SummaryToluene is anoxically degraded to CO 2 by the denitrifying bacterium Thauera aromatica. The initial reaction in this pathway is the addition of fumarate to the methyl group of toluene, yielding benzylsuccinate as the first intermediate. We purified the enzyme catalysing this reaction, benzylsuccinate synthase (EC 4.1.99-), and studied its properties. The enzyme was highly oxygen sensitive and contained a redox-active flavin cofactor, but no iron centres. The native molecular mass was 220 kDa; four subunits of 94 (␣), 90 (␣Ј), 12 (␤) and 10 kDa (␥) were detected on sodium dodecyl sulphate (SDS) gels. The N-terminal sequences of the ␣-and ␣Ј-subunits were identical, suggesting a C-terminal degradation of half of the ␣-subunits to give the ␣Ј-subunit. The composition of native enzyme therefore appears to be ␣ 2 ␤ 2 ␥ 2 . A 5 kb segment of DNA containing the genes for the three subunits of benzylsuccinate synthase was cloned and sequenced. The masses of the predicted gene products correlated exactly with those of the subunits, as determined by electrospray mass spectrometry. Analysis of the derived amino acid sequences revealed that the large subunit of the enzyme shares homology to glycyl radical enzymes, particularly near the predicted radical site. The highest similarity was observed with pyruvate formate lyases and related proteins. The radical-containing subunit of benzylsuccinate synthase is oxygenolytically cleaved at the site of the glycyl radical, producing the ␣Ј-subunit. The predicted cleavage site was verified using electrospray mass spectrometry. In addition, a gene coding for an activating protein catalysing glycyl radical formation was found. The four genes for benzylsuccinate synthase and the activating enzyme are organized as a single operon; their transcription is induced by toluene. Synthesis of the predicted gene products was achieved in Escherichia coli in a T7-promotor/polymerase system.

The sample fractionation steps conducted prior to mass detection are critically important for the comprehensive analysis of complex protein mixtures. This paper illustrates the effectiveness of OFFGEL electrophoresis with the Agilent 3100 OFFGEL Fractionator for the fractionation of peptides. An Escherichia coli tryptic digest was separated in 24 fractions, and peptides were identified by reversed-phase liquid chromatography on a microfluidic device with mass spectrometric detection. About 90% of the identified individual peptides were found in only one or two fractions. The distribution of the calculated isoelectric points for the peptides identified in each fraction was especially narrow in the acidic pH range. Standard deviations approached the size of the pH segment covered by the respective fraction. The experimental peptide isoelectric point measured by OFFGEL electrophoresis was used as an additional filter for validation of peptide identifications. Molecular & Cellular Proteomics 5:1968 -1974, 2006.Comprehensive analysis of whole proteomes is an extraordinary challenge because of the complexity and wide range of protein concentrations. The success of proteome analysis projects is highly dependent on the quality of the sample fractionation employed prior to analysis by MS. Reducing sample complexity through efficient fractionation allows more complete in-depth analysis of the sample with MS/MS. For peptide-level analysis, cation-exchange (SCX) 1 and reversedphase chromatography (RP) are typically combined (1). The use of IPG IEF instead of SCX has been described before (2, 3). Compared with cation-exchange chromatography, IPG IEF provides higher resolution separation and experimentally derived pI information, which can be used as a filter criterion for tandem mass spectral data validation (3, 4). However, a major limitation of this method is the tedious post-IEF sample processing that requires cutting the IPG gel strip into sections and then extracting and cleaning up peptides from the gel sections.In the present work, an Escherichia coli total protein digest was fractionated by OFFGEL electrophoresis with the Agilent 3100 OFFGEL Fractionator and by reversed-phase chromatography and then analyzed by mass detection (RP-LC/MS). OFFGEL electrophoresis is a recent advance in separation technology that fractionates proteins or peptides according to their pI (5-8). This technique achieves the same high resolution as IPG gels but recovers the sample in the liquid phase. Therefore, it fits nicely into the LC/MS workflow. After fractionation and acidification, a portion of the sample was injected directly onto a chip-based reversed-phase column without additional sample preparation, eliminating the need for tedious and error-prone peptide isolation from the IPG gel. The distribution of the calculated isoelectric points for the identified peptides in the respective fractions was especially narrow in the acidic pH range, with standard deviations in the range of the expected fraction width. About 90% of the identifie...

As a result, the GTP binding properties are unusual, since the triphosphate moiety of GTP is bound with higher affinity than the purine base. Purified Toc34 hydrolyses GTP at a low rate, which could regulate the receptor function. The rate of hydrolysis is greatly stimulated by a precursor protein.

The design and chemical synthesis of de novo metalloproteins on cellulose membranes with the structure of an antiparallel four-helix bundle is described. All possible combinations of three different sets of amphiphilic helices were assembled on cyclic peptide templates which were bound by a cleavable linker to the cellulose. In the hydrophobic interior, the four-helix bundle proteins carry a cysteine and several histidines at various positions for copper ligation. This approach was used successfully to synthesize, for the first time, copper proteins based on a four-helix bundle. UV-vis spectra monitored on the solid support showed ligation of copper(II) by about one-third out of the 96 synthesized proteins and tetrahedral complexes of cobalt(II) by most of these proteins. Three of the most stable copper-binding proteins were synthesized in solution and their structural properties analyzed by spectroscopic methods. Circular dichroism, one-dimensional NMR, and size-exclusion chromatography indicate a folding into a compact state containing a high degree of secondary structure with a reasonably ordered hydrophobic core. They displayed UV-vis absorption, resonance Raman, and EPR spectra intermediate between those of type 1 and type 2 copper centers. The present approach provides a sound basis for further optimizing the copper binding and its functional properties by using combinatorial protein chemistry guided by rational principles.