Proteomics in mixtures: Study results of ABRF-PRG02.

The trend in proteomics is to work with increasingly complex protein mixtures, limiting the protein separation steps prior to analysis. This is due in part to the difficulties encountered with detecting low abundance proteins, protein losses during SDS PAGE, and the limited separation capability of even 2D PAGE where a single protein spot may still contain multiple proteins. Hence, the ABRF-PRG02 sample was designed to study a simple protein mixture of five proteins at the approximately 2 pmol and approximately 200 fmol levels. The sample, after a tryptic digestion, was sent out by the Proteomics Research Group of the ABRF to interested member labs. A total of 41 labs participated in this study, with each participant using some type of mass spectrometric analysis. Laboratories that used microLC-NSI (microLC with nanospray ionization) with MS/MS analysis had a higher percent accuracy than labs using MALDI-MS (matrix assisted laser desorption ionization mass spectrometry).

Identification of the autophosphorylation sites and characterization of their effects in the protein kinase DYRK1A.

Protein kinases of the DYRK ('dual-specificity tyrosine-regulated kinase') family are characterized by a conserved Tyr-Xaa-Tyr motif (Tyr-319-Tyr-321) in a position exactly corresponding to the activation motif of the mitogen-activated protein kinase (MAP kinase) family (Thr-Xaa-Tyr). In a molecular model of the catalytic domain of DYRK1A, the orientation of phosphorylated Tyr-321 is strikingly similar to that of Tyr-185 in the known structure of the activated MAP kinase, extracellular-signal-regulated kinase 2. Consistent with our model, substitution of Tyr-321 but not of Tyr-319 by phenylalanine markedly reduced the enzymic activity of recombinant DYRK1A expressed in either Escherichia coli or mammalian cells. Direct identification of phosphorylated residues by tandem MS confirmed that Tyr-321, but not Tyr-319, was phosphorylated. When expressed in COS-7 cells, DYRK1A was found to be fully phosphorylated on Tyr-321. A catalytically inactive mutant of DYRK1A contained no detectable phosphotyrosine, indicating that Tyr-321 is autophosphorylated by DYRK1A. MS identified Tyr-111 and Ser-97 as additional autophosphorylation sites in the non-catalytic N-terminal domain of bacterially expressed DYRK1A. Enzymic activity was not affected in the DYRK1A-Y111F mutant. The present experimental data and the molecular model indicate that the activity of DYRK1A is dependent on the autophosphorylation of a conserved tyrosine residue in the activation loop.

HMG-1 enhances HMG-I/Y binding to an A/T-rich enhancer element from the pea plastocyanin gene.

High-mobility-group proteins HMG-1 and HMG-I/Y bind at overlapping sites within the A/T-rich enhancer element of the pea plastocyanin gene. Competition binding experiments revealed that HMG-1 enhanced the binding of HMG-I/Y to a 31-bp region (P31) of the enhancer. Circularization assays showed that HMG-1, but not HMG-I/Y, was able to bend a linear 100-bp DNA containing P31 so that the ends could be ligated. HMG-1, but not HMG-I/Y, showed preferential binding to the circular 100-bp DNA compared with the equivalent linear DNA, indicating that alteration of the conformation of the DNA by HMG-1 was not responsible for enhanced binding of HMG-I/Y. Direct interaction of HMG-I/Y and HMG-1 in the absence of DNA was demonstrated by binding of 35S-labeled proteins to immobilized histidine-tagged proteins, and this was due to an interaction of the N-terminal HMG-box-containing region of HMG-1 and the C-terminal AT-hook region of HMG-I/Y. Kinetic analysis using the IAsys biosensor revealed that HMG-1 had an affinity for immobilized HMG-I/Y (Kd = 28 nM) similar to that for immobilized P31 DNA. HMG-1-enhanced binding of HMG-I/Y to the enhancer element appears to be mediated by the formation of an HMG-1-HMG-I/Y complex, which binds to DNA with the rapid loss of HMG-1.

A proteomic analysis of organelles from Arabidopsis thaliana.

We introduce the use of Arabidopsis thaliana callus culture as a system for proteomic analysis of plant organelles using liquid-grown callus. This callus is relatively homogeneous, reproducible and cytoplasmically rich, and provides organelles in sufficient quantities for proteomic studies. A database was generated of mitochondrial, endoplasmic reticulum (ER), Golgi/prevacuolar compartment and plasma membrane (PM) markers using two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2-D SDS-PAGE) and peptide sequencing or mass spectrometric methods. The major callus membrane-associated proteins were characterised as being integral or peripheral by Triton X-114 phase partitioning. The database was used to define specific proteins at the Arabidopsis callus plasma membrane. This database of organelle proteins provides the basis for future characterisation of the expression and localisation of novel plant proteins.

