MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it pH dependence of listeriolysin O aggregation and pore-forming ability. pH dependence of listeriolysin O aggregation and pore-forming ability. FEBS J. (1742-464X) 2012 imex curation Bavdek A., Kostanjšek R., Antonini V., Lakey JH., Dalla Serra M., Gilbert RJ., Anderluh G. Only protein-protein interactions in vitro In vitro solution sedimentati cosedimentation in solution solution sedimentati predetermined predetermined participant predetermined Bavdek A., Kostanjšek R., Antonini V., Lakey JH., Dalla Serra M., Gilbert RJ., Anderluh G. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation pH dependence of listeriolysin O aggregation and pore-forming ability. pH dependence of listeriolysin O aggregation and pore-forming ability. FEBS J. (1742-464X) 2012 imex curation Bavdek A., Kostanjšek R., Antonini V., Lakey JH., Dalla Serra M., Gilbert RJ., Anderluh G. Only protein-protein interactions in vitro In vitro fluorescence fluorescence technology fluorescence predetermined predetermined participant predetermined Bavdek A., Kostanjšek R., Antonini V., Lakey JH., Dalla Serra M., Gilbert RJ., Anderluh G. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation pH dependence of listeriolysin O aggregation and pore-forming ability. pH dependence of listeriolysin O aggregation and pore-forming ability. FEBS J. (1742-464X) 2012 imex curation Bavdek A., Kostanjšek R., Antonini V., Lakey JH., Dalla Serra M., Gilbert RJ., Anderluh G. Only protein-protein interactions in vitro In vitro electron microscopy electron microscopy Electron crystallography Electron cryomicroscopy Electron crystallography Electron cryomicroscopy predetermined predetermined participant predetermined Bavdek A., Kostanjšek R., Antonini V., Lakey JH., Dalla Serra M., Gilbert RJ., Anderluh G. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation pH dependence of listeriolysin O aggregation and pore-forming ability. pH dependence of listeriolysin O aggregation and pore-forming ability. FEBS J. (1742-464X) 2012 imex curation Bavdek A., Kostanjšek R., Antonini V., Lakey JH., Dalla Serra M., Gilbert RJ., Anderluh G. Only protein-protein interactions in vitro In vitro light scattering light scattering predetermined predetermined participant predetermined Bavdek A., Kostanjšek R., Antonini V., Lakey JH., Dalla Serra M., Gilbert RJ., Anderluh G. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation tacy_lismo Listeriolysin O LLO Thiol-activated cytolysin hlyA lisA lmo0202 hly protein protein lismo Listeria monocytogenes serovar 1/2a (strain ATCC BAA-679 / EGD-e) MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASPPASPKTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRTVIDDRNLPLVKNRNISIWGTTLYPKYSNKVDNPIE 83B6B0C11C033FB1 hly-hly 1 LLO 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined LLO 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f2 mint Overall the conclusion for LLO at pH 5.5 is that it is predominately undergoing rapidly interconverting interactions resulting in a complex distribution of apparent sedimentation coefficients in the region of 7 S. At pH 7.5, however, the migration of LLO was very different; instead of well-defined peaks a single, broader c(s) distribution was observed which, like that around 7 S at pH 5.5 could not be accurately described by a single Gaussian but could be deconvoluted as two Gaussians. One was distributed around a sedimentation coefficient of 2.9 S, similar to the 3.1 S seen for LLO at pH 5.5, and one was distributed around 3.6 S. The merging of these two peaks in a single overall distribution suggests that they are representative of a rapid interconversion of two different conformations of the LLO monomer. Overall, the results at pH 7.5 indicate metastability in the LLO structure. To further investigate the relationship between the solution states of LLO and pH we observed the behaviour of the protein as the temperature was raised within the analytical ultracentrifuge (Fig. 2b and c). As the temperature is raised at pH 5.5 the ~7 S peak, visible at 20 °C (Fig. 2a), decreased in amplitude and first larger and then smaller species were visible in an increasingly complex sedimentation profile. This indicates the formation of LLO oligomers in solution, and the stabilisation of the rapidly interconverting interactions seen at 20°C in a series of discrete higher order oligomers. At pH 7.5 the rapidly equilibrating ~7 S peak is absent at a constant 20 °C but as the temperature is raised there is initially an apparent formation of the same well-defined oligomers seen at pH 5.5 but then, when 37 °C is reached, all the higher-order oligomers form a single, broad peak at 11 S. hly-hly-1 3 LLO 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined LLO 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction mint We have therefore also checked the presence of high-molecular weight aggregates or dimers by transmission electron microscopy (TEM) (Fig. 3). When LLO in buffer with pH 5.5 was deposited on grids and negatively stained, we noted slightly curved ~20 nm long particles, but no larger structures (Fig. 3a). These were visible even if LLO was preincubated for 30 min at 37 °C (Fig. 3b). CDCs are slightly elongated rod-shaped molecules, measuring 115 Å × 30 Å × 55 Å [16]. The observed particles may thus correspond to self-associated aggregates of a couple of LLO molecules and we do not believe they represent nascent, arc-shaped oligomers. We could not observe these particles when LLO was imaged at pH 7.5 below 20 °C. At higher temperatures amorphous aggregates were visible f3 f5b hly-hly-2 2 LLO 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined LLO 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction mint Aggregates are not ordered or fibrillar in structure, but are amorphous μm-sized structures as revealed by TEM (Fig. 5b) and bright-field microscopy images (Fig. 5c). We have also checked whether aggregated state of LLO at pH 7.5 affect the absorbance and the fluorescence of Congo red and Thioflavine T dyes, respectively, which are routinely used to stain β-sheet amyloid aggregates [33-36]. The LLO aggregates were easily stained in the presence of Congo red and were brightly fluorescent (Fig. 5c). We have observed a characteristic shift in the Congo red absorbance spectrum to approximately 540 nm (solid thick line in Fig. 5d) [33,35]. We have also noted an increase in the Thioflavin T emission maximum in the presence of LLO aggregates (Fig. 5e). f5c f5d f5e f6a f6b t1 hly-hly-3 4 LLO 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined LLO 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction mint f6a f6b t1