The number of proteins being included in the lipocalin structural family has been steadily increasing since the solution of the structures of beta -lactoglobulin and retinol- binding protein. The family now comprises some 23 proteins and these come from many different sources and display a variety of characteristics. The work described here focused on two of those proteins, beta -lactoglobulin (Big) and apolipoprotein D (apo D).
The structure of bovine beta -lactoglobulin (Monaco, et al., 1987)from the trigonal crystals grown at pH 7.8, space group P3221, presents several aspects that are in disagreement with previous work, in particular the positioning of the bound retinol in the surface of the protein and the observation that the protein was in its monomeric form. We have checked these findings independently by extending the resolution of a 6 A x -ray structure (Green, et al., 1979). A 3.0 A model is now available though still in the refinement stages. Not surprisingly it was found that the protein is indeed in the dimeric form and that the arrangement of the dimer is very similar to the one found in BIgY (orthorhombic crystal form)(Papiz, et al., 1986), the other independently determined structure. Changes were observed in the threading of the sequence, in particular between residues 75 and 32 where movements of as much as five residues are found, and in the C- terminus between 141 and 150 where a shift of two residues is observed along a n- strand. These changes result in an overall increase of the hydrophobicity of the pocket. In parallel, cocrystallization of Blg with a variety of ligands has been successful in at least one case where density is evident in the binding cavity.
Human apolipoprotein D is present in the fluid of cysts formed during gross- cystic- disease, which is the most common breast disease in premenopausal women, and is a potential biological marker for breast cancer. The protein is found as well, associated with the lipoproteic system in the blood and is produced in high amounts in regenerating rat peripherial nerves. Several structural aspects of the protein from breast cysts were investigated; four of the cysteines were shown to be forming disulphide bridges and the fifth is not present as a free -cysteine. The influence of the pH on the conformational transitions (investigated by CD) and monomer -multimer balance (investigated by gel filtration) shows occurring parallelism with changes on Blg and in particular the transition seen between pH 6.5 and 5.5 could be due to the onset of the dissociation of the tetramer that populates the higher pHs. From the CD studies a 15% a -helix content was determined, more than the 7 -10% found for Blg or the 7% for Rbp but very close to the content on insectocyanin, with which shares 30 -40% residue identity. We examined too, the association of this globular protein with lipid vesicles, and electron- micrographs showed the formation of rouleaux structures, an effect commonly observed with other apolipoproteins; CD showed a small change in the amount of a -helix of the bound -protein .
The binding of several hydrophobic molecules was monitored by fluorescence and it was shown that arachidonic acid, a fatty acid with intense biological activity, binds with a association constant of 4.1x107M -1. This ligand provides a possible link between the diverse biological situations where apo D is present. A synthetic antagonist to the thromboxane A2 receptor was shown to bind too but with slightly less affinity.