Kinetic analysis of high-mobility-group proteins HMG-1 and HMG-I/Y binding to cholesterol-tagged DNA on a supported lipid monolayer.

High-mobility-group proteins HMG-1 and HMG-I/Y bind to multiple sites within a 268 bp A/T-rich enhancer element of the pea plastocyanin gene ( PetE ). Within a 31 bp region of the enhancer, the binding site for HMG-1 overlaps with the binding site for HMG-I/Y. The kinetics of binding and the affinities of HMG-1 and HMG-I/Y for the 31 bp DNA were determined using surface plasmon resonance. Due to very high non-specific interactions of the HMG proteins with a carboxymethyl-dextran matrix, a novel method using a cholesterol tag to anchor the DNA in a supported lipid monolayer on a thin gold film was devised. The phosphatidylcholine monolayer produced a surface that reduced background interactions to a minimum and permitted the measurement of highly reproducible protein-DNA interactions. The association rate constant ( k (a)) of HMG-I/Y with the 31 bp DNA was approximately 5-fold higher than the rate constant for HMG-1, whereas the dissociation constant ( K (D)) for HMG-I/Y (3.1 nM) was approximately 7-fold lower than that for HMG-1 (20.1 nM). This suggests that HMG-I/Y should bind preferentially at the overlapping binding site within this region of the PetE enhancer.

Autonomous folding of a peptide corresponding to the N-terminal beta-hairpin from ubiquitin.

The N-terminal 17 residues of ubiquitin have been shown by 1H NMR to fold autonomously into a beta-hairpin structure in aqueous solution. This structure has a specific, native-like register, though side-chain contacts differ in detail from those observed in the intact protein. An autonomously folding hairpin has previously been identified in the case of streptococcal protein G, which is structurally homologous with ubiquitin, but remarkably, the two are not in topologically equivalent positions in the fold. This suggests that the organization of folding may be quite different for proteins sharing similar tertiary structures. Two smaller peptides have also been studied, corresponding to the isolated arms of the N-terminal hairpin of ubiquitin, and significant differences from simple random coil predictions observed in the spectra of these subfragments, suggestive of significant limitation of the backbone conformational space sampled, presumably as a consequence of the strongly beta-structure favoring composition of the sequences. This illustrates the ability of local sequence elements to express a propensity for beta-structure even in the absence of actual sheet formation. Attempts were made to estimate the population of the folded state of the hairpin, in terms of a simple two-state folding model. Using published "random coil" values to model the unfolded state, and values derived from native ubiquitin for the putative unique, folded state, it was found that the apparent population varied widely for different residues and with different NMR parameters. Use of the spectra of the subfragment peptides to provide a more realistic model of the unfolded state led to better agreement in the estimates that could be obtained from chemical shift and coupling constant measurements, while making it clear that some other approaches to population estimation could not give meaningful results, because of the tendency to populate the beta-region of conformational space even in the absence of the hairpin structure.

Involvement of a flavin iminoquinone methide in the formation of 6-hydroxyflavin mononucleotide in trimethylamine dehydrogenase: a rationale for the existence of 8alpha-methyl and C6-linked covalent flavoproteins.

In trimethylamine dehydrogenase, substrate is bound in the active site via cation-pi bonding to three aromatic residues (Tyr-60, Trp-264, and Trp-355). Mutation of one of these residues (Trp-355 --> Leu, mutant W355L) influences the chemistry of the flavin mononucleotide in the active site, enabling derivatization to 6-hydroxy-FMN. The W355L mutant is purified as a mixture of deflavo, natural 6-S-cysteinyl-FMN, and inactive 6-hydroxy-FMN forms, and the enzyme is severely compromised in its ability to oxidatively demethylate trimethylamine. Analysis of samples of the native and recombinant wild-type trimethylamine dehydrogenases also revealed the presence of 6-hydroxy-FMN, but at much reduced levels compared with that of the W355L enzyme. Unlike that for a C30A mutant of trimethylamine dehydrogenase, addition of substrate to the W355L trimethylamine dehydrogenase is not required for the production of 6-hydroxy-FMN. A mechanism is proposed for the 6-hydroxylation of FMN in trimethylamine dehydrogenase that involves an electrophilic flavin iminoquinone methide. The proposed mechanism involving the flavin iminoquinone methide could apply to the flavinylation of trimethylamine dehydrogenase at the C6 position but also to the flavinylation of enzymes via the 8alpha position, thus providing a rationale for the evolution of covalent flavoproteins in general. Covalent linkage at C6 or the 8alpha-methyl prevents 6-hydroxylation by direct modification at the C6 atom or by preventing formation of the flavin iminoquinone methide, respectively.

