Systems structural biology measurements by in vivo cross-linking with mass spectrometry.

This protocol describes a workflow for utilizing large-scale cross-linking with mass spectrometry (XL-MS) to make systems-level structural biology measurements in complex biological samples, including cells, isolated organelles, and tissue samples. XL-MS is a structural biology technique that provides information on the molecular structure of proteins and protein complexes using chemical probes that report the proximity of probe-reactive amino acids within proteins, typically lysine residues. Information gained through XL-MS studies is often complementary to more traditional methods, such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy. The use of MS-cleavable cross-linkers, including protein interaction reporter (PIR) technologies, enables XL-MS studies on protein structures and interactions in extremely complex biological samples, including intact living cells. PIR cross-linkers are designed to contain chemical bonds at specific locations within the cross-linker molecule that can be selectively cleaved by collision-induced dissociation or UV light. When broken, these bonds release the intact peptides that were cross-linked, as well as a reporter ion. Conservation of mass dictates that the sum of the two released peptide masses and the reporter mass equals the measured precursor mass. This relationship is used to identify cross-linked peptide pairs. Release of the individual peptides permits accurate measurement of their masses and independent amino acid sequence determination by tandem MS, allowing the use of standard proteomics search engines such as Comet for peptide sequence assignment, greatly simplifying data analysis of cross-linked peptide pairs. Search results are processed with XLinkProphet for validation and can be uploaded into XlinkDB for interaction network and structural analysis.

Insights in luteovirid structural biology guided by chemical cross-linking and high resolution mass spectrometry.

Interactions among plant pathogenic viruses in the family Luteoviridae and their plant hosts and insect vectors are governed by the topology of the viral capsid, which is the sole vehicle for long distance movement of the viral genome. Previous application of a mass spectrometry-compatible cross-linker to preparations of the luteovirid Potato leafroll virus (PLRV; Luteoviridae: Polerovirus) revealed a detailed network of interactions between viral structural proteins and enabled generation of the first cross-linking guided coat protein models. In this study, we extended application of chemical cross-linking technology to the related Turnip yellows virus (TuYV; Luteoviridae: Polerovirus). Remarkably, all cross-links found between sites in the viral coat protein found for TuYV were also found in PLRV. Guided by these data, we present two models for the TuYV coat protein trimer, the basic structural unit of luteovirid virions. Additional cross-links found between the TuYV coat protein and a site in the viral protease domain suggest a possible role for the luteovirid protease in regulating the structural biology of these viruses.

Dynamic Proteome Response of Pseudomonas aeruginosa to Tobramycin Antibiotic Treatment.

Genetically susceptible bacteria become antibiotic tolerant during chronic infections, and the mechanisms responsible are poorly understood. One factor that may contribute to differential sensitivity in vitro and in vivo is differences in the time-dependent tobramycin concentration profile experienced by the bacteria. Here, we examine the proteome response induced by subinhibitory concentrations of tobramycin in Pseudomonas aeruginosa cells grown under planktonic conditions. These efforts revealed increased levels of heat shock proteins and proteases were present at higher dosage treatments (0.5 and 1 µg/ml), while less dramatic at 0.1 µg/ml dosage. In contrast, many metabolic enzymes were significantly induced by lower dosages (0.1 and 0.5 µg/ml) but not at 1 µg/ml dosage. Time course proteome analysis further revealed that the increase of heat shock proteins and proteases was most rapid from 15 min to 60 min, and the increased levels sustained till 6 h (last time point tested). Heat shock protein IbpA exhibited the greatest induction by tobramycin, up to 90-fold. Nevertheless, deletion of ibpA did not enhance sensitivity to tobramycin. It seemed possible that the absence of sensitization could be due to redundant functioning of IbpA with other proteins that protect cells from tobramycin. Indeed, inactivation of two heat shock chaperones/proteases in addition to ibpA in double mutants (ibpA/clpB, ibpA/PA0779 and ibpA/hslV) did increase tobramycin sensitivity. Collectively, these results demonstrate the time- and concentration-dependent nature of the P. aeruginosa proteome response to tobramycin and that proteome modulation and protein redundancy are protective mechanisms to help bacteria resist antibiotic treatments.

Cross-linking measurements of the Potato leafroll virus reveal protein interaction topologies required for virion stability, aphid transmission, and virus-plant interactions.

Protein interactions are critical determinants of insect transmission for viruses in the family Luteoviridae. Two luteovirid structural proteins, the capsid protein (CP) and the readthrough protein (RTP), contain multiple functional domains that regulate virus transmission. There is no structural information available for these economically important viruses. We used Protein Interaction Reporter (PIR) technology, a strategy that uses chemical cross-linking and high resolution mass spectrometry, to discover topological features of the Potato leafroll virus (PLRV) CP and RTP that are required for the diverse biological functions of PLRV virions. Four cross-linked sites were repeatedly detected, one linking CP monomers, two within the RTP, and one linking the RTP and CP. Virus mutants with triple amino acid deletions immediately adjacent to or encompassing the cross-linked sites were defective in virion stability, RTP incorporation into the capsid, and aphid transmission. Plants infected with a new, infectious PLRV mutant lacking 26 amino acids encompassing a cross-linked site in the RTP exhibited a delay in the appearance of systemic infection symptoms. PIR technology provided the first structural insights into luteoviruses which are crucially lacking and are involved in vector-virus and plant-virus interactions. These are the first cross-linking measurements on any infectious, insect-transmitted virus.