The three-dimensional solution structure of the recombinant B domain (FB) of staphylococcal protein A, which specifically binds to the Fc portion of immunoglobulin G, was determined by NMR spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. On the basis of 692 experimental constraints including 587 distance constraints obtained from the nuclear Overhauser effect (NOE), 57 torsion angle (phi, chi 1) constraints, and 48 constraints associated with 24 hydrogen bonds, a total of 10 converged structures of FB were obtained. The atomic root mean square difference among the 10 converged structures is 0.52 +/- 0.10 A for the backbone atoms and 0.98 +/- 0.08 A for all heavy atoms (excluding the N-terminal segment from Thr1 to Glu9 and the C-terminal segment from Gln56 to Ala60, which are partially disordered). FB is composed of a bundle of three alpha-helices, i.e., helix I (Gln10-His19), helix II (Glu25-Asp37), and helix III (Ser42-Ala55). Helix II and helix III are antiparallel to each other, whereas the long axis of helix I is tilted at an angle of about 30 degrees with respect to those of helix II and helix III. Most of the hydrophobic residues of FB are buried in the interior of the bundle of the three helices. It is suggested that the buried hydrophobic residues form a hydrophobic core, contributing to the stability of FB.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiserum raised against a Fos peptide (ami-

ABSTRACTcDNA clones encoding chum salmon (Oncorhynchus keta) growth hormone (sGH) have been isolated from a cDNA library prepared from chum salmon pituitary gland poly(A)+ RNA. Synthetic oligodeoxynucleotide mixtures based on amino acid residues 23-28 of sGH were used as hybridization probes to select recombinant plasmids carrying the sGH coding sequence. The complete nucleotide sequence of sGH cDNA has been determined. The cDNA sequence codes for a polypeptide of 210 amino acids, including a putative signal sequence of 22 amino acids. The 5' and 3' untranslated regions of the message were 64 and 426 bases long, respectively. Mature sGH was efficiently expressed in Escherichia coli carrying a plasmid in which the sGH cDNA was under control of the E. coli trp promoter; sGH comprised about 15% of the total cellular protein in such bacteria. The partially purified sGH from E. coli stimulated the growth of rainbow trout and the activity was indistinguishable from that of natural sGH.Human growth hormone now can be produced by genetically engineered organisms and can be used as a therapeutic agent. Salmon growth hormone (sGH) can be synthesized by use of similar techniques, and the massive supply of sGH may be extremely important to fish culture. Growth hormone (GH), together with prolactin and chorionic somatomammotropin (placental lactogen), forms a set of proteins that are structurally related and have partially overlapping biological activities (1). Primary structure analysis of the peptides and of the genes suggests that these hormone genes evolved from a common ancestral origin (1-5). Therefore, these genes provide an excellent model system for studying structurefunction relationships, evolution, and regulation of expression. GH genes have been isolated from several mammalian species and characterized in detail (3,(6)(7)(8). To obtain information about the evolution and the mechanisms of organization of this set of genes, it is essential to compare the structures of these hormone genes isolated from many organisms at various evolutionary stages. No information, however, has been available about lower vertebrates such as fish.

A signal peptidase specifically required for the secretion of the lipoprotein of the Escherichia coli outer membrane cleaves off the signal peptide at the bond between a glycine and a cysteine residue. This cysteine residue was altered to a glycine residue by guided site-specific mutagenesis using a synthetic oligonucleotide and a plasmid carrying an inducible lipoprotein gene. The induction of mutant lipoprotein production was lethal to the cells. A large amount of the prolipoprotein was accumulated in the outer membrane fraction. No protein of the size of the mature lipoprotein was detected.These results indicate that the prolipoprotein signal peptidase requires a glyceride modified cysteine residue at the cleavage site.

Mixtures consisting of polyamine (branched polyethyleneimine (PEI) and polyallylamine (PAA)) and n-alkanoic acid (C n: n = the carbon number of alkanoic acid) were prepared and their thermal and liquid crystalline properties were examined. The occurrence of proton transfer from the carboxylic acid to the amine groups was found by IR measurement of the mixtures. The polyamines and C n exhibited no mesophase. Also, PEI/C n with n = 3, 4, 5, which are mixtures of PEI and C n, did not show a liquid crystalline phase. However, PEI/C n with n> 5 clearly displayed a lamellar fluid liquid crystal phase having a layer structure with low order inside the layer, due to ion–ion interactions. In the lamellar mesophase, PEI/C n showed a focal-conic fan texture which resembles the fan-shaped textures in smectic A and C phases. PEI/C n with 5 < n < 13 formed a bilayer structure in the lamellar mesophase. On the other hand, a tilted bilayer structure is proposed as a packing model of PEI/C n with 13 < n< 19. PAA/C18 consisting of polyallylamine and C18 also showed a lamellar fluid mesophase and formed a similar layer structure to PEI/C18.

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The recombinant B domain (FB) of staphylococcal protein A, which specifically binds to the Fc portion of immunoglobulin G (IgG), has been investigated with the use of two-dimensional proton nuclear magnetic resonance spectroscopy. All backbone and side-chain proton resonances of FB (60 amino acid residues), except the amide proton resonance of Ala2, were assigned by the sequential assignment procedures by using double-quantum-filtered correlated spectroscopy (DQF-COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA), and nuclear Overhauser enhancement spectroscopy (NOESY). On the basis of the NOESY data, three helical regions, Glu9-His19, Glu25-Asp37, and Ser42-Ala55, were identified in the free FB in solution. Existence of two of the three helical regions, Glu9-His19 and Glu25-Asp37, in consistent with the X-ray crystallographic structure of the Fc-bound FB [Deisenhofer, J. (1981) Biochemistry 20, 2361-2370]. By contrast, in the Fc-bound FB as revealed by the X-ray analysis, the Ser42-Glu48 segment is extended and no structural information has been available in the Ala49-Ala55 segment. We suggest that a significant conformation change is induced in the C-terminal region of FB when it is bound to the Fc portion of IgG.

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