Agonist-induced changes in the structure of the acetylcholine receptor M2 regions revealed by photoincorporation of an uncharged nicotinic noncompetitive antagonist.

To characterize structural changes induced in the nicotinic acetylcholine receptor (AChR) by agonists, we have mapped the sites of photoincorporation of the cholinergic noncompetitive antagonist 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (]125I]TID) in the presence and absence of 50 microM carbamylcholine. [125I]TID binds to the AChR with similar affinity under both these conditions, but agonist inhibits photoincorporation into all subunits by greater than 75% (White, B. H., Howard, S., Cohen, S. G., and Cohen, J. B. (1991) J. Biol. Chem. 266, 21595-21607). [125I]TID-labeled sites on the beta- and delta-subunits were identified by amino-terminal sequencing of both cyanogen bromide (CNBr) and tryptic fragments purified by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reversed-phase high-performance liquid chromatography. In the absence of agonist, [125I]TID specifically labels homologous aliphatic residues (beta L-257, delta L-265, beta V-261, and delta V-269) in the M2 region of both subunits. In the presence of agonist, labeling of these residues is reduced approximately 90%, and the distribution of labeled residues is broadened to include a homologous set of serine residues at the amino terminus of M2. In the beta-subunit residues beta S-250, beta S-254, beta L-257, and beta V-261 are all labeled in the presence of carbamylcholine. This pattern of labeling supports an alpha-helical model for M2 with the labeled face forming the ion channel lumen. The observed redistribution of label in the resting and desensitized states provides the first direct evidence for an agonist-dependent rearrangement of the M2 helices. The efficient labeling of the resting state channel in a region capable of structural change also suggests a plausible model for AChR gating in which the aliphatic residues labeled by [125I]TID form a permeability barrier to the passage of ions. We also report increased labeling of the M1 region of the delta-subunit in the presence of agonist.     

Amino acid preferences of small proteins. Implications for protein stability and evolution.

The dependence of amino acid frequency on sequence length has been examined for the 20 natural amino acids using a set of 2275 protein sequences with little sequence identity. As expected, the frequency of cysteine increases dramatically for sequences shorter than 100 amino acids with a length-dependence that corresponds to an average of two Cys per sequence independent of length. Surprisingly dramatic changes were also observed for the frequencies of arginine, lysine, aspartic acid, and glutamic acid: Arg and Lys frequencies increase for short sequences whereas Asp and Glu frequencies decrease. These changes do not appear to be due to an over-abundance of DNA- and membrane-binding proteins in the database and may, therefore, be related to protein stability. Possible stabilizing mechanisms include increased hydrogen bonding by Arg and increased hydrophobic stabilization due to the amphiphilic character of Arg and Lys. These observations suggest that amino acid composition played an important role in the evolution of small proteins.     

Structure of a fluid dioleoylphosphatidylcholine bilayer determined by joint refinement of x-ray and neutron diffraction data. III. Complete structure.

We present in this paper the complete structure of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in the L alpha phase (66% RH, 23 degrees C) obtained by the joint refinement of neutron and x-ray lamellar diffraction data. The structural details obtained have previously required a large number of neutron diffraction experiments, using numerous specifically-deuterated phospholipid isomorphs (Büldt et al., 1978. Nature (Lond.). 271:182-184). The joint-refinement approach minimizes specific deuteration by utilizing independent neutron and x-ray data sets. The method yields a quasimolecular structure consisting of a series of multiatomic fragments that are each represented by one or several Gaussian distributions whose positions and widths can be determined to within 0.06 to 0.52 A exclusive of the methylene region. The image of DOPC at 66% RH (5.36 +/- 0.08 waters per lipid) is consistent with many aspects of bilayer structure previously determined by structural and spectroscopic studies. The most striking feature of the structure is the large amount of transbilayer thermal motion suggested by the widths and overlaps of the Gaussian envelopes of the quasimolecular fragments. We discuss the "dynamic bilayer thickness" which describes the minimum effective thickness of the hydrocarbon permeability barrier in terms of the thermal motion of the water. A gradient of thermal motion exists that increases in either direction away from the glycerol backbone which is the most constrained portion of the bilayer. The steric interactions between headgroups of apposed bilayers, expected at the hydration level of our experiments, are clearly revealed. A useful consequence of the quasimolecular structure is that average boundaries within bilayers calculated using composition and volumetric data and ad hoc assumptions can be related to the positions of the principal structural groups. Several measures of "bilayer thickness" in common use can be identified as the positions of the cholines for Luzzati's d1 (Luzzati and Husson. 1962. J. Cell Biol. 12:207-219) and the glycerols for Small's dL (Small. 1967. J. Lipid Res. 8:551-556). We do not know if these relations will be true at other hydrations or for other lipids. Of particular interest is the fact that the position of the carbonyl groups marks the average hydrocarbon/headgroup boundary. It must be emphasized, however, that this region of the bilayer must be generally characterized as one of tumultuous chemical heterogeneity because of the thermal motion of the bilayer.     

Proteolytic degradation of heterologous proteins expressed inAspergillus niger

Summary Proteolytic degradation of heterologous proteins expressed in the filamentous fungusAspergillus niger reduces the yield of authentic target protein. The activities ofA. niger proteases are differentiated by their effects on two proteins expressed and secreted fromA. niger: hen egg-white lysozyme and porcine pancreatic phospholipase A₂.     

