To fulfill their biological functions, proteins must interact with their specific binding partners and often function as large assemblies composed of multiple proteins or proteins plus other biomolecules. Structural characterization of these complexes, including identification of all binding partners, their relative binding affinities, and complex topology, is integral for understanding function. Understanding how proteins assemble and how subunits in a complex interact is a cornerstone of structural biology. Here we report a native mass spectrometry (MS)-based method to characterize subunit interactions in globular protein complexes. We demonstrate that dissociation of protein complexes by surface collisions, at the lower end of the typical surface-induced dissociation (SID) collision energy range, consistently cleaves the weakest protein:protein interfaces, producing products that are reflective of the known structure. We present here combined results for multiple complexes as a training set, two validation cases, and four computational models. We show that SID appearance energies can be predicted from structures via a computationally derived expression containing three terms (number of residues in a given interface, unsatisfied hydrogen bonds, and a rigidity factor).protein complex | native mass spectrometry | protein interactions | structural biology | surface-induced dissociation N ative mass spectrometry (MS) has emerged as a powerful structural biology tool. By using "soft" ionization techniques such as nanoelectrospray ionization, noncovalent interactions can be retained, enabling the study of intact protein:protein, protein: ligand, and protein:RNA complexes in the gas phase (1-4). Native MS overcomes many of the barriers associated with traditional protein characterization methods; it requires low sample volumes (3-10 μL) and micromolar or lower concentrations, while also having a broad mass range for analysis, allowing study of small monomeric proteins up to large megadalton assemblies (1,5).Typical MS experiments to study subunit interactions of protein complexes involve first preparing the sample in an aqueous solution at near neutral pH, typically 100-200 mM ammonium acetate. The complex is then introduced intact into the mass spectrometer to measure the mass of the native complex. To obtain subunit connectivity information on the sample, the complex can be disrupted in solution, typically either with small volumes of organic solvent or through alteration of the ionic strength; this destabilizes the protein:protein interfaces and allows measurement of stable subcomplexes (6, 7). This approach, however, targets all species present in solution and can therefore be problematic for heterogeneous samples where it may not be possible to decipher which subcomplex came from which precursor. Alternatively, the complex can be isolated and then dissociated in the gas phase. The most commonly used dissociation method for such studies is collision-induced dissociation (CID). In CID protein ions are accelerated i...

Salmonella enterica serovar Typhimurium (Salmonella) is one of the most significant food-borne pathogens affecting both humans and agriculture. We have determined that Salmonella encodes an uptake and utilization pathway specific for a novel nutrient, fructose-asparagine (F-Asn), which is essential for Salmonella fitness in the inflamed intestine (modeled using germ-free, streptomycin-treated, ex-germ-free with human microbiota, and IL10−/− mice). The locus encoding F-Asn utilization, fra, provides an advantage only if Salmonella can initiate inflammation and use tetrathionate as a terminal electron acceptor for anaerobic respiration (the fra phenotype is lost in Salmonella SPI1− SPI2− or ttrA mutants, respectively). The severe fitness defect of a Salmonella fra mutant suggests that F-Asn is the primary nutrient utilized by Salmonella in the inflamed intestine and that this system provides a valuable target for novel therapies.

The trimethylamine methyltransferase MttB is the first described member of a superfamily comprising thousands of microbial proteins. Most members of the MttB superfamily are encoded by genes that lack the codon for pyrrolysine characteristic of trimethylamine methyltransferases, raising questions about the activities of these proteins. The superfamily member MtcB is found in the human intestinal isolate Eubacterium limosum ATCC 8486, an acetogen that can grow by demethylation of L-carnitine. Here, we demonstrate that MtcB catalyzes L-carnitine demethylation. When growing on L-carnitine, E. limosum excreted the unusual biological product norcarnitine as well as acetate, butyrate, and caproate. Cellular extracts of E. limosum grown on L-carnitine, but not lactate, methylated cob(I)alamin or tetrahydrofolate using L-carnitine as methyl donor. MtcB, along with the corrinoid protein MtqC, and the methyl-corrinoid:tetrahydrofolate methyltransferase MtqA were much more abundant in E. limosum cells grown on L-carnitine than on lactate. Recombinant MtcB methylates either cob(I)alamin or Co(I)-MtqC in the presence of L-carnitine, and to a much lesser extent, γ-butyrobetaine. Other quaternary amines were not substrates. Recombinant MtcB, MtqC, and MtqA methylated tetrahydrofolate via L-carnitine, forming a key intermediate in the acetogenic Wood-Ljungdahl pathway. To our knowledge MtcB methylation of cobalamin or Co(I)-MtqC represents the first described mechanism of biological L-carnitine demethylation. The conversion of L-carnitine and its derivative γ-butyrobetaine to trimethylamine by the gut microbiome has been linked to cardiovascular disease. The activities of MtcB and related proteins in E. limosum might demethylate proatherogenic quaternary amines and contribute to the perceived health benefits of this human gut symbiont.

