Throughout December 2010 and January 2011, Queensland experienced widespread flooding due to unusually protracted and heavy rainfalls. In mid-January 2011, four individuals from a small community in Central Queensland were hospitalized with leptospirosis. A further five cases were subsequently identified from around Central Queensland, bringing the total to nine. Microscopic agglutination testing found that serovar Arborea (Leptospira borgpetersenii serovar Arborea) was presumptively responsible for leptospirosis in seven of nine confirmed cases. Serovars Hardjo and Australis were identified in samples from two remaining cases. All cases had exposure to flood water. No single exposure source was identified. This is the first reported outbreak of leptospirosis in Central Queensland and the first report of leptospirosis cases associated with flood water inundation in Queensland. Public health authorities should continue to promote awareness of leptospirosis in flood-affected populations. Healthcare providers must maintain a high level of suspicion for leptospirosis during and after flood events.

The CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was cross-linked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived.

Intramolecular cross-linking coupled with mass spectrometric identification of cross-linked amino acids is a rapid method for elucidating low-resolution protein tertiary structures or fold families. However, previous cross-linking studies on model proteins, such as cytochrome c and ribonuclease A, identified a limited number of peptide cross-links that are biased toward only a few of the potentially reactive lysine residues. Here, we report an approach to improve the diversity of intramolecular protein cross-linking starting with a systematic quantitation of the reactivity of lysine residues of a model protein, bovine cytochrome c. Relative lysine reactivities among the 18 lysine residues of cytochrome c were determined by the ratio of d0 and acetyl-d3 groups at each lysine after partial acetylation with sulfosuccinimidyl acetate followed by denaturation and quantitative acetylation of remaining unmodified lysines with acetic-d6 anhydride. These lysine reactivities were then compared with theoretically derived pKa and relative solvent accessibility surface values. To ascertain if partial N-acetylation of the most reactive lysine residues prior to cross-linking can redirect and increase the observable Lys-Lys cross-links, partially acetylated bovine cytochrome c was cross-linked with the amine-specific, bis-functional reagent, bis(sulfosuccinimidyl)suberate. After proteolysis and mass spectrometry analysis, partial acetylation was shown to significantly increase the number of observable peptides containing Lys-Lys cross-links, shifting the pattern from the most reactive lysine residues to less reactive ones. More importantly, these additional cross-linked peptides contained novel Lys-Lys cross-link information not seen in the non-acetylated protein and provided additional distance constraints that were consistent with the crystal structure and facilitated the identification of the proper protein fold.

Modern biomedical research is evolving with the rapid growth of diverse data types, biophysical characterization methods, computational tools and extensive collaboration among researchers spanning various communities and having complementary backgrounds and expertise. Collaborating researchers are increasingly dependent on shared data and tools made available by other investigators with common interests, thus forming communities that transcend the traditional boundaries of the single research laboratory or institution. Barriers, however, remain to the formation of these virtual communities, usually due to the steep learning curve associated with becoming familiar with new tools, or with the difficulties associated with transferring data between tools. Recognizing the need for shared reference data and analysis tools, we are developing an integrated knowledge environment that supports productive interactions among researchers. Here we report on our current collaborative environment, which focuses on bringing together structural biologists working in the area of mass spectrometric based methods for the analysis of tertiary and quaternary macromolecular structures (MS3D) called the Collaboratory for MS3D (C-MS3D). C-MS3D is a Web-portal designed to provide collaborators with a shared work environment that integrates data storage and management with data analysis tools. Files are stored and archived along with pertinent meta data in such a way as to allow file handling to be tracked (data provenance) and data files to be searched using keywords and modification dates. While at this time the portal is designed around a specific application, the shared work environment is a general approach to building collaborative work groups. The goal of this is to not only provide a common data sharing and archiving system, but also to assist in the building of new collaborations and to spur the development of new tools and technologies.

A systematic study of the dissociation patterns of crosslinked peptides analyzed by tandem mass spectrometry is reported. A series of 11-mer peptides was designed around either a polyalanine or polyglycine scaffold with arginine at the C terminus. One or two lysine residues were included at various locations within the peptides to effect inter- or intra-molecular crosslinking, respectively. Crosslinked species were generated with four commonly used amine-specific chemical crosslinking reagents: disuccinimidyl suberate (DSS), disuccinimidyl tartarate (DST), dithiobis(succinimidylpropionate) (DSP), and disuccinimidyl glutarate (DSG). The influence of precursor charge state, location of crosslink, and specific crosslinking reagent on the MS/MS dissociation pattern was examined. Observed trends in the dissociation patterns obtained for these species will allow for improvements to software used in the automated interpretation of crosslinked peptide MS/MS data.