Promoter methylation and progressive transgene inactivation inArabidopsis

Agrobacterium-transformedArabidopsis plants were generated and the stability of their T-DNA-encoded resistance to kanamycin was examined. Of seven families, each homozygous for a single insertion event, two showed progressive inactivation of resistance over four generations of inbreeding. Loss of resistance was associated with methylation of anSst II site in thenos promoter of the kanamycin resistance gene. Treatment of plant roots from inactive lines with the demethylating agent 5-azacytidine restored the ability of such lines to form callus on kanamycin-containing media. These observations are consistent with the view that methylation is a factor in the progressive inactivation of transgenes inArabidopsis.     

Genes required for formation of the apoMoFe protein of Klebsiella pneumoniae nitrogenase in Escherichia coli

A binary plasmid system was used to produce nitrogenase components in Escherichia coli and subsequently to define a minimum set of nitrogen fixation (nif) genes required for the production of the iron-molybdenum cofactor (FeMoco) reactivatable apomolybdenum-iron (apoMoFe) protein of nitrogenase. The active MoFe protein is an alpha 2 beta 2 tetramer containing two FeMoco clusters and 4 Fe4S4 P centers (for review see, Orme-Johnson, W.H. (1985) Annu. Rev. Biophys. Biophys. Chem. 14, 419-459). The plasmid pVL15, carrying a tac-promoted nifA activator gene, was coharbored in E. coli with the plasmid pGH1 which contained nifHDKTYENXUSVWZMF' derived from the chromosome of the nitrogen fixing bacterium Klebsiella pneumoniae. The apoMoFe protein produced in E. coli by pGH1 + VL15 was identical to the apoprotein in derepressed cells of the nifB- mutant of K. pneumoniae (UN106) in its electrophoretic properties on nondenaturing polyacrylamide gels as well as in its ability to be activated by FeMoco. The constituent peptides migrated identically to those from purified MoFe protein during electrophoresis on denaturing gels. The concentrations of apoMoFe protein produced in nif-transformed strains of E. coli were greater than 50% of the levels of MoFe protein observed in derepressed wild-type K. pneumoniae. Systematic deletion of individual nif genes carried by pGH1 has established the requirements for the maximal production of the FeMoco-reactivatable apoMoFe protein to be the following gene products, NifHDKTYUSWZM+A. It appears that several of the genes (nifT, Y, U, W, and Z) are only required for maximal production of the apoMoFe protein, while others (nifH, D, K, and S) are absolutely required for synthesis of this protein in E. coli. One curious result is that the nifH gene product, the peptide of the Fe protein, but not active Fe protein itself, is required for formation of the apoMoFe protein. This suggests the possibility of a ternary complex of the NifH, D, and K peptides as the substrate for the processing to form the apoMoFe protein. We also find that nifM, the gene which processes the nifH protein into Fe protein (Howard, K.S., McLean, P.A., Hansen, F. B., Lemley, P.V., Kobla, K.S. & Orme-Johnson, W.H. (1986) J. Biol. Chem. 261, 772-778) can, under certain circumstances, partially replace other processing genes (i.e. nifTYU and/or WZ) although it is not essential for apoMoFe protein formation. It also appears that nifS and nifU, reported to play a role in Fe protein production in Azotobacter vinelandii, play no such role in K. pneumoniae, although these genes are involved in apoMoFe formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

The asymmetric transmembrane distribution of phosphatidylethanolamine,phosphatidylserine, and fatty acids of the bovine retinal rod outer segment disk membrane

Summary The transmembrane distribution of the major aminophospholipids in the bovine retinal rod outer segment disk membrane, phosphatidylethanolamine and phosphatidylserine, was determined using a novel pair of permeable and impermeable covalent modification reagents. The values for the percentages of phosphatidylethanolamine and phosphatidylserine in the outer monolayer were calculated from a simple expression which takes into account the leakage of impermeable reagent into the disk lumen as monitored by the extent of labeling of lysine entrapped in the lumen. We infer from our results that at least 73 to 87% of the disk phosphatidylethanolamine and 77 to 88% of the disk phosphatidylserine are in the outer disk membrane monolayer. The fatty acid composition of the inner aminophospholipids is slightly more saturated than the outer aminophospholipids. Calculations using the lateral surface areas occupied by the disk membrane lipids suggest that 65 to 100% of the disk phosphatidylcholine is on the inner membrane surface. Since the disk phosphatidylcholine is also somewhat more saturated than the phosphatidylethanolamine and phosphatidylserine of the outer monolayer, the total inner membrane monolayer fatty acid composition is more saturated than that of the outer monolayer fatty acid composition.     

Comparison of content with synthesis,uptake and breakdown of GABA in substantia nigra of unoperated,hemitransected and kainate-treated rats

The synthesis, high affinity uptake and degradation of GABA were compared with GABA content measured simultaneously in the nigras of unoperated, hemitransected and kainate-treated rats. In intact animals nigral GABA concentration was linearly related to glutamate decarboxylase activity, but not to GABA aminotransferase (GABA-T) or GABA uptake. One week after hemitransection all three parameters showed a linear reduction with GABA content, suggesting striatonigral GABA neurones may account for as much as 90% of the GABA and GAD, 31% of the GABA-T and 58% of GABA uptake sites in whole nigra. Nigral kainate treatment unexpectedly reduced all four parameters in parallel by as much as 50%, consistent with the formation of an unselective lesion.