β-methylamino-L-alanine (BMAA), a neurotoxic nonprotein amino acid produced by most cyanobacteria, has been proposed to be the causative agent of devastating neurodegenerative diseases on the island of Guam in the Pacific Ocean. Because cyanobacteria are widespread globally, we hypothesized that BMAA might occur and bioaccumulate in other ecosystems. Here we demonstrate, based on a recently developed extraction and HPLC-MS/MS method and long-term monitoring of BMAA in cyanobacterial populations of a temperate aquatic ecosystem (Baltic Sea, 2007–2008), that BMAA is biosynthesized by cyanobacterial genera dominating the massive surface blooms of this water body. BMAA also was found at higher concentrations in organisms of higher trophic levels that directly or indirectly feed on cyanobacteria, such as zooplankton and various vertebrates (fish) and invertebrates (mussels, oysters). Pelagic and benthic fish species used for human consumption were included. The highest BMAA levels were detected in the muscle and brain of bottom-dwelling fishes. The discovery of regular biosynthesis of the neurotoxin BMAA in a large temperate aquatic ecosystem combined with its possible transfer and bioaccumulation within major food webs, some ending in human consumption, is alarming and requires attention.

It is established that noncovalent complexes can be maintained both during and after electrospray and that assemblies of increasing size and complexity often lead to broadened peaks in mass spectra. This broadening arises from the tendency of large protein assemblies to form adducts with salts and is compounded when complexes are isolated directly from cells, without the full protein complement. To investigate the origins of this broadening in mass spectral peaks and to develop the optimal method for analyzing mass spectra of large protein complexes, we have carried out a systematic investigation of a series of noncovalent complexes representing a range of different sizes and architectures. We establish a positive correlation between peak width and the increased mass observed and show that this correlation is independent of the instrumental parameters employed. Using this relationship we show that we can determine masses of both 30S subunits and intact 2.3 MDa 70S ribosomes from Thermus thermophilus. The masses of both particles are consistent with multiple populations of ribosomes. To identify these various populations we combine simulated mass spectra of ribosomes, with and without the full protein complement, and estimate the extent of adducts from our study of known complexes. The results allow us to determine the contribution of the different subpopulations to the overall mass spectrum. We confirm the existence of these subpopulations using tandem mass spectrometry of intact 30S subunits. Overall, the results show that, rather than uniform particles, gas-phase ribosomes consist of a number of discrete populations. More generally, the results establish a rigorous procedure for accurate mass measurement and spectral analysis of heterogeneous macromolecular assemblies.

The hydrolytic endoribonuclease RNase E, which is widely distributed in bacteria and plants, plays key roles in mRNA degradation and RNA processing in Escherichia coli. The enzymatic activity of RNase E is contained within the conserved amino-terminal half of the 118 kDa protein, and the carboxy-terminal half organizes the RNA degradosome, a multi-enzyme complex that degrades mRNA co-operatively and processes ribosomal and other RNA. The study described herein demonstrates that the carboxy-terminal domain of RNase E has little structure under native conditions and is unlikely to be extensively folded within the degradosome. However, three isolated segments of 10 -40 residues, and a larger fourth segment of 80 residues, are predicted to be regions of increased structural propensity. The larger of these segments appears to be a protein -RNA interaction site while the other segments possibly correspond to sites of self-recognition and interaction with the other degradosome proteins. The carboxy-terminal domain of RNase E may thus act as a flexible tether of the degradosome components. The implications of these and other observations for the organization of the RNA degradosome are discussed.

Protein complexes are an intrinsic aspect of life in the membrane. Knowing which proteins are assembled in these complexes is therefore essential to understanding protein function(s). Unfortunately, recent high throughput protein interaction studies have failed to deliver any significant information on proteins embedded in the membrane, and many membrane protein complexes remain ill defined. In this study, we have optimized the blue native-PAGE technique for the study of membrane protein complexes in the inner and outer membranes of Escherichia coli. In combination with second dimension SDS-PAGE and mass spectrometry, we have been able to identify 43 distinct protein complexes. In addition to a number of well characterized complexes, we have identified known and orphan proteins in novel oligomeric states. For two orphan proteins, YhcB and YjdB, our findings enable a tentative functional assignment. We propose that YhcB is a hitherto unidentified additional subunit of the cytochrome bd oxidase and that YjdB, which co-localizes with the ZipA protein, is involved in cell division. Our reference two-dimensional blue native-SDS-polyacrylamide gels will facilitate future studies of the assembly and composition of E. coli membrane protein complexes during different growth conditions and in different mutant backgrounds.It has been suggested that nearly all biochemical processes are performed by protein complexes (1). This is particularly true in cellular membranes, where many well characterized proteins assemble into complexes that carry out important tasks in energy generation, protein trafficking, and small molecule transport. Many uncharacterized proteins ("orphans") are also predicted to be localized in cell membranes (2, 3), and it is probable that they also often assemble into complexes. Identifying the interacting partners of these proteins is critical to understanding their function.Unfortunately, our knowledge of protein complexes in cellular membranes is poor, because membrane proteins are incompatible with commonly used protein interaction assays. High throughput studies on model systems (4 -11) have therefore consistently disregarded membrane proteins (12). Although genetic tools specific for membrane protein interactions have been developed (13-15), they have not yet been pursued past proof of principle.A related and elusive aspect of membrane biology pertains to how proteins are assembled into complexes following their insertion into the membrane. Although some folding chaperones have been identified for model substrates, the ubiquity of their roles is not known, and little is known about the assembly process. Robust and effective experimental assays are required to tackle the question of membrane protein assembly.Blue native (BN) 3 -PAGE (16, 17) offers an attractive proteomic solution for the analysis of membrane protein complexes. It has been successfully applied to respiratory complexes in mitochondria and Paracoccus denitrificans (18 -24) and photosynthetic complexes of chloroplasts and Synechocystis ...

Ribosomes are universal translators of the genetic code into protein and represent macromolecular structures that are asymmetric, often heterogeneous, and contain dynamic regions. These properties pose considerable challenges for modern-day structural biology. Despite these obstacles, high-resolution x-ray structures of the 30S and 50S subunits have revealed the RNA architecture and its interactions with proteins for ribosomes from Thermus thermophilus, Deinococcus radiodurans, and Haloarcula marismortui. Some regions, however, remain inaccessible to these highresolution approaches because of their high conformational dynamics and potential heterogeneity, specifically the so-called L7͞ L12 stalk complex. This region plays a vital role in protein synthesis by interacting with GTPase factors in translation. Here, we apply tandem MS, an approach widely applied to peptide sequencing for proteomic applications but not previously applied to MDa complexes. Isolation and activation of ions assigned to intact 30S and 50S subunits releases proteins S6 and L12, respectively. Importantly, this process reveals, exclusively while attached to ribosomes, a phosphorylation of L12, the protein located in multiple copies at the tip of the stalk complex. Moreover, through tandem MS we discovered a stoichiometry for the stalk protuberance on Thermus thermophilus and other thermophiles and contrast this assembly with the analogous one on ribosomes from mesophiles. Together with evidence for a potential interaction with the degradosome, these results show that important findings on ribosome structure, interactions, and modifications can be discovered by tandem MS, even on well studied ribosomes from Thermus thermophilus.bacterial ribosomes ͉ L7͞L12 stalk complex ͉ mass spectrometry T he advent of atomic structures of the 30S and 50S subunits from Thermus thermophilus (1, 2), Deinococcus radiodurans (3), and Haloarcula marismortui (4) has revealed detailed information on the proteins that interact with rRNA. However, protein-protein interactions, particularly those in the stalk complex, are not well defined. In the 5.5-Å 70S structure of ribosomes from Thermus thermophilus density could not be assigned to L10, and only two of the four L7͞L12 proteins that have been proposed for Escherichia coli (5) were tentatively placed at the base of the stalk (6). Interestingly, the stalk complex is readily studied by MS where dissociation of proteins is governed primarily by the extent of protein-RNA interaction (7).The process of electrospray MS, first applied to the study of intact proteins in 1989 (8), is carried out by evaporation of protein-containing droplets to form multiply charged ions in the gas phase. Although not readily applied to MDa particles such as ribosomes, the dissociation of individual proteins and stalk complexes from the intact particle has been shown (7, 9, 10). Such spectra are extremely difficult to interpret in part because of the number of proteins (Ͼ50), their numerous posttranslational modifications, and the presence ...