Recent work using chemical cross-linking to define interresidue distance constraints in proteins has shown that these constraints are useful for testing tertiary structural models. We applied this approach to the G-protein-coupled receptor bovine rhodopsin in its native membrane using lysine- and cysteine-targeted bifunctional cross-linking reagents. Cross-linked proteolytic peptides of rhodopsin were identified by combined liquid chromatography and FT-ICR mass spectrometry with automated data-reduction and assignment software. Tandem mass spectrometry was used to verify cross-link assignments and locate the exact sites of cross-link attachment. Cross-links were observed to form between 10 pairs of residues in dark-state rhodopsin. For each pair, cross-linkers with a range of linker lengths were tested to determine an experimental distance-of-closest-approach (DCA) between reactive side-chain atoms. In all, 28 cross-links were identified using seven different cross-linking reagents. Molecular mechanics procedures were applied to published crystal structure data to calculate energetically achievable theoretical DCAs between reactive atoms without altering the position of the protein backbone. Experimentally measured DCAs are generally in good agreement with the theoretical DCAs. However, a cross-link between C316 and K325 in the C-terminal region cannot be rationalized by DCA simulations and suggests that backbone reorientation relative to the crystal coordinates occurs on the timescale of cross-linking reactions. Biochemical and spectroscopic data from other studies have found that the C-terminal region is highly mobile in solution and not fully represented by X-ray crystallography data. Our results show that chemical cross-linking can provide reliable three-dimensional structural information and insight into local conformational dynamics in a membrane protein.

We have developed a new approach for the analysis of interacting interfaces in protein complexes and protein quaternary structure based on cross-linking in the solid state. Protein complexes are freeze-dried under vacuum, and cross-links are introduced in the solid phase by dehydrating the protein in a nonaqueous solvent creating peptide bonds between amino and carboxyl groups of the interacting peptides. Cross-linked proteins are digested into peptides with trypsin in both H2(16)O and H(2)18O and then readily distinguished in mass spectra by characteristic 8 atomic mass unit (amu) shifts reflecting incorporation of two 18O atoms into each C terminus of proteolytic peptides. Computer analysis of mass spectrometry (MS) and MS/MS data is used to identify the cross-linked peptides. We demonstrated specificity and reproducibility of our method by cross-linking homo-oligomeric protein complexes of glutathione-S-transferase (GST) from Schistosoma japonicum alone or in a mixture of many other proteins. Identified cross-links were predominantly of amide origin, but six esters and thioesters were also found. The cross-linked peptides were validated against the GST monomer and dimer X-ray structures and by experimental (MS/MS) analyses. Some of the identified cross-links matched interacting peptides in the native 3D structure of GST, indicating that the structure of GST and its oligomeric complex remained primarily intact after freeze-drying. The pattern of oligomeric GST obtained in solid state was the same as that obtained in solution by Ru (II) Bpy(3)2+ catalyzed, oxidative "zero-length" cross-linking, confirming that it is feasible to use our strategy for analyzing the molecular interfaces of interacting proteins or peptides.

Motivation: Specific and sensitive ligand-based protein detection assays that employ antibodies or small molecules such as peptides, aptamers or other small molecules require that the corresponding surface region of the protein be accessible and that there be minimal cross-reactivity with non-target proteins. To reduce the time and cost of laboratory screening efforts for diagnostic reagents, we developed new methods for evaluating and selecting protein surface regions for ligand targeting.Results: We devised combined structure-and sequence-based methods for identifying 3D epitopes and binding pockets on the surface of the A chain of ricin that are conserved with respect to a set of ricin A chains and unique with respect to other proteins. We (1) used structure alignment software to detect structural deviations and extracted from this analysis the residue-residue correspondence, (2) devised a method to compare corresponding residues across sets of ricin structures and structures of closely related proteins, (3) devised a sequence-based approach to determine residue infrequency in local sequence context and (4) modified a pocket-finding algorithm to identify surface crevices in close proximity to residues determined to be conserved/unique based on our structure- and sequence-based methods. In applying this combined informatics approach to ricin A, we identified a conserved/ unique pocket in close proximity ( but not overlapping) the active site that is suitable for bi- dentate ligand development. These methods are generally applicable to identification of surface epitopes and binding pockets for development of diagnostic reagents, therapeutics and vaccines.

In this work we introduce a postprocessing filter (PostDOCK) that distinguishes true binding ligand-protein complexes from docking artifacts (that are created by DOCK 4.0.1). PostDOCK is a pattern recognition system that relies on (1) a database of complexes, (2) biochemical descriptors of those complexes, and (3) machine learning tools. We use the protein databank (PDB) as the structural database of complexes and create diverse training and validation sets from it based on the "families of structurally similar proteins" (FSSP) hierarchy. For the biochemical descriptors, we consider terms from the DOCK score, empirical scoring, and buried solvent accessible surface area. For the machine-learners, we use a random forest classifier and logistic regression. Our results were obtained on a test set of 44 structurally diverse protein targets. Our highest performing descriptor combinations obtained approximately 19-fold enrichment (39 of 44 binding complexes were correctly identified, while only allowing 2 of 44 decoy complexes), and our best overall accuracy was 92%.

Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurement using liquid chromatography-fourier transform mass spectrometry and an automated data analysis pipeline. Assignments were confirmed and, if necessary, resolved, by tandem MS. The relative reactivity of lysine residues participating in cross-links was evaluated by labeling with NHS-esters. A distinct pattern of cross-link formation within the C-terminal domain, and between loop I and the C-terminal domain, emerged. Theoretical distances based on cross-linking were compared to inter-atomic distances determined from the energy-minimized X-ray crystal structure and Monte Carlo conformational search procedures. In general, the observed cross-links can be explained by re-positioning participating side-chains without significantly altering backbone structure. One exception, between C3 16 and K325, requires backbone motion to bring the reactive atoms into sufficient proximity for cross-linking. Evidence from other studies suggests that residues around K325 for a region of high backbone mobility. These findings show that cross-linking studies can provide insight into the structural dynamics of membrane proteins in their native environment.

We have developed a novel approach to modeling the transmembrane spanning helical bundles of integral membrane proteins using only a sparse set of distance constraints, such as those derived from MS3-D, dipolar-EPR and FRET experiments. Algorithms have been written for searching the conformational space of membrane protein folds matching the set of distance constraints, which provides initial structures for local conformational searches. Local conformation search is achieved by optimizing these candidates against a custom penalty function that incorporates both measures derived from statistical analysis of solved membrane protein structures and distance constraints obtained from experiments. This results in refined helical bundles to which the interhelical loops and amino acid side-chains are added. Using a set of only 27 distance constraints extracted from the literature, our methods successfully recover the structure of dark-adapted rhodopsin to within 3.2 Å of the crystal structure.

Chemical crosslinking is an important tool for probing protein structure and protein-protein interactions. The approach usually involves crosslinking of specific amino acids within a folded protein or protein complex, enzymatic digestion of the crosslinked protein(s), and identification of the resulting crosslinked peptides by liquid chromatography/mass spectrometry (LC/MS). In this manner, distance constraints are obtained for residues that must be in close proximity to one another in the native structure or complex. As the complexity of the system under study increases, for example, a large multi-protein complex, simply measuring the mass of a crosslinked species will not always be sufficient to determine the identity of the crosslinked peptides. In such a case, tandem mass spectrometry (MS/MS) could provide the required information if the data can be properly interpreted. In MS/MS, a species of interest is isolated in the gas phase and allowed to undergo collision induced dissociation (CID). Because the gas-phase dissociation pathways of peptides have been well studied, methods are established for determining peptide sequence by MS/MS. However, although crosslinked peptides dissociate through some of the same pathways as isolated peptides, the additional dissociation pathways available to the former have not been studied in detail. Software such as MS2Assign hasmore » been written to assist in the interpretation of MS/MS from crosslinked peptide species, but it would be greatly enhanced by a more thorough understanding of how these species dissociate. We are thus systematically investigating the dissociation pathways open to crosslinked peptide species. A series of polyalanine and polyglycine model peptides have been synthesized containing one or two lysine residues to generate defined inter- and intra-molecular crosslinked species, respectively. Each peptide contains 11 total residues, and one arginine residue is present at the carboxy terminus to mimic species generated by tryptic digestion. The peptides have been allowed to react with a series of commonly used crosslinkers such as DSS, DSG, and DST. The tandem mass spectra acquired for these crosslinked species are being examined as a function of crosslinker identity, site(s) of crosslinking, and precursor charge state. Results from these model studies and observations from actual experimental systems are being incorporated into the MS2Assign software to enhance our ability to effectively use chemical crosslinking in protein complex determination.« less

Fourier transform tandem mass spectrometry (FT-MS/MS) can be used to unambiguously assign intramolecular chemical cross-links to specific amino acid residues even when two or more possible cross-linking sites are adjacent in the cross-linked protein. Bovine rhodopsin (Rho) in its dark-adapted state was intramolecularly cross-linked with lysine-cysteine (K-C) or lysine-lysine (K-K) cross-linkers to obtain interatomic distance information. Large, multiply charged, cross-linked peptide ions containing adjacent lysines, corresponding to Rho(50-86) (K(66) or K(67)) cross-linked to Rho(310-317) (C(316)) or Rho(318-348) (K(325) or K(339)), were fragmented by collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD), and electron capture dissociation (ECD). Complementary sequence-specific information was obtained by combining cross-link assignments; however, only ECD revealed full palmitoylation of adjacent cysteines (C(322) and C(323)) and cross-linking of K(67) (and not K(66)) to C(316), K(325), and K(339). ECD spectra contained crucial c- and z-ions resulting from cleavage of the bond between K(66) and K(67). To our knowledge, this work also presents the first demonstration that ECD can be used to characterize S-linked fatty acid acylation on cysteines. The comprehensive fragmentation of large peptides by CID, IRMPD, and particularly ECD, in conjunction with the high resolution and mass accuracy of FT-MS/MS, is shown to be a valuable means of characterizing mammalian membrane proteins with both chemical and posttranslational modifications.