High mobility group proteins HMG-1 and HMG-I/Y bind to a positive regulatory region of the pea plastocyanin gene promoter.

A 268 bp region (P268) of the pea plastocyanin gene promoter responsible for high-level expression has been shown to interact with the high mobility group proteins HMG-1 and HMG-I/Y isolated from pea shoot chromatin. cDNAs encoding an HMG-1 protein of 154 amino acid residues containing a single HMG-box and a C-terminal acidic tail and an HMG-I/Y-like protein of 197 amino acid residues containing four AT-hooks have been isolated and expressed in Escherichia coli to provide large amounts of full-length proteins. DNase I footprinting identified eight binding sites for HMG-I/Y and six binding sites for HMG-1 in P268. Inhibition of binding by the antibiotic distamycin, which binds in the minor groove of A/T-rich DNA, revealed that HMG-I/Y binding was 400-fold more sensitive than HMG-1 binding. Binding-site selection from a pool of random oligonucleotides indicated that HMG-I/Y binds to oligonucleotides containing stretches of five or more A/T bp and HMG-1 binds preferentially to oligonucleotides enriched in dinucleotides such as TpT and TpG.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry as a rapid quality control method in oligonucleotide synthesis.

Oligonucleotide synthesis is a major activity for a large number of laboratories throughout the world. Dimethoxytrityl-deprotection monitors on instruments give the operator a measure of synthetic efficiency, but cannot be considered as a reliable indicator of the final quality of the cleaved and deprotected oligonucleotide. We have therefore adapted and developed methods of analyzing oligonucleotides by matrix-assisted laser desorption ionization mass spectrometry to the quality control situation, resulting in the ability to analyze 20 oligonucleotides up to at least 40 bases long in less than an hour. Most oligonucleotides can be analyzed automatically using the automatic scanning feature of a Kratos MALDI III analyzer.

Casein kinase II phosphorylates Ser468 in the PEST domain of the Drosophila IkappaB homologue cactus.

Cactus protein is a Drosophila homologue of the mammalian IkappaB family of cytoplasmic anchor proteins. In unstimulated cells they function to retain rel/NFkappaB transcription factors in the cytoplasm but are rapidly degraded in response to signalling. The destruction of cactus or IkappaBalpha allows the rel/NFkappaB transcription factor to relocalise to the nucleus. Cactus is a phosphoprotein and has in its C-terminus a PEST protein stability domain. In this paper we show that, like mammalian IkappaBalpha, the PEST domain of cactus is phosphorylated by casein kinase II. We have localised the site of modification to a single residue, Ser468, and find no evidence for additional phosphorylation sites. The conservation of these sites in mammalian and invertebrate cytoplasmic anchor proteins suggests that phosphorylation by casein kinase II may play a critical functional role, plausibly in the regulation of constitutive or inducible proteolysis.

Photoaffinity labelling of a DNA-binding site on the globular domain of histone H5.

We have labelled a DNA-binding site on the globular domain of histone H5 (GH5) by ultraviolet-activated cross-linking of a self-complementary 5-bromodeoxyuridine (5BrU)-substituted oligonucleotide with the sequence 5'-AGCGA5BrUATCGCT-3'. Cross-linking was to His62, mainly to the protein backbone. This observation provides further support for the mode of binding of GH5 to DNA proposed on the basis of the similarity between the X-ray crystal structure of GH5 and the DNA-bound structures of catabolite activator protein and hepatic nuclear factor 3 gamma [Ramakrishnan, V. (1994) Curr. Opin. Struct. Biol. 4. 44-50].

Native-like beta-hairpin structure in an isolated fragment from ferredoxin: NMR and CD studies of solvent effects on the N-terminal 20 residues.

The conformational properties of protein fragments have been widely studied as models of the earliest initiation events in protein folding. While native-like alpha-helices and beta-turns have been identified, less is known about the factors that underly beta-sheet formation, in particular beta-hairpins, where considerably greater long-range order is required. The N-terminal 20 residue sequence of native ferredoxin I (from the blue-green alga Aphanothece sacrum) forms a beta-hairpin in the native structure and has been studied in isolation by NMR and CD spectroscopy. Local native-like interactions alone are unable to stabilize significantly a folded conformation of the 20-residue fragment in purely aqueous solution. However, we show that the addition of low levels of organic co-solvents promotes formation of native-like beta-hairpin structure. The results suggest an intrinsic propensity of the peptide to form a native-like beta-hairpin structure, and that the organic co-solvent acts in lieu of the stabilizing influence of tertiary interactions (probably hydrophobic contacts) which occur in the folding of the complete ferredoxin sequence. The structure of the isolated hairpin, including the native-like register of interstrand hydrogen bonding interactions, appears to be determined entirely by the amino acid sequence. The solvent conditions employed have enabled this intrinsic property to be established.