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Insertions in the Salmonella enterica fra locus, which encodes the fructose-asparagine (F-Asn) utilization pathway, are highly attenuated in mouse models of inflammation (>1000-fold competitive index). Here, we report that F-Asn is bacteriostatic to a fraB mutant (IC50 19 μM), but not to the wild-type or a fra island deletion mutant. We hypothesized that the presence of FraD kinase and absence of FraB deglycase causes build-up of a toxic metabolite: 6-phosphofructose-aspartate (6-P-F-Asp). We used biochemical assays to assess FraB and FraD activities, and mass spectrometry to confirm that the fraB mutant accumulates 6-P-F-Asp. These results, together with our finding that mutants lacking fraD or the fra island are not attenuated in mice, suggest that the extreme attenuation of a fraB mutant stems from 6-P-F-Asp toxicity. Salmonella FraB is therefore an excellent drug target, a prospect strengthened by the absence of the fra locus in most of the gut microbiota.

Phenyl methylphosphonic acid (PMP) and p-nitrophenyl methylphosphonic acid (p-NPMP) have been synthesized. The hydrolysis of PMP and its anion has been studied in acid, neutral, and basic solutions; the hydrolysis of the anion of p-NPMP was examined in neutral and basic media. We have observed nucleophilic attack by water on p-NPMP in neutral solution. In basic media, both PMP and p-NPMP react via nucelophilic attack by hydroxide ion at phosphorus. Near pH 9, calcium ions catalyze the hydrolysis of p-NPMP. PMP hydrolysis shows a rate maximum in moderately concentrated acid solutions.

A phenolate anion reacts with persulfate ion in alkaline solution to yield a product in which a sulfate group enters the ring para or ortho to the phenolic group. Para substitution predominates. Subsequent acid‐catalyzed hydrolysis yields the dihydric phenol. The reaction was discovered by Karl Elbs in 1893 and named the Elbs persulfate oxidation . The reaction is generally applicable to ortho ‐, meta ‐, and para ‐substituted phenols with isomer distributions. The yields are not very high, particularly from para ‐substituted phenols, but the major contaminant is usually unchanged starting material that can be separated easily from the intermediate sulfate ester by solvent extraction. Other generally oxidizable groups such as an aldehyde or a double bond are usually not affected under the reaction conditions. The reaction was last thoroughly reviewed in 1951. T. R. Seshadri has made major contributions to the development of the Elbs oxidation. Nearly 30% of the references in this chapter are due to him and his colleagues. By analogy with the Elbs persulfate oxidation of phenols, it might be expected that aromatic amines would react with persulfate to give p ‐aminoaryl sulfates. Although the Elbs reaction had been known since 1893, it was not until 60 years later that Boyland et al. reported the extension of this reaction to aromatic amines. In accordance with expectations, aminoaryl sulfates were indeed the major products of the reaction, but, unexpectedly, the substitution took place exclusively ortho to the amino group rather than predominantly in the para position as in the phenol oxidation. Para substitution takes place only if the ortho positions are occupied by substituents other than hydrogen. Boyland and Sims explored the preparative aspects of this reaction in a series of papers. It seems appropriate to name the reaction the Boyland–Sims oxidation. Primary, secondary, and tertiary aromatic amines are all converted to the corresponding o ‐aminoaryl sulfates under conditions similar to those used for the Elbs oxidation, that is, room temperature or below, aqueous alkali, and equimolar quantities of amine and persulfate.