The amino terminus of the aspartate chemoreceptor is formylmethionine

The amino terminus of the Salmonella typhimurium aspartate receptor has been identified as formylmethionine by mass spectral analysis of the amino-terminal tryptic peptide. Purification and analysis of the blocked amino-terminal peptide was facilitated by the use of a mutant aspartate receptor which has a cysteine residue at position 3. The sequence of this peptide confirms the translational start site predicted from the nucleotide sequence of the tar gene. Furthermore, in vivo labeling experiments reveal that the formyl group is present on chemotaxis receptors produced at wild-type levels in Escherichia coli, indicating that the presence of the formyl group is not a consequence of over-production of the receptor. The stability of the amino-terminal formyl group on the receptor may be a consequence of the membrane localization of the receptor and the dependence of this localization on the membrane transport machinery of the cell.     

A mechanism for simultaneous sensing of aspartate and maltose by the Tar chemoreceptor of Escherichia coli

The Tar chemoreceptor of Escherichia coli exhibits partial sensory additivity. Tar can mediate simultaneous responses to two disparate ligands, aspartate and substrate-loaded maltose-binding protein (MBP). To investigate how one receptor generates concurrent signals to two stimuli, ligand-binding asymmetry was imposed on the rotationally symmetric Tar homodimer. Mutations causing specific defects in aspartate or maltose chemotaxis were introduced pairwise into plasmid-borne tar genes. The doubly mutated tar genes did not restore aspartate or maltose chemotaxis in a strain containing a chromosomal deletion of tar (Δ tar ). However, when Tar proteins with complementing sets of mutations were co-expressed from compatible plasmids, the resulting heterodimeric receptors enabled Δ tar cells to respond to aspartate or maltose. The effect of one attractant on the response to the other depended on the relative orientations of the functional binding sites for aspartate and MBP. When the sites were in the 'same' orientation, saturating levels of one attractant strongly inhibited chemotaxis to the other. In the 'opposite' orientation, such inhibitory effects were negligible. These data demonstrate that opposing subunits of Tar can transmit signals to aspartate and maltose independently if the ligands are restricted to the 'opposite' binding orientation. When aspartate and MBP bind in the 'same' orientation, they compete for signalling through one subunit. In the wild-type Tar dimer, aspartate and MBP can bind in either the 'same' or the 'opposite' orientation, a freedom that can explain the partial additivity of the aspartate and maltose responses that is seen with tar ⁺ cells.     

The activity of the murine DNA methyltransferase Dnmt1 is controlled by interaction of the catalytic domain with the N-terminal part of the enzyme leading to an allosteric activation of the enzyme after binding to methylated DNA

The mammalian DNA methyltransferase Dnmt1 is responsible for the maintenance of the pattern of DNA methylation in vivo. It is a large multidomain enzyme comprising 1620 amino acid residues. We have purified and characterized individual domains of Dnmt1 (NLS-containing domain, NlsD, amino acid residues: 1-343; replication foci-directing domain, 350-609; Zn-binding domain (ZnD), 613-748; polybromo domain, 746-1110; and the catalytic domain (CatD), 1124-1620). CatD, ZnD and NlsD bind to DNA, demonstrating the existence of three independent DNA-binding sites in Dnmt1. CatD shows a preference for binding to hemimethylated CpG-sites; ZnD prefers methylated CpGs; and NlsD specifically binds to CpG-sites, but does not discriminate between unmethylated and methylated DNA. These results are not compatible with the suggestion that the target recognition domain of Dnmt1 resides in the N terminus of the enzyme. We show by protein-protein interaction assays that ZnD and CatD interact with each other. The isolated catalytic domain does not methylate DNA, neither alone nor in combination with other domains. Full-length Dnmt1 was purified from baculovirus-infected insect cells. Under the experimental conditions, Dnmt1 has a strong (50-fold) preference for hemimethylated DNA. Dnmt1 is stimulated to methylate unmodified CpG sites by the addition of fully methylated DNA. This effect is dependent on Zn, suggesting that binding of methylated DNA to ZnD triggers the allosteric activation of the catalytic center of Dnmt1. The allosteric activation model can explain kinetic data obtained by others. It suggests that Dnmt1 might be responsible for spreading of methylation, a process that is observed during aging and carcenogenesis but may be important for de novo methylation of DNA.     