Flavinylation in wild-type trimethylamine dehydrogenase and differentially charged mutant enzymes: a study of the protein environment around the N1 of the flavin isoalloxazine.

In wild-type trimethylamine dehydrogenase, residue Arg-222 is positioned close to the isoalloxazine N1/C2 positions of the 6S-cysteinyl FMN. The positively charged guanidino group of Arg-222 is thought to stabilize negative charge as it develops at the N1 position of the flavin during flavinylation of the enzyme. Three mutant trimethylamine dehydrogenases were constructed to alter the nature of the charge at residue 222. The amount of active flavinylated enzyme produced in Escherichia coli is reduced when Arg-222 is replaced by lysine (mutant R222K). Removal or reversal of the charge at residue 222 (mutants R222V and R222E, respectively) leads to the production of inactive enzymes that are totally devoid of flavin. A comparison of the CD spectra for the wild-type and mutant enzymes revealed no major structural change following mutagenesis. Like the wild-type protein, each mutant enzyme contained stoichiometric amounts of the 4Fe-4S cluster and ADP. Electrospray MS also indicated that the native and recombinant wild-type enzymes were isolated as a mixture of deflavo and holo enzyme, but that each of the mutant enzymes have masses expected for deflavo trimethylamine dehydrogenase. The MS data indicate that the lack of assembly of the mutant proteins with FMN is not due to detectable levels of post-translational modification of significant mass. The experiments reported here indicate that simple mutagenic changes in the FMN-binding site can reduce the proportion of flavinylated enzyme isolated from Escherichia coli and that positive charge is required at residue 222 if flavinylation is to proceed.

Protein recognition of ammonium cations using side-chain aromatics: a structural variation for secondary ammonium ligands.

A model for the structure of dimethylamine dehydrogenase was generated using the crystal coordinates of trimethylamine dehydrogenase. Substrate is bound in trimethylamine dehydrogenase by cation-pi bonding, but modeling of dimethylamine dehydrogenase suggests that secondary amines are bound by a mixture of cation-pi and conventional hydrogen bonding. In dimethylamine dehydrogenase, binding is orientationally more specific and distinct from those proteins that bind tertiary and quaternary amine groups.

A short linear peptide derived from the N-terminal sequence of ubiquitin folds into a water-stable non-native beta-hairpin.

A 16-residue peptide derived from the N-terminal sequence of ubiquitin forms a stable monomeric beta-hairpin that is estimated to be approximately 80% populated in aqueous solution. The peptide sequence has been modified from native ubiquitin by replacing the five residues found in a type I G1 bulged turn (Thr-Leu-Thr-Gly-Lys) with four residues (Asn-Pro-Asp-Gly) to maximize the probability of forming a beta-turn. Unexpectedly, the bulged turn conformation is re-established in the beta-hairpin in solution with two consequences: a one-amino acid frameshift in the alignment of the peptide main chain occurs relative to the native hairpin, and side chains formerly on opposite faces of the hairpin are brought together on the same face. The presence of the bulged turn in native ubiquitin may help in the avoidance of the stable non-native register of amino acids found here which would be unproductive for folding.

The primary structure of Hyphomicrobium X dimethylamine dehydrogenase. Relationship to trimethylamine dehydrogenase and implications for substrate recognition.

The gene encoding dimethylamine dehydrogenase from Hyphomicrobium X has been cloned and over-expressed in Escherichia coli. Using the chemically determined protein sequence, primers were designed to amplify DNA fragments encoding the proximal and distal parts of the gene. These fragments were used to synthesise two probes and the dmd gene was cloned as part of two BamHI fragments isolated from digested genomic DNA. The sequence of the complete open reading frame was determined on both strands and contained 2211 bp coding for a protein of 736 amino acids, including the N-terminal methionine residue that is removed when expressed in the native host. The molecular mass of the processed apoprotein predicted from the DNA sequence is 82,523 Da. Dimethylamine dehydrogenase is closely related to the trimethylamine dehydrogenase of Methylophilus methylotrophus W3A1 (63.5% identical) and other class I FMN-binding beta 8 alpha 8 barrel flavoproteins. Residues in the active site of trimethylamine dehydrogenase that are known, or implicated, to be important in catalysis are conserved in dimethylamine dehydrogenase. Sequence alignment of dimethylamine and trimethylamine dehydrogenases suggests that the specificity for secondary and tertiary amines resides in a single amino acid substitution in a substrate-binding aromatic bowl located in the active site of the enzymes.

