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)

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Quantitative oligosaccharide profiles were determined for each of 18 human IgG paraproteins representing the four subclasses. Each paraprotein exhibits a unique profile that may be substantially different from that observed for polyclonal IgG. The IgG2 and some IgG3 proteins analysed exhibit a predominance of oligosaccharide moieties having galactose on the Man(alpha 1----3) arm rather than the Man(alpha 1----6) arm; it was previously held that galactosylation of the Man(alpha 1----6) arm is preferred, as observed for IgG1, IgG4 and polyclonal IgG. An IgG4 protein is reported that has galactosylated Man(alpha 1----3) and Man(alpha 1----6) arms on both Fc-localized carbohydrate moieties; previous findings suggested that such fully glycosylated structures could not be accommodated within the internal space of the C gamma 2 domains. Unusual monoantennary oligosaccharides present in IgG2 and IgG3 proteins were isolated and their structures determined.

Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation, and gene expression. ERK5 is approximately twice the size of ERK1/2, and its amino-terminal half contains the kinase domain that shares homology with ERK1/2 and TEY activation motif, whereas the carboxyl-terminal half is unique. In this study, we examined a physiological role of ERK5 in rat pheochromocytoma cells (PC12), comparing it with ERK1/2. Nerve growth factor (NGF) induced phosphorylation of both ERK5 and ERK1/2, whereas the cAMP analog dibutyryl cAMP (Bt 2 cAMP) caused only ERK1/2 phosphorylation. U0126, at 30 M, that blocks ERK1/2 signaling selectively attenuated neurite outgrowth induced by NGF and Bt 2 cAMP, but BIX02188 and BIX02189, at 30 M, that block ERK5 signaling and an ERK5 dominant-negative mutant suppressed only NGF-induced neurite outgrowth. Next, we examined the expression of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis. Both NGF and Bt 2 cAMP increased tyrosine hydroxylase gene promoter activity in an ERK1/2-dependent manner but was ERK5-independent. However, when both ERK5 and ERK1/2 signalings were inhibited, tyrosine hydroxylase protein up-regulation by NGF and Bt 2 cAMP was abolished, because of the loss of stabilization of tyrosine hydroxylase protein by ERK5. Taking these results together, ERK5 is involved in neurite outgrowth and stabilization of tyrosine hydroxylase in PC12 cells, and ERK5, along with ERK1/2, plays essential roles in the neural differentiation process.

The solution conformation of an antibacterial protein sapecin has been detennined by 'H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4-12), one helix (residues 1523), and two extended strands (residues 2431 and 3W). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP-5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three-dimensional structure determination has revealed that a basic-residue rich region and the hydrophobic surface face each other on the surface of sapecin.

The structure of carbohydrates in acetylcholine receptor (AChR) from Torpedo californica is reported. Oligosaccharides released quantitatively from the whole molecule by N-oligosaccharide glycopeptidase digestion were fractionated by thin-layer chromatography and further purified by high-performance liquid chromatography. We show that more than 70% of the total oligosaccharide chains in Torpedo AChR are of the high-mannose type with the structures (Man)8(GlcNAc)z and (Man)g(GlcNAc),. The structure of these oligosaccharides were determined by proton nuclear magnetic resonance spectroscopy. These two types of oligosaccharides were shown to be distributed in different proportions in all subunits of Torpedo AChR. We also show that several kinds of complex-type oligosaccharides comprising the rest of the carbohydrate in the protein exist mainly in the y and 6 subunits. The structure of the carbohydrate moiety that is distributed on the four subunits of AChR was also examined by susceptibility to endo-P-N-acetylglucosaminidase and sialidase and by binding affinity to lectins, e.g. concanavalin A, leucoagglutinating phytohemagglutinin, and wheat germ agglutinin.The nicotinic acetylcholine receptor (AChR) is distributed in mammalian skeletal muscles and electroplaques of electric fishes [l-31. It is known that AChR is composed of four different homologous glycosylated subunits (a, P, y, and 8) in a molar stoichiometry of aZPy6.

A 13C NMR study is reported of switch variant anti-dansyl antibodies developed by Dangl et al. [(1982) Cytometry 2, 395-401], who had used the fluorescence-activated cell sorter to select and clone these variants. These switch variant antibodies possess the identical VH, VL, and CL domains in conjunction with different heavy chain constant regions. In the present study, switch variant antibodies of IgG1, IgG2a, and IgG2b subclasses were used along with a short-chain IgG2a antibody, in which the entire CH1 domain is deleted. The switch variant antibodies were specifically labeled with [1-13C]methionine by growing hybridoma cells in serum-free medium. Assignments of all the methionyl carbonyl carbon resonances have been completed by using the intact antibodies along with their fragments and recombined proteins in which either heavy or light chain is labeled. A double labeling method [Kainosho, M., & Tsuji, T. (1982) Biochemistry 21, 6273-6279] has played a crucial role in the process of the spectral assignments. The strategy used for the assignments has been described in detail. In incorporating 15N-labeled amino acids into the antibodies for the double labeling, isotope dilution caused a serious problem except in the cases of [alpha-15N]lysine and [15N]threonine, both of which cannot become the substrate of transaminases. It was found that beta-chloro-L-alanine is most effective in suppressing the isotope scrambling. So far, spectral assignments by the double labeling method have been possible with 15N-labeled Ala, His, Ile, Lys, Met, Ser, Thr, Tyr, and Val.(ABSTRACT TRUNCATED AT 250 WORDS)