axis (cross-beta-structures) have hitherto been confined to fibrous proteins and to some synthetic polydisperse polypeptides of rather low molecular weight. This structure has now been found in some monodisperse linear homo-oligopeptides with aliphatic hydrocarbon side chains. X-ray diffraction photographs of t-Boc-(D-Ala)7-OMe and HCl.H(D-Ala)7-OMe show characteristic reflections for this form. In addition, the good orientation of suitably prepared specimens has enabled a fairly complete determination of the unit cell of the heptapeptides to be made. t-Boc-(D-Ala)7-OMe and HCl.H(D-Ala)7-OMe molecules are packed in a monoclinic lattice with a = 4.80, b = 34.5, c = 5.82 A, beta = 65.6 degrees and a = 4.80, b = 29.5, c = 5.82 A, beta = 65.6 degrees, respectively. It has not been possible to establish whether the arrangement of the chains within the sheets is parallel or antiparallel.

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

The three-dimensional structure of chicken liver basic fatty acid-binding protein has been determined at 2.7 A 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-beta-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 alpha-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.