Chemotaxis kinase CheA is activated by three neighbouring chemoreceptor dimers as effectively as by receptor clusters

Chemoreceptors are central to bacterial chemotaxis. These transmembrane homodimers form trimers of dimers. Trimers form clusters of a few to thousands of receptors. A crucial receptor function is 100‐fold activation, in signalling complexes, of sensory histidine kinase CheA. Significant activation has been shown to require more than one receptor dimer but the number required for full activation was unknown. We investigated this issue using Nanodiscs, soluble, nanoscale (～10 nm diameter) plugs of lipid bilayer, to limit the number of neighbouring receptors contributing to activation. Utilizing size‐exclusion chromatography, we separated primary preparations of receptor‐containing Nanodiscs, otherwise heterogeneous for number and orientation of inserted receptors, into fractions enriched for specific numbers of dimers per disc. Fractionated, clarified Nanodiscs carrying approximately five dimers per disc were as effective in activating kinase as native membrane vesicles containing many neighbouring dimers. At five independently inserted dimers per disc, every disc would have at least three dimers oriented in parallel and thus able act together as they would in native membrane. We conclude full kinase activation involves interaction of CheA with groups of three receptor dimers, presumably as a trimer of dimers, and that more extensive interactions among receptors are not necessary for full kinase activation.     

The binding of anionic and nonionic surfactants to collagen through the hydrophobic effect

The adsorption of nonionic surfactants on hide powder previously treated with anionic surfactants has been studied. The adsorption of nonionic surfactants takes place through hydrophobic interactions. A mechanism has been proposed for this interaction, assuming that the nonionic surfactant has been fixed by means of secondary adsorption (hydrophobic interaction) after the primary adsorption of the anionic surfactant (ionic and hydrophobic interaction) which makes it possible.     

The residue composition of the aromatic anchor of the second transmembrane helix determines the signaling properties of the aspartate/maltose chemoreceptor Tar of Escherichia coli

Repositioning of the tandem aromatic residues (Trp-209 and Tyr-210) at the cytoplasmic end of the second transmembrane helix (TM2) modulates the signal output of the aspartate/maltose chemoreceptor of Escherichia coli (Tar(Ec)). Here, we directly assessed the effect of the residue composition of the aromatic anchor by studying the function of a library of Tar(Ec) variants that possess all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. We identified three important properties of the aromatic anchor. First, a Trp residue at position 209 was required to maintain clockwise (CW) signal output in the absence of adaptive methylation, but adaptive methylation restored the ability of all of the mutant receptors to generate CW rotation. Second, when the aromatic anchor was replaced with tandem Ala residues, signaling was less compromised than when an Ala residue occupied position 209 and an aromatic residue occupied position 210. Finally, when Trp was present at position 209, the identity of the residue at position 210 had little effect on baseline signal output or aspartate chemotaxis, although maltose taxis was significantly affected by some substitutions at position 210. All of the mutant receptors we constructed supported some level of aspartate and maltose taxis in semisolid agar swim plates, but those without Trp at position 209 were overmethylated in their baseline signaling state. These results show the importance of the cytoplasmic aromatic anchor of TM2 in maintaining the baseline Tar(Ec) signal output and responsiveness to attractant signaling.     

The ribosomal l1 protuberance in yeast is methylated on a lysine residue catalyzed by a seven-beta-strand methyltransferase

Modification of proteins of the translational apparatus is common in many organisms. In the yeast Saccharomyces cerevisiae, we provide evidence for the methylation of Rpl1ab, a well conserved protein forming the ribosomal L1 protuberance of the large subunit that functions in the release of tRNA from the exit site. We show that the intact mass of Rpl1ab is 14 Da larger than its calculated mass with the previously described loss of the initiator methionine residue and N-terminal acetylation. We determined that the increase in mass of yeast Rpl1ab is consistent with the addition of a methyl group to lysine 46 using top-down mass spectrometry. Lysine modification was confirmed by detecting (3)H-N-ε-monomethyllysine in hydrolysates of Rpl1ab purified from yeast cells radiolabeled in vivo with S-adenosyl-l-[methyl-(3)H]methionine. Mass spectrometric analysis of intact Rpl1ab purified from 37 deletion strains of known and putative yeast methyltransferases revealed that only the deletion of the YLR137W gene, encoding a seven-β-strand methyltransferase, results in the loss of the +14-Da modification. We expressed the YLR137W gene as a His-tagged protein in Escherichia coli and showed that it catalyzes N-ε-monomethyllysine formation within Rpl1ab on ribosomes from the ΔYLR137W mutant strain lacking the methyltransferase activity but not from wild-type ribosomes. We also showed that the His-tagged protein could catalyze monomethyllysine formation on a 16-residue peptide corresponding to residues 38-53 of Rpl1ab. We propose that the YLR137W gene be given the standard name RKM5 (ribosomal lysine (K) methyltransferase 5). Orthologs of RKM5 are found only in fungal species, suggesting a role unique to their survival.     

