Structure of a 16 kDa integral membrane protein that has identity to the putative proton channel of the vacuolar H(+)-ATPase.

A 16 kDa protein has been isolated in a homogeneous form as the major component of a paracrystalline paired membrane structure closely resembling the gap junction. The primary structure of this protein from arthropod and vertebrate species has been determined by protein and cDNA sequencing. The amino acid sequences are highly conserved and virtually identical to the amino acid sequence of the proteolipid subunit of the vacuolar H(+)-ATPases. The disposition of the protein in the membrane has been studied using proteases and the N,N'-dicyclohexylcarbodiimide reactive site identified. These data, together with secondary structure predictions, suggest that the 16 kDa protein is for the most part buried in the membrane, arranged in a bundle of four hydrophobic alpha-helices. Using computer graphics, a model has been constructed based on this arrangement and on the electron microscopic images of the paracrystalline arrays.

Complete sequence and model for the C1 subunit of the carotenoprotein, crustacyanin, and model for the dimer, beta-crustacyanin, formed from the C1 and A2 subunits with astaxanthin.

The complete sequence has been determined for the C1 subunit of crustacyanin, an astaxanthin-binding protein from the carapace of the lobster Homarus gammarus (L.). The polypeptide, 181 residues long, is similar (38% identity) to the other main subunit, A2 and to plasma retinol-binding protein. The tertiary structure of the C1 subunit has been modelled on that derived for the A2 subunit from the coordinates of retinol-binding protein. Residues lining the putative binding cavities and at the putative carotenoid binding sites of the two subunits are highly conserved. The carotenoid environments are characterized by a preponderance of aromatic and polar residues and the absence of charged side-chains. A tentative model for the dimer, beta-crustacyanin, formed between the two subunits with their associated carotenoid ligands, is discussed. The model is based on the crystal structure of the dimer of bilin-binding protein, a member of the same superfamily. This structure has enabled us to examine mechanisms for the bathochromic spectral shift of the protein-bound carotenoid and to identify likely contact regions between dimers in octameric alpha-crustacyanin.

Complete sequence and model for the A2 subunit of the carotenoid pigment complex, crustacyanin.

The complete sequence has been determined for the A2 subunit of crustacyanin, an astaxanthin-binding protein from the carapace of the lobster Homarus gammarus. The polypeptide chain is 174 residues long and is similar to proteins of the retinol-binding protein superfamily. Some regions of the sequence are most similar to the retinol-binding protein, beta-lactoglobulin subgroup, while the disulphide bonding pattern is more akin to that seen in the porphyrin binding proteins insecticyanin and bilin-binding protein. It is beginning to appear as though this superfamily of proteins, characterized by a similar gross structural framework, may be further subdivided into interrelated subclasses. Model building based on the coordinates of the known structure of human plasma retinol-binding protein and on empirical prediction algorithms has allowed the putative identification of side-chains which line the binding cavity. This pocket is larger than in retinol binding protein and beta-lactoglobulin but does not allow the carotenoid to adopt a folded conformation. The amino acid composition of the pocket does not support a 'charge-shift'-type hypothesis to support the bathochromic shift phenomenon which takes place on interaction of the chromophore with the protein. Instead aromatic side-chains may play a prominent role.

Multiple sequence alignment of protein families showing low sequence homology: a methodological approach using database pattern-matching discriminators for G-protein-linked receptors.

A multiple alignment has been constructed, containing 37 sequences from related families of membrane-bound receptors believed to share the same structural framework as rhodopsin. Sequence homology within families was high (occasionally greater than 90%), but homology between them was generally low (20% or less). Database pattern-scanning methods were therefore used to construct a set of discriminators to aid both the task of alignment and the identification of distantly related sequences showing similar rhodopsin-like transmembrane helices. The results indicate that these discriminators are uniquely able to identify each of the transmembrane helices without major cross-reaction with similar regions in unrelated integral membrane proteins. This ability engenders more accurate alignments of the sequences and facilitates structural analysis and model building of the receptors.

The lobster carapace carotenoprotein, alpha-crustacyanin. A possible role for tryptophan in the bathochromic spectral shift of protein-bound astaxanthin.

Crustacyanin, cross-linked with dimethyl pimelimidate to stabilize the protein against denaturation, was used to test the effects of tryptophan modification with BNPS-skatole [3-bromo-3-methyl-2-(nitrophenylmercaptol)-3H-indole] on the ability of the apoprotein to recombine with astaxanthin. The cross-linked apoprotein re-forms alpha-crustacyanin with astaxanthin in reasonable yield following incubation of the protein under the conditions for tryptophan modification in the absence of BNPS-skatole. The BNPS-skatole-treated protein reconstitutes with astaxanthin to give a carotenoprotein with lambda max. at 472 nm, that of the carotenoid in hexane, in a yield similar to that of the BNPS-skatole-untreated control. The implied involvement of tryptophan residues at the sites of astaxanthin attachment in crustacyanin and their possible roles in the binding sites of vitamin A in vitamin A-proteins are discussed in relation to the bathochromic spectral shifts of the chromophores.

Alternative ligands as probes for the carotenoid-binding site of lobster carapace crustacyanin.

The apoproteins of the lobster carotenoprotein, crustacyanin, show single high-affinity binding sites for the hydrophobic fluorescence probes 8-anilo-1-naphthalenesulphonic acid and cis-parinaric acid, and exhibit fluorescence transfer from tryptophan to the ligands. These results, together with information from the amino acid sequences, infer that the native carotenoid, astaxanthin, is bound to each apoprotein within an internal hydrophobic pocket, or calyx.

Complete amino acid sequence of pyrazine-binding protein from cow nasal mucosa.

The sequence is reported of the pyrazine-binding protein from cow olfactory/respiratory mucosa. The protein consists of 159 amino acids and clearly belongs to the retinol-binding protein family. It is most closely related to the urinary proteins from mice and rats and to the odour-binding protein from rat nasal epithelium. It is unique however, in that only one of the otherwise conserved features of the family is still present--namely a single tryptophan. Most surprisingly the protein contains no cysteine and, therefore, does not rely for its structural stability on the disulphide bond(s) present in other members of this group. A model for the protein has been constructed based on the co-ordinates of beta-lactoglobulin. From this, it is possible to identify residues which may line the binding site. The impression gained is of a much larger pocket than occurs with retinol-binding protein or beta-lactoglobulin. The character of the binding pocket remains essentially hydrophobic but with a significant reduction in its aromatic content and an increase in H-bonding side chains.

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.

The structure of beta-lactoglobulin and its similarity to plasma retinol-binding protein.

Since its first isolation, bovine beta-lactoglobulin (BLG) has been an enigma: although it is abundant in the whey fraction of milk, its function is still not clear. The results of the many physicochemical studies on the protein need a structural interpretation. We report here the structure of the orthorhombic crystal form of cow BLG at pH 7.6, at a resolution of 2.8 A. It has an unusual protein fold, composed of two slabs of antiparallel beta-sheet, which shows a remarkable similarity to plasma retinol-binding protein. A possible binding site for retinol in BLG has been identified by model-building. This suggests a role for BLG in vitamin A transport and we have discovered specific receptors for the BLG-retinol complex in the intestine of neonate calves.