The flavinylation reaction of trimethylamine dehydrogenase. Analysis by directed mutagenesis and electrospray mass spectrometry.

The flavinylation reaction products of wild-type and mutant forms of trimethylamine dehydrogenases purified from Methylophilus methylotrophus (bacterium W3A1) and Escherichia coli were studied by electrospray mass spectrometry (ESMS). The ESMS analyses demonstrated for the first time that wild-type enzyme expressed in M. methylotrophus is predominantly in the holoenzyme form, although a small proportion is present as the deflavo enzyme. ESMS demonstrated that the deflavo forms of the recombinant wild-type and mutant enzymes are not post-translationally modified and therefore prevented from assembling with flavin mononucleotide (FMN) because of previously unrecognized modifications. The data suggest that the higher proportion of deflavo enzyme observed for the recombinant wild-type enzyme is a consequence of the higher expression levels in E. coli. Mutagenesis of the putative flavinylation base (His-29 to Gln-29) did not prevent flavinylation, but the relative proportion of flavinylated product was substantially less than that seen for the recombinant wild-type enzyme. No flavinylation products were observed for a double mutant (His-29 to Cys-29; Cys-30 to His-30), in which the positions of the putative flavinylation base and cysteine nucleophile were exchanged. Taken together, the data indicate that the assembly of trimethylamine dehydrogenase with FMN occurs during the folding of the enzyme, and in the fully folded form, deflavo enzyme is unable to recognize FMN. Results of site-directed mutagenesis experiments in the FMN-binding site suggest that following mutation the affinity for FMN during the folding process is reduced. Consequently, in the folded mutant enzymes, less flavin is trapped in the active site, and reduced levels of flavinylated product are obtained.

Electron tunneling in substrate-reduced trimethylamine dehydrogenase: kinetics of electron transfer and analysis of the tunneling pathway.

The reoxidation of substrate-reduced trimethylamine dehydrogenase by the artificial electron acceptor ferricenium hexafluorophosphate was studied by stopped-flow spectroscopy. The rate constants for the two sequential one-electron transfers from the reduced 4Fe-4S center to ferricenium ions were measured, the first (ka = 49 s-1) being about 7 times greater than the second (kb = 7.3 s-1) at 20 degrees C and neutral pH. The temperature dependence of the second electron transfer was studied over the range 10-40 degrees C, and the rate constant ranged from 5.7 to 19.2 s-1. Analysis of the temperature perturbation of kb by Marcus theory yielded values for the reorganizational energy of 1.95 eV and the electronic coupling matrix element of 0.26 cm-1. An electron tunneling pathway distance of 13 +/- 0.7 A was calculated which correlates with the shortest pathway measured from the 4Fe-4S center to the protein surface using the crystallographic coordinates of trimethylamine dehydrogenase. Tyr-442 is implicated in facilitating electron transfer from the enzyme to ferricenium ions. The data suggest a location for the docking site on the surface of trimethylamine dehydrogenase for the physiological electron acceptor (ETF).

A reactive, surface cysteine residue of the class-II fructose-1,6-bisphosphate aldolase of Escherichia coli revealed by electrospray ionisation mass spectrometry.

The state of post-translational modification of the class-II fructose-1,6-bisphosphate aldolase (FBP-aldolase) purified from Escherichia coli was examined by electrospray ionisation mass spectrometry (ESI-MS). The mass was larger than that expected from the known DNA sequence by approximately 80 +/- 6 Da, suggesting the presence of a covalent modification on the protein. Phosphorylation (+ 80 Da), a known modification in an FBP-aldolase from Bacillus subtilis and a suspected modification in this E. coli aldolase, was ruled out as the extra mass was readily removed by treatment with dithiothreitol. Purification of aldolase by a protocol which omitted 2-mercaptoethanol from all buffers resulted in the purified protein having the expected mass (39016 Da). The extra mass was therefore established as a covalent adduct of the protein with 2-mercaptoethanol (+ 76 Da). Reduction and alkylation studies, followed by isolation of tryptic peptides, established that the site of attachment was Cys36. Although no significant effect of the modification on the activity of the protein was observed, the study underlines the ease with which a protein can be modified covalently by a simple and mild purification procedure; such labelling, which may not always be benign, would be undetectable without the routine use of mass spectrometric analysis.