Dual recognition of the bacterial chemoreceptor by chemotaxis-specific domains of the CheR methyltransferase

Adaptation to persisting stimulation is required for highly sensitive detection of temporal changes of stimuli, and often involves covalent modification of receptors. Therefore, it is of vital importance to understand how a receptor and its cognate modifying enzyme(s) modulate each other through specific protein-protein interactions. In the chemotaxis of Escherichia coli, adaptation requires methylation of chemoreceptors (e.g. Tar) catalyzed by the CheR methyltransferase. CheR binds to the C-terminal NWETF sequence of a chemoreceptor that is distinct from the methylation sites. However, little is known about how CheR recognizes its methylation sites or how it is distributed in a cell. In this study, we used comparative genomics to demonstrate that the CheR chemotaxis methyltransferase contains three structurally and functionally distinct modules: (i) the catalytic domain common to a methyltransferase superfamily; (ii) the N-terminal domain; and (iii) the beta-subdomain of the catalytic domain, both of which are found exclusively in chemotaxis methyltransferases. The only evolutionary conserved motif specific to CheR is the positively charged face of helix alpha2 in the N-terminal domain. The disulfide cross-linking analysis suggested that this face interacts with the methylation helix of Tar. We also demonstrated that CheR localizes to receptor clusters at cell poles via interaction of the beta-subdomain with the NWETF sequence. Thus, the two chemotaxis-specific modules of CheR interact with distinct regions of the chemoreceptor for targeting to the receptor cluster and for recognition of the substrate sites, respectively.     

Electrostatic and hydrophobic interactions governing the interaction and binding of beta-lactoglobulin to membranes

The role of electrostatic and hydrophobic interactions in the binding and penetration of beta-lactoglobulin (betaLG) to preformed lipid membranes was studied using various phospholipid micelles and vesicles. Zwitterionic lysophospholipid micelles are able to induce the beta-sheet to alpha-helix transition, as judged by circular dichroism (CD), but the degree of transition is dramatically below and the amount of lipid required above that for anionic phospholipids with equivalent hydrocarbon chains. Anionic phospholipids with short hydrocarbon chains induce only low alpha-helical content in betaLG as compared to phospholipids with the same head group but longer hydrocarbon chains. These results suggest that both electrostatic and hydrophobic interactions are indispensable in betaLG-lipid interaction. Furthermore, air-water interface monolayer surface pressure and fluorescence anisotropy studies reveal that the membrane insertion of betaLG strongly depends on the nature of phospholipids, given the identical headgroup, particularly lipid packing. These results are supported by urea denaturation and acrylamide fluorescence quenching tests and by the FTIR-ATR polarization results for betaLG in multilayers on a surface. Under the same experimental conditions, the membrane binding and insertion of betaLG as well as the stability of the betaLG-lipid complexes can be enhanced by lowering the pH. Collectively, electrostatic interactions play a crucial role in all the processes involved in the betaLG-lipid interaction, while the presence of hydrophobic interaction remains necessary. Finally, betaLG biological function in the transport of fatty acids was tested by demonstrating the release of 2-AS from a 2-AS-betaLG complex on binding to lipids.     

The X-linked methylated DNA binding protein,Mecp2, is subject to X inactivation in the mouse

DNA methylation at the promoter region of X-linked genes is associated with the maintenance of X inactivation in mammals. One of the methylated DNA binding proteins, MECP2, that binds to methylated bases in DNA is encoded by a gene (Mecp2) located on the mouse X Chromosome (Chr). To determine whether this gene was expressed from the inactive X Chr, and X-autosome translocation (T(X;16)16H) system in which expression from the Mecp2 allele on the inactive X Chr could be assayed was used. Results from these experiments indicate that Mecp2 is subject to X inactivation in mouse.     

Peptide specificity of protein prenyltransferases is determined mainly by reactivity rather than binding affinity

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase I) catalyze the attachment of lipid groups from farnesyl diphosphate and geranylgeranyl diphosphate, respectively, to a cysteine near the C-terminus of protein substrates. FTase and GGTase I modify several important signaling and regulatory proteins with C-terminal CaaX sequences ("C" refers to the cysteine residue that becomes prenylated, "a" refers to any aliphatic amino acid, and "X" refers to any amino acid). In the CaaX paradigm, the C-terminal X-residue of the protein/peptide confers specificity for FTase or GGTase I. However, some proteins, such as K-Ras, RhoB, and TC21, are substrates for both FTase and GGTase I. Here we demonstrate that the C-terminal amino acid affects the binding affinity of K-Ras4B-derived hexapeptides (TKCVIX) to FTase and GGTase I modestly. In contrast, reactivity, as indicated by transient and steady-state kinetics, varies significantly and correlates with hydrophobicity, volume, and structure of the C-terminal amino acid. The reactivity of FTase decreases as the hydrophobicity of the C-terminal amino acid increases whereas the reactivity of GGTase I increases with the hydrophobicity of the X-group. Therefore, the hydrophobicity, as well as the structure of the X-group, determines whether peptides are specific for farnesylation, geranylgeranylation, or dual prenylation.     

Proteome wide evaluation of the separation ability of hydrophobic interaction chromatography by fluorescent dye binding analysis

Hydrophobic interaction chromatography (HIC) is an important tool in the industrial purification of proteins from various sources. The HIC separation behavior of individual (or model) proteins has been widely researched by others. On the contrary, this study focused on the fractionation ability of HIC when it is challenged with whole proteomes. The impact of the nature of three different proteomes, that is, yeast, soybean, and Chinese hamster ovary cells, on HIC separation was investigated. In doing so, chromatography fractions obtained under standardized conditions were evaluated in terms of their overall hydrophobicity—as measured by fluorescence dye binding. This technique allowed for the calculation of an average protein surface hydrophobicity (S₀) for each fraction; a unique correlation between S₀ and the observed chromatographic behavior was established in each case. Following a similar strategy, the effect of three different ligands (polypropylene glycol, phenyl, and butyl) and two adsorbent particle sizes (65 and 100 µm) on the chromatographic behavior of the yeast proteome was evaluated. As expected, the superficial hydrophobicity of the proteins eluted is correlated with the salt concentration of its corresponding elution step. The findings reveled how—and in which extent—the type of ligand and the size of the beads actually influenced the fractionation of the complex biological mixture. Summarizing, the approach presented here can be instrumental to the study of the performance of chromatography adsorbents under conditions close to industrial practice and to the development of downstream processing strategies. Copyright © 2013 John Wiley & Sons, Ltd.     

The O‐methyltransferase gene MdoOMT1 is required for biosynthesis of methylated phenylpropenes in ripe apple fruit

Phenylpropenes, such as eugenol and trans‐anethole, are important aromatic compounds that determine flavour and aroma in many herbs and spices. Some apple varieties produce fruit with a highly desirable spicy/aromatic flavour that has been attributed to the production of estragole, a methylated phenylpropene. To elucidate the molecular basis for estragole production and its contribution to ripe apple flavour and aroma we characterised a segregating population from a Royal Gala (RG, estragole producer) × Granny Smith (GS, non‐producer) apple cross. Two quantitative trait loci (QTLs; accounting for 9.2 and 24.8% of the variation) on linkage group (LG) 1 and LG2 were identified that co‐located with seven candidate genes for phenylpropene O‐methyltransferases (MdoOMT1–7). Of these genes, only expression of MdoOMT1 on LG1 increased strongly with ethylene and could be correlated with increasing estragole production in ripening RG fruit. Transient over‐expression in tobacco showed that MdoOMT1 utilised a range of phenylpropene substrates and catalysed the conversion of chavicol to estragole. Royal Gala carried two alleles (MdoOMT1a, MdoOMT1b) whilst GS appeared to be homozygous for MdoOMT1b. MdoOMT1a showed a higher affinity and catalytic efficiency towards chavicol than MdoOMT1b, which could account for the phenotypic variation at the LG1 QTL. Multiple transgenic RG lines with reduced MdoOMT1 expression produced lower levels of methylated phenylpropenes, including estragole and methyleugenol. Differences in fruit aroma could be perceived in these fruit, compared with controls, by sensory analysis. Together these results indicate that MdoOMT1 is required for the production of methylated phenylpropenes in apple and that phenylpropenes including estragole may contribute to ripe apple fruit aroma.     

Sequence-specific DNA binding by the MspI DNA methyltransferase.

The MspI methyltransferase (M.MspI) recognizes the sequence CCGG and catalyzes the formation of 5-methylcytosine at the fist C-residue. We have investigated the sequence-specific DNA-binding properties of M.MspI under equilibrium conditions, using gel-mobility shift assays and DNasel footprinting. M.MspI binds to DNA in a sequence-specific manner either alone or in the presence of the normal methyl donor S-adenosyl-L-methionine as well as the analogues, sinefungin and S-adenosyl-L-homocysteine. In the presence of S-adenosyl-L-homocysteine, M.MspI shows the highest binding affinity to DNA containing a hemimethylated recognition sequence (Kd = 3.6 x 10(-7) M), but binds less well to unmethylated DNA (Kd = 8.3 x 10(-7) M). Surprisingly it shows specific, although poor, binding to fully methylated DNA (Kd = 4.2 x 10(-6) M). M.MspI binds approximately 5-fold more tightly to DNA containing its recognition sequence, CCGG, than to nonspecific sequences in the absence of cofactors. In the presence of S-adenosyl-L-methionine, S-adenosyl-L-homocysteine or sinefungin the discrimination between specific and non-specific sequences increases up to 100-fold. DNasel footprinting studies indicate that 16 base pairs of DNA are covered by M.MspI, with the recognition sequence CCGG located asymmetrically within the footprint.     

Mbd1 is recruited to both methylated and nonmethylated CpGs via distinct DNA binding domains

MBD1 is a vertebrate methyl-CpG binding domain protein (MBD) that can bring about repression of methylated promoter DNA sequences. Like other MBD proteins, MBD1 localizes to nuclear foci that in mice are rich in methyl-CpG. In methyl-CpG-deficient mouse cells, however, Mbd1 remains localized to heterochromatic foci whereas other MBD proteins become dispersed in the nucleus. We find that Mbd1a, a major mouse isoform, contains a CXXC domain (CXXC-3) that binds specifically to nonmethylated CpG, suggesting an explanation for methylation-independent localization. Transfection studies demonstrate that the CXXC-3 domain indeed targets nonmethylated CpG sites in vivo. Repression of nonmethylated reporter genes depends on the CXXC-3 domain, whereas repression of methylated reporters requires the MBD. Our findings indicate that MBD1 can interpret the CpG dinucleotide as a repressive signal in vivo regardless of its methylation status.     

The effects of arginine on protein binding and elution in hydrophobic interaction and ion-exchange chromatography

Arginine is effective in suppressing aggregation of proteins and may be beneficial to be included during purification processes. We have shown that arginine reduces non-specific protein binding in gel permeation chromatography and facilitates elution of antibodies from Protein-A columns. Here we have examined the effects of arginine on binding and elution of the proteins during hydrophobic interaction (HIC) and ion- exchange chromatographies (IEC) using recombinant monoclonal antibodies (mAbs) and human interleukin-6. In the case of HIC, the proteins were bound to a phenyl-Sepharose column in the presence of ammonium sulfate (AS) with or without arginine and eluted with a descending concentration of AS. While use of 1 M AS in the loading buffer resulted in complete binding of the mAb, inclusion of 1 M arginine in loading and equilibration buffer, only when using low-substituted phenyl-Sepharose, resulted in weaker binding of the proteins. While decreasing AS concentration to 0.75 M resulted in partial elution of the mAB, elution was facilitated with inclusion of 0.5-1 M arginine. In the case of IEC, arginine was included in the loading samples. Inclusion of arginine during binding to the IEC columns resulted in a greater recovery and less aggregation even when elution was done in the absence of arginine. These results indicate that arginine enhances elution of proteins bound to the resin, suggesting its effectiveness as a solvent for elution in HIC and IEC.     

The role of tryptophan residues and hydrophobic interaction in the binding of riboflavin in egg-yolk flavoprotein.

Egg-yolk flavoprotein has 7.2 tryptophan residues exposed, while the apoprotein shows an apparent exposure of 80 percent of these (5.7 residues) with dimethylsulphoxide as the perturbant. In the apoprotein at pH 6.9 only 4 groups are perturbed to ethylene glycol, 3.2 to glycerol and 1.4 to sucrose. Diminishing estimates of exposure obtained with increasing molecular diameter of the perturbant suggests that part of indole chromophores of apoprotein are located in "crevices" of the protein molecule. The apoprotein was treated with 2-hydroxy-5-nitrobenzyl bromide, H2O2 and N-bromosuccinimide under conditions designed to accomplish modification of tryptophan residues. Five to six of the eight tryptophans present in the protein were modified. Under these conditions the apoprotein completely looses its capacity for binding riboflavin and the fluorescent intensity of the protein at 360 nm is quenched at the same time to about 80 percent of its initial value. The presence of nonpolar amino acid residues on the surface of the apoprotein suggested the importance of hydrophobic interactions as the dominant factor controlling the binding of riboflavin. The hydrophobic probes Indocyanine green and 4-benzoylamide-4-aminostilbene-2,2-disulphonic acid bound to the apoprotein giving equimolar complexes with dissocation constants, KD 6.5-10(-7) M and 1.8-10(-6) M, respectively, Addition of an equimolar amount of riboflavin quantitatively displaced these dyes from their complexes with apoprotein as shown by spectrophotometric and spectrofluorometric studies.