BEGIN:VCALENDAR VERSION:2.0 PRODID:-//CERN//INDICO//EN BEGIN:VEVENT SUMMARY:Short Talk 9\, Annika Söderholm - Structure and substrate specifi city of the tryptophan biosynthesis enzyme IGPS from Pseudomonas aeruginos a DTSTART;VALUE=DATE-TIME:20191010T143000Z DTEND;VALUE=DATE-TIME:20191010T145000Z DTSTAMP;VALUE=DATE-TIME:20260525T152650Z UID:indico-contribution-141-768@lindico453.srv.lu.se DESCRIPTION:Speakers: Annika Söderholm (Uppsala university)\nIn bacteria\ , tryptophan synthesis is performed by the enzymes encoded in the trp oper on. The product of the\ntrpC gene\, indole-3-glycerol phosphate synthase ( IGPS) catalyzes the indole-forming reaction of tryptophan\nsynthesis. The reaction mechanism includes a decarboxylation step of the substrate 1-(o-c arboxyphenylamino)\n1-deoxyribulose 5-phosphate (CdRP). The decarboxylatio n has been assumed to constitute an essential step\nof the mechanism since no activity with the decarboxylated variant of the substrate\, phenylamin odeoxyribulosephosphate\n(PAdRP)\, was observed in an early study on IGPS from Escherichia coli (Smith and Yanofsky\,\n1962).\nIn this study\, we de monstrate enzyme-catalyzed formation of the native product IGP from decarb oxylated\nsubstrate PAdRP using IGPS from Pseudomonas aeruginosa. Moreover \, the crystal structure of P. aeruginosa\nIGPS in complex with a substrat e analogue was solved to 2.1 Å resolution. By structural comparison to E. coli\nIGPS (Wilmanns et al.\, 1992)\, we provide structure-based hypothese s on the difference in substrate specificity\nbetween the E.coli and P. ae ruginosa homologs.\n\nReferences:\nSmith\, B. O. H. and Yanofsky\, C. (196 2) ‘Enzymes Involved in the Biosynthesis of Tryptophan’\, Methods Enzy mol.\,\n5\, pp. 794–806.\nWilmanns\, M. et al. (1992) ‘Three-dimension al structure of the bifunctional enzyme phosphoribosylanthranilate\nisomer ase: Indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 Å resolution’\, Journal\nof Molecular Biology\, 223(2)\, pp. 477–507 .\n\nhttps://lindico453.srv.lu.se/event/125/contributions/768/ LOCATION:Kulturen Auditorium URL:https://lindico453.srv.lu.se/event/125/contributions/768/ END:VEVENT BEGIN:VEVENT SUMMARY:Short Talk 8\, Yong Wang - Integrative Ensemble Modeling of a Larg e Membrane Protein Complex Using Diverse and Ambiguous Information DTSTART;VALUE=DATE-TIME:20191010T141000Z DTEND;VALUE=DATE-TIME:20191010T143000Z DTSTAMP;VALUE=DATE-TIME:20260525T152650Z UID:indico-contribution-141-790@lindico453.srv.lu.se DESCRIPTION:Speakers: Yong Wang (University of Copenhagen)\nMitochondria c ontain approximately 1200 different proteins\, 99% of which are synthesize d on cytosolic ribosomes and need to be delivered into the right destinati on through the intermembrane space by transport machineries\, such as the TIM chaperone. Currently\, the mechanistic and structural details of how t he TIM chaperone binds to these mitochondrial proteins remain elusive. To gain structural insight into the binding and chaperone mechanisms\, we foc used on the complex of the TIM9/10 chaperone and the mitochondrial GDP/GTP carrier membrane protein (Ggc1). Such complexes are difficult to study be cause they consist of a transiently formed\, dynamic complex between two f olded proteins and a membrane protein that should be solubilized and bound by the chaperone. X-ray crystallography has revealed the core structure o f the free chaperone protein\, but because of the dynamic nature and large size (~1400 amino acids) of the complex its structural features have rema ined elusive. Using an integrative approach that combines biochemical assa ys\, NMR spectroscopy and SAXS it was\, however\, able to obtain detailed but ambiguous information on the structures of the complex. In particular\ , the experiments showed that the complex consists of two well-structured (TIM9)3/(TIM10)3 hexamers bound to a mostly disordered Ggc1. In this work\ , we developed a protocol to integrate such heterogeneous experimental dat a with a coarse-grained molecular model to provide a description of the co nformational ensemble of the TIM9/10-Ggc1 complex. In particular\, we used a hybrid structure-based model (to describe the intra-molecular interacti ons within the folded chaperone)\, an NMR-derived contact potential for ch aperone-client interactions and a knowledge-based potential (to describe t he inter-molecular interactions between the chaperones and chaperone-clien t interactions). We used molecular dynamics (MD) simulations to sample the conformational landscape of the complex\, and the resulting coarse-graine d conformational ensemble was subsequently converted into all-atom resolut ion and refined using a Bayesian/Maximum Entropy re-weighting approach usi ng the SAXS data. This allows us to generate a weighted ensemble in agreem ent with experimental measurement. Such integrative structural modeling me thod is useful to generate a structural ensemble of large and dynamic prot eins in a both efficient and reliable way.\n\nReference:\nKatharina Weinh äupl\, Caroline Lindau\, Audrey Hessel\, Yong Wang\, Conny Schütze\, To bias Jores\,\nLaura Melchionda\, Birgit Schönfisch\, Hubert Kalbacher\, B eate Bersch\, Doron Rapaport\, Martha\nBrennich\, Kresten Lindorff-Larsen\ , Nils Wiedemann* and Paul Schanda*. Structural Basis of\nMembrane Protein Chaperoning Through the Mitochondrial Intermembrane Space. Cell\, 175\, 1 365-\n1379\, (2018)\n\nhttps://lindico453.srv.lu.se/event/125/contribution s/790/ LOCATION:Kulturen Auditorium URL:https://lindico453.srv.lu.se/event/125/contributions/790/ END:VEVENT BEGIN:VEVENT SUMMARY:Keynote 8\, Prof. Henning Tidow: Structural studies of integral me mbrane proteins using stealth carrier nanodiscs DTSTART;VALUE=DATE-TIME:20191010T145000Z DTEND;VALUE=DATE-TIME:20191010T153000Z DTSTAMP;VALUE=DATE-TIME:20260525T152650Z UID:indico-contribution-141-769@lindico453.srv.lu.se DESCRIPTION:Speakers: Henning Tidow (Hamburg University)\nStructural studi es of integral membrane proteins (IMPs) are challenging\, as many of them are inactive or insoluble in the absence of a lipid environment. We pionee red an approach making use of fractionally deuterium labelled ‘stealth c arrier’ nanodiscs that are effectively invisible to low-resolution neutr on diffraction and enable structural studies of IMPs in a lipidic native-l ike solution environment. We show the potential of the method in a joint s mall-angle neutron scattering (SANS) and X-ray scattering (SAXS) study of the ATP-binding cassette (ABC) transporter protein MsbA solubilized in the stealth nanodiscs. The data allow for a direct observation of the signal from the solubilized protein without contribution from the surrounding lip id nanodisc. Not only the overall shape but also differences between confo rmational states of MsbA can be reliably detected from the scattering data \, demonstrating the sensitivity of the approach and its general applicabi lity to structural studies of IMPs. In a follow-up project\, we could also apply this method to investigate the structural basis for the activation of an essential Ca2+-pump by its regulator calmodulin.\nThis methodology c an be applied to other classes of integral membrane proteins and paves the way for low-resolution structure determination of IMPs in solution using both ab initio and rigid body analysis approaches.\n\nhttps://lindico453.s rv.lu.se/event/125/contributions/769/ LOCATION:Kulturen Auditorium URL:https://lindico453.srv.lu.se/event/125/contributions/769/ END:VEVENT END:VCALENDAR