The most universal cell-cell signaling mechanism in Gram-negative bacteria occurs via the production and response to a class of small diffusible molecules called N-acylhomoserine lactones (AHLs). This communication is called quorum sensing and is responsible for the regulation of several physiological processes and many virulence factors in pathogenic bacteria. The detection of these molecules has been rendered possible by the utilization of genetically engineered bacterial biosensors which respond to the presence of exogenously supplied AHLs. In this study, using diverse bacterial biosensors, several biosensor activating fractions were purified by organic extraction, HPLC and TLC of cell-free culture supernatants of plant growth-promoting Pseudomonas putida WCS358. Surprisingly, it was observed that the most abundant compounds in these fractions were cyclic dipeptides (diketopiperazines, DKPs), a rather novel finding in Gram-negative bacteria. The purification, characterization, chemical synthesis of four DKPs are reported and their possible role in cell-cell signaling is discussed.

The formation of a disulfide bond between adjacent cysteine residues is accompanied by the formation of a tight turn of the protein backbone. In nearly 90% of the structures analyzed a type VIII turn was found. The peptide bond between the two cysteines is in a distorted trans conformation, the omega torsion angle ranges from 159 to -133 degrees, with an average value of 171 degrees. The constrained nature of the vicinal disulfide turn and the pronounced difference observed between the oxidized and reduced states, suggests that vicinal disulfides may be employed as a 'redox-activated' conformational switch.

Protein methylation at arginine residues is a prevalent posttranslational modification in eukaryotic cells that has been implicated in processes from RNA-binding and transporting to protein sorting and transcription activation. Three main forms of methylarginine have been identified: N G -monomethylarginine (MMA), asymmetric N G ,N G -dimethylarginine (aDMA), and symmetric N G ,NЈ G -dimethylarginine (sDMA). To investigate gas-phase fragmentations and characteristic ions arising from methylated and unmodified arginine residues in detail, we subjected peptides containing these residues to electrospray triple-quadrupole tandem mass spectrometry. A variety of low mass ions including (methylated) ammonium, carbodiimidium, and guanidinium ions were observed. Fragment ions resulting from the loss of the corresponding neutral fragments (amines, carbodiimide, and guanidine) from intact molecular ions as well as from N-and C-terminal fragment ions were also identified. Furthermore, the peptides containing either methylated or unmodified arginines gave rise to abundant fragment ions at m/z 70, 112, and 115, for which cyclic ion structures are proposed. Electrospray ionization tandem mass spectra revealed that dimethylammonium (m/z 46) is a specific marker ion for aDMA. A precursor ion scanning method utilizing this fragment ion was developed, which allowed sensitive and specific detection of aDMA-containing peptides even in the presence of a five-fold excess of phosphorylase B digest. Interestingly, regular matrix-assisted laser desorption/ionization mass spectra recorded from aDMA-or sDMA-containing peptides showed metastable fragment ions resulting from cleavages of the arginine side chains. The neutral losses of mono-and dimethylamines permit the differentiation between aDMA and sDMA. (J Am Soc Mass Spectrom 2004, 15, 142Ϫ149)

The oxidative folding of the Amaranthus ␣-amylase inhibitor, a 32-residue cystine-knot protein with three disulfide bridges, was studied in vitro in terms of the disulfide content of the intermediate species. A nonnative vicinal disulfide bridge between cysteine residues 17 and 18 was found in three of five fully oxidized intermediates. One of these, the most abundant folding intermediate (MFI), was further analyzed by 1 H NMR spectroscopy and photochemically induced dynamic nuclear polarization, which revealed that it has a compact structure comprising slowly interconverting conformations in which some of the amino acid side chains are ordered. NMR pulsed-field gradient diffusion experiments confirmed that its hydrodynamic radius is indistinguishable from that of the native protein. Molecular modeling suggested that the eight-membered ring of the vicinal disulfide bridge in MFI may be located in a loop region very similar to those found in experimentally determined 3D structures of other proteins. We suggest that the structural constraints imposed on the folding intermediates by the nonnative disulfides, including the vicinal bridge, may play a role in directing the folding process by creating a compact fold and bringing the cysteine residues into close proximity, thus facilitating reshuffling to native disulfide bridges.T he cystine knot, or the knottin fold, is a compact structure consisting of a short triple-stranded antiparallel ␤-sheet reinforced by three disulfide bridges that form a topological knot [see Amaranthus ␣-amylase inhibitor (AAI) structure, Fig. 1A Inset]. It is found both in small peptides and as a domain of larger proteins (1) and in addition, it is a recurrent substructure of larger cysteine-rich motifs such as the well known conotoxin fold (2). Despite their small size, cystine knots can fulfill a large variety of functions ranging from ion-channel blocking to enzyme inhibition, which makes them ideal protein engineering scaffolds for industrial applications (3, 4). Because cystine knots occur in many unrelated species (fungi, plants, snails, and spiders, etc.), it is presumed that this fold has emerged via convergent evolution (2). The compactness and versatility of this fold are best illustrated by the fact that both the shortest peptide inhibitor of ␣-amylases, AAI [32 amino acids (Fig. 1 A)] (5-8) and the shortest lectin molecule known so far (9), are members of this family.Cystine knot peptides are known to form readily in vitro under the conditions of oxidative folding (4). In this work, we describe an analysis of the oxidative folding intermediates of AAI, which shows that the native protein (N) forms via several fully oxidized intermediates with nonnative disulfide bonds, including the vicinal disulfide 17-18. NMR spectroscopy revealed that the main folding intermediate has a structure as compact as the completely folded peptide, comprising a number of slowly interconverting backbone conformations. On the basis of the experimental data, we suggest that the compactness, a